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ePubs

Here is a listing of electronic publications authored by NAVBO members and of relevance to the entire Vascular Biology Community. 

ePubs

Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice

Authors:
Zhu W, Saw D, Weiss M, Sun Z, Wei M, Shaligram S, Wang S, Su H

Abstract:
Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.

Citation:
Transl Stroke Res. 2018 Dec 3. doi: 10.1007/s12975-018-0676-1. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/30511203

Partial Deletion of Tie2 Affects Microvascular Endothelial Responses to Critical Illness in a Vascular Bed and Organ-Specific Way

Authors:
Jongman RM, Zwiers PJ, van de Sluis B, van der Laan M, Moser J Zijlstra JG, Dekker D, Huijkman N, Moorlag HE, Popa ER, Molema G, van Meurs M

Abstract:
Tie2 is a tyrosine kinase receptor that is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behaviour are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly-generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, VCAM-1, and ICAM-1, and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared to Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared to controls. In contrast to the hypothesis that a dysbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.

Citation:
Shock. 2018 Jul 30. doi: 10.1097/SHK.0000000000001226. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/30520765

Early Heterogenic Response of Renal Microvasculature to Hemorrhagic Shock/Resuscitation and the Influence of NF-κB Pathway Blockade

Authors:
Yan R, van Meurs M, Popa ER, Li R, Zwiers PJ, Zijlstra JG, Molema G.

Abstract:
Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with an NF-κB inhibitor on these responses. We found that HS/R disturbed the balance of the Angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, pro-inflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Citation:
Shock. 2018 Feb 21. doi: 10.1097/SHK.0000000000001126. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29470361

Phenotypic characterization of murine models of cerebral cavernous malformations

Authors:
Zeineddine HA, Girard R, Saadat L, Shen L, Lightle R, Moore T, Cao Y, Hobson N, Shenkar R, Avner K, Chaudager K, Koskimäki J, Polster SP, Fam MD, Shi C, Lopez-Ramirez MA, Tang AT, Gallione C, Kahn ML, Ginsberg M, Marchuk DA, Awad IA

Abstract:
Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.

Citation:
Lab Invest. 2018 Jun 26. doi: 10.1038/s41374-018-0030-y. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29946133

Cerebral cavernous malformations form an anticoagulant vascular domain

Authors:
Lopez-Ramirez MA, Pham A, Girard R, Wyseure T, Hale P, Yamashita A, Koskimäki J, Polster S, Saadat L, Romero IA, Esmon CT, Lagarrigue F, Awad IA, Mosnier LO, Ginsberg MH

Abstract:
Cerebral cavernous malformations (CCM) are common brain vascular dysplasias prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here we show that central nervous system (CNS) hemorrhage in CCM is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions and in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO ) or Pdcd10 (Pdcd10ECKO ), which cause CCM formation, result in increased levels of vascular TM and EPCR, and in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10 Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of one or two copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial co-factors that generate anticoagulant APC can contribute to bleeding in CCMs and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

Citation:
Blood. 2018 Nov 15. pii: blood-2018-06-856062. doi: 10.1182/blood-2018-06-856062. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/30442679

Association of Somatic GNAQ Mutation With Capillary Malformations in a Case of Choroidal Hemangioma

Authors:
Colette A. Bichsel, Jeremy Goss, Mohammed Alomari , Sanda Alexandrescu, Richard Robb, Lois E. Smith , Marcelo Hochman, Arin Greene, Joyce Bischoff

Abstract:
Importance Choroidal hemangiomas are defined by a thickened choroid owing to vessel overgrowth, which may increase the intraocular pressure and lead to glaucoma. Choroidal hemangioma and glaucoma often co-occur in patients with Sturge-Weber syndrome, a rare neurocutaneous disorder characterized by capillary malformations. Objective To determine whether the mutation found in most capillary malformations, GNAQ R183Q (c.548G>A), was present in the choroidal hemangioma of a patient with Sturge-Weber syndrome. Design, Setting, and Participant Using laser-capture microdissection, choroidal blood vessels were isolated from paraffin-embedded tissue sections, and genomic DNA was extracted for mutational analysis. Choroidal sections were analyzed in parallel. A patient with choroidal hemangioma and Sturge-Weber syndrome who had undergone enucleation was analyzed in this study at Boston Children’s Hospital. Negative controls were choroidal tissue from an eye with retinoblastoma and unaffected lung tissue; brain tissue from a different patient with Sturge-Weber syndrome served as a positive control. Infantile hemangioma was analyzed as well. Data were analyzed in 2018. Main Outcomes and Measures The mutant allelic frequency of GNAQ R183 and GNAQ Q209L/H/P was determined by droplet digital polymerase chain reaction on isolated genomic DNA. The infantile hemangioma marker glucose transporter-1 was visualized by immunofluorescent staining of tissue sections. Results The GNAQ R183Q mutation was present in the patient’s choroidal vessels (21.1%) at a frequency similar to that found in brain tissue from a different patient with Sturge-Weber syndrome (25.1%). In contrast, choroidal vessels from a case of retinoblastoma were negative for the mutation (0.5%), as was lung tissue (0.2%). The patient’s choroidal tissue was negative for the 3 GNAQ mutations associated with congenital hemangioma and for the infantile hemangioma marker glucose transporter-1. Conclusions and Relevance The results suggest that a more accurate description for choroidal hemangioma in patients with Sturge-Weber syndrome is choroidal capillary malformation. This finding may explain why propranolol, used to treat infantile hemangiomas, has been largely ineffective in patients with choroidal hemangioma. Further studies are needed to corroborate this finding.

Citation:
JAMA Ophthalmol. Published online October 25, 2018. doi:10.1001/jamaophthalmol.2018.5141

https://jamanetwork.com/journals/jamaophthalmology/article-abstract/2708000?utm_source=jps&utm_medium=email&utm_campaign=author_alert-jamanetwork&utm_content=author-author_engagement&utm_term=1m

HIV-Nef Protein Persists in the Lungs of Aviremic HIV Patients and Induces Endothelial Cell Death

Authors:
Chelvanambi S, Bogatcheva N, Bednorz M, Agarwal S, Maier B, Alves NJ, Li W, Syed F, Saber MM, Dahl N, Lu H, Day RB, Smith P, Jolicoeur P, Yu Q, Dhillon NK, Weissmann N, Twigg Iii HL, Clauss M

Abstract:
It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV encoded proteins in latently HIV-infected T cells and macrophages. The pro-apoptotic protein HIV-Nef persists in the blood of ART treated patients within extracellular vesicles (EV) and PBMC. Here we demonstrate that HIV-Nef is present in cells and extracellular vesicles (EV) isolated from bronchoalveolar lavage (BAL) of patients on ART. We hypothesize that HIV-Nef persistence in lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. HIV-Nef presence in HIV patients correlates with the surface expression of the pro-apoptotic Endothelial-Monocyte Activating Polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in HEK293T, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EV and increases the amount of EV secreted from Nef-expressing T cells and Nef-transfected HEK293T cells. EV from BAL of HIV-positive patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in VE-Cadherin-positive endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis using an EMAPII dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

Citation:
Am J Respir Cell Mol Biol. 2018 Oct 15. doi: 10.1165/rcmb.2018-0089OC. [Epub ahead of print]

https://www.atsjournals.org/doi/10.1165/rcmb.2018-0089OC

Lysophosphatidic acid acts on LPA1 receptor to increase H2O2 during flow‐induced dilation in human adipose arterioles

Authors:
Chabowski DS, Kadlec AO, Ait-Aissa K, Hockenberry JC, Pearson PJ, Beyer AM, Gutterman DD

Abstract:
BACKGROUND AND PURPOSE: Nitric oxide (NO) produces arteriolar flow-induced dilation (FID) in healthy subjects but is replaced by mitochondria-derived hydrogen peroxide (mtH2 O2 ) in patients with coronary artery disease (CAD). Lysophosphatidic acid (LPA) is elevated in patients with risk factors for CAD but its functional effect in arterioles is unknown. We tested whether elevated LPA changes the mediator of FID from NO to mtH2 O2 in human visceral and subcutaneous adipose arterioles.

EXPERIMENTAL APPROACH: Arterioles were cannulated on glass micropipettes and pressurized to 60 mmHg. We recorded lumen diameter after graded increases in flow in the presence of either nitric oxide synthase (NOS) inhibition (L-NAME) or H2 O2 scavenging (Peg-Cat) ± LPA (10 μM, 30 min), ± LPA1 /LPA3 receptor antagonist (Ki16425) or LPA2 receptor antagonist (H2L5186303). We analyzed LPA receptor RNA and protein levels in human arterioles and cultured human endothelial cells. KEY RESULTS: FID was inhibited by L-NAME but not Peg-Cat in untreated vessels. In vessels treated with LPA, FID was of similar magnitude but inhibited by Peg-Cat while L-NAME had no effect. Rotenone attenuated FID in vessels treated with LPA indicating mitochondria as a source of ROS. RNA transcripts from LPA1 and LPA2 but not LPA3 receptor were detected in arterioles. LPA1 but not LPA3 receptor protein was detected by Western blot. Pretreatment of vessels with an LPA1 /LPA3 , but not LPA2 , receptor antagonist prior to LPA preserved NO-mediated dilation. CONCLUSIONS AND IMPLICATIONS: These findings suggest an LPA1 receptor-dependent pathway by which LPA increases arteriolar release of mtH2 O2 as a mediator of FMD.

Citation:
Br J Pharmacol. 2018 Aug 28. doi: 10.1111/bph.14492. [Epub ahead of print]

https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14492

Cerebral Cavernous Malformations Develop Through Clonal Expansion of Mutant Endothelial Cells

Authors:
Matthew R Detter, Daniel A Snellings, and Douglas A Marchuk

Abstract:
Rationale: Vascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations. Objective: We utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multi-color fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation. Methods and Results: We combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with one of four possible colors. We acquired Z-series confocal images from serial brain sections and created 3D reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3D distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (p<0.001). Many of the small (<100m diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100m diameter) CCMs contained both the clonally dominant mutant cells and wild type ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wild type ECs to increase the size of the malformation. Conclusions: This is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wild type ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.

Citation:
Originally published 18 Sep 2018 Circulation Research. 2018

https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.118.313970

Gene expression profiles of rat MMECs with different glucose levels and fgl2 gene silencing

Authors:
Zheng Z, Zhang F, Gao D, Wu Y, Wu H

Abstract:
BACKGROUND: Cardiac microvascular endothelial cells is one of the key factors in the process of diabetic cardiomyopathy, a common chronic complication of diabetes. Fibrinogen-like protein 2 (FGL2) is linked to apoptosis, angiogenesis, and inflammatory response, all of which also occur in diabetes. Thus, we investigate the role of FGL 2 and other genes in the pathology of diabetic cardiomyopathy.

METHODS: In the present study, we used high-throughput microarray to profile gene expression in rat myocardial microvascular endothelial cells (MMEC) with or without silencing the fgl2 gene and in different glucose environments. We use volcanic maps to isolate genes with significantly different expression levels between conditions, using the standard statistical criteria of fold changes ≥ 1.5 and P-values ≤ 0.05. From this list, we identified genes with the most signicant changes in RNA levels and confirmed their protein-level changes with Western blot. Furthermore, bioinformatic analysis predicts possible pathophysiology and clinical relevance of these proteins in diabetic cardiomyopathy. RESULTS: We identified 17 upregulated and 15 downregulated genes caused by silencing fgl2 gene. Most of them are involved in metabolism, ion transport, cell membrane surface recognition signal modification, inflammatory response, and immune response. Using Western blott, we were able to confirm protein-level expression changes of 3 genes. Specifically, in both normal and high glucose conditions, silencing fgl2 significantly decreased the expression levels of CCL3 and PLAGL1 while increasing the expression level of CTSC. Significantly, bioinformatic analyses show that CCL3 is related to type 1 diabetes mellitus, PLAGL1 to cardiomyocytes, and CTSC to albuminuria in type 2 diabetes. CONCLUSIONS: Our study provides clues for further studies on the mechanism of diabetic cardiomyopathy as well as function of FGL2 in this process, potentially offering new therapeutic strategies for treating diabetic cardiomyopathy.

Citation:
Diabetes Metab Res Rev. 2018 Aug 11:e3058. doi: 10.1002/dmrr.3058. [Epub ahead of print]

https://onlinelibrary.wiley.com/doi/pdf/10.1002/dmrr.3058

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes

Authors:
Wu H, Rahman HNA, Dong Y, Liu X, Lee Y, Wen A, To KH, Xiao L, Birsner AE, Bazinet L, Wong S, Song K, Brophy ML, Mahamud MR, Chang B, Cai X, Pasula S, Kwak S, Yang W, Bischoff J, Xu J, Bielenberg DR, Dixon JB, D'Amato RJ, Srinivasan RS, Chen H

Abstract:
Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

Citation:
J Clin Invest. 2018 Aug 13. pii: 96063. doi: 10.1172/JCI96063. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/30102256

Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

Authors:
Paik DT, Tian L, Lee J, Sayed N, Chen IY, Rhee S, Rhee JW, Kim Y, Wirka RC, Buikema JW, Wu SM, Red-Horse K, Quertermous T, Wu JC

Abstract:
Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology.

Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

Citation:
Circ Res. 2018 Jul 9. pii: CIRCRESAHA.118.312913. doi: 10.1161/CIRCRESAHA.118.312913. [Epub ahead of print]

https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.118.312913

Estrogen Inhibits LDL (Low-Density Lipoprotein) Transcytosis by Human Coronary Artery Endothelial Cells via GPER (G-Protein–Coupled Estrogen Receptor) and SR-BI

Authors:
Ghaffari S, Naderi Nabi F, Sugiyama MG, Lee WL

Abstract:
Objective - The atheroprotective effects of estrogen are independent of circulating lipid levels. Whether estrogen regulates transcytosis of LDL (low-density lipoprotein) across the coronary endothelium is unknown. Approach and Results - Using total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells from multiple donors. LDL transcytosis was significantly higher in cells from men compared with premenopausal women. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male but not female donors; transcytosis of albumin was not affected. Estrogen caused downregulation of endothelial SR-BI, and overexpression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen’s effect on LDL transcytosis was blocked by depletion of the GPER (G-protein–coupled estrogen receptor). GPER was found to be enriched in endothelial cells compared with hepatocytes and is reported to signal via transactivation of the EGFR (epidermal growth factor receptor); inhibition of EGFR prevented the effect of estrogen on LDL transcytosis and SR-BI mRNA. Last, SR-BI expression was significantly higher in human coronary artery endothelial cells from male compared with premenopausal female donors. Conclusions - Estrogen significantly inhibits LDL transcytosis by downregulating endothelial SR-BI; this effect requires GPER.

Citation:
Arteriosclerosis, Thrombosis, and Vascular Biology. 2018;0:ATVBAHA.118.310792

https://www.ahajournals.org/doi/abs/10.1161/ATVBAHA.118.310792

Near‐Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution

Authors:
Zhuoran Ma, Mingxi Zhang, Jingying Yue, Cynthia Alcazar, Yeteng Zhong, Timothy C. Doyle, Hongjie Dai Ngan F. Huang

Abstract:
Real‐time optical imaging is a promising approach for visualizing in vivo hemodynamics and vascular structure in mice with experimentally induced peripheral arterial disease (PAD). The application of a novel fluorescence‐based all‐optical imaging approach in the near‐infrared IIb (NIR‐IIb, 1500–1700 nm emission) window, for imaging hindlimb microvasculature and blood perfusion in a mouse model of PAD is reported. In phantom studies, lead sulfide/cadmium sulfide (PbS/CdS) quantum dots show better retention of image clarity, in comparison with single‐walled nanotube (SWNT) NIR‐IIa (1000–1400 nm) dye, at varying depths of penetration. When systemically injected to mice, PbS/CdS demonstrates improved clarity of the vasculature, compared to SWNTs, as well as higher spatial resolution than in vivo microscopic computed tomography. In a mouse model of PAD, NIR‐IIb imaging of the ischemic hindlimb vasculature shows significant improvement in blood perfusion over the course of 10 days (P < 0.05), as well as a significant increase in microvascular density over the first 7 days after induction of PAD. In conclusion, NIR‐IIb imaging of PbS/CdS vascular contrast agent is a useful multifunctional imaging approach for high spatial resolution imaging of the microvasculature and quantification of blood perfusion recovery.

Citation:
Advanced Functional Materials,First published: 23 July 2018, https://doi.org/10.1002/adfm.201803417

https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201803417

Role of complement C5a and histones in septic cardiomyopathy

Authors:
Fattahi F, Frydrych LM, Bian G, Kalbitz M, Herron TJ, Malan EA, Delano MJ, Ward PA

Abstract:
Polymicrobial sepsis (after cecal ligation and puncture, CLP) causes robust complement activation with release of C5a. Many adverse events develop thereafter and will be discussed in this review article. Activation of complement system results in generation of C5a which interacts with its receptors (C5aR1, C5aR2). This leads to a series of harmful events, some of which are connected to the cardiomyopathy of sepsis, resulting in defective action potentials in cardiomyocytes (CMs), activation of the NLRP3 inflammasome in CMs and the appearance of extracellular histones, likely arising from activated neutrophils which form neutrophil extracellular traps (NETs). These events are associated with activation of mitogen-activated protein kinases (MAPKs) in CMs. The ensuing release of histones results in defective action potentials in CMs and reduced levels of [Ca2+]i-regulatory enzymes including sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and Na+/Ca2+ exchanger (NCX) as well as Na+/K+-ATPase in CMs. There is also evidence that CLP causes release of IL-1β via activation of the NLRP3 inflammasome in CMs of septic hearts or in CMs incubated in vitro with C5a. Many of these events occur after in vivo or in vitro contact of CMs with histones. Together, these data emphasize the role of complement (C5a) and C5a receptors (C5aR1, C5aR2), as well as extracellular histones in events that lead to cardiac dysfunction of sepsis (septic cardiomyopathy).

Citation:
Mol Immunol. 2018 Jun 15. pii: S0161-5890(18)30194-9. doi: 10.1016/j.molimm.2018.06.006. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29914696

Endothelial cells in the innate response to allergens and initiation of atopic asthma

Authors:
Asosingh K, Weiss K, Queisser K, Wanner N, Yin M, Aronica M, Erzurum S

Abstract:
Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Citation:
J Clin Invest. 2018 Jun 18. pii: 97720. doi: 10.1172/JCI97720. [Epub ahead of print]

https://www.jci.org/articles/view/97720

Oscillatory Strain Promotes Vessel Stabilization and Alignment through Fibroblast YAP‐Mediated Mechanosensitivity

Authors:
Shira Landau, Shahar Ben‐Shaul, Shulamit Levenberg

Abstract:
Endothelial cells form the interior layer of blood vessels and, as such, are constantly exposed to shear stress and mechanical strain. While the impact of shear stress on angiogenesis is widely studied, the role of mechanical strain is less understood. To this end, endothelial cells and fibroblasts are cocultured under oscillatory strain to create a vessel network. The two cell types show distinctly different sensitivities to the mechanical stimulation. The fibroblasts, sense the stress directly, and respond by increased alignment, proliferation, differentiation, and migration, facilitated by YAP translocation into the nucleus. In contrast, the endothelial cells form aligned vessels by tracking fibroblast alignment. YAP inhibition in constructs under mechanical strain results in vessel destruction whereas less damage is observed in the YAP‐inhibited static control. Moreover, the mechanical stimulation enhances vessel development and stabilization. Additionally, vessel orientation is preserved upon implantation into a mouse dorsal window chamber and promotes the invading host vessels to orient in the same manner. This study sheds light on the mechanisms by which mechanical strain affects the development of blood vessels within engineered tissues. This can be further utilized to engineer a more organized and stable vasculature suitable for transplantation of engineered grafts.

Citation:
Advanced Science, First published: 15 July 2018, https://doi.org/10.1002/advs.201800506

https://onlinelibrary.wiley.com/doi/abs/10.1002/advs.201800506

Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis

Authors:
Lino M, Wan MH, Rocca AS, Ngai D, Shobeiri N, Hou G, Ge C, Franceschi RT, Bendeck MP

Abstract:
OBJECTIVE: Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. APPROACH AND RESULTS: Ddr1+/+; Ldlr-/- (single knock-out) and Ddr1-/-; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. CONCLUSIONS: These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.

Citation:
Arterioscler Thromb Vasc Biol. 2018 Jun 21. pii: ATVBAHA.118.311238. doi: 10.1161/ATVBAHA.118.311238. [Epub ahead of print]

http://atvb.ahajournals.org/content/early/2018/06/20/ATVBAHA.118.311238

Use of 4-phenylbutyrate to define therapeutic parameters for reducing intracerebral hemorrhage and myopathy in Col4a1 mutant mice

Authors:
Hayashi G, Labelle-Dumais C, Gould DB

Abstract:
Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers that constitute a major component of nearly all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that includes variable cerebrovascular and skeletal muscle manifestations. The pathogenicity of COL4A1 and COL4A2 mutations is generally attributed to impaired secretion into basement membranes. Sodium 4-phenylbutyrate (4PBA) is an FDA-approved drug that promotes mutant heterotrimer secretion in vitro and in vivo Here, we use different 4PBA treatment paradigms to define therapeutic parameters for preventing cerebrovascular and muscular pathologies in Col4a1 mutant mice. We show efficacy of long-term 4PBA treatment in reducing the severity of intracerebral hemorrhages (ICH) in Col4a1 mutant mice aged up to 8 months. In addition, we demonstrate that maximal efficacy of 4PBA on ICH and myopathy was achieved when treatment was initiated prenatally, whereby even transient 4PBA administration had lasting benefits after being discontinued. Importantly, postnatal treatment with 4PBA also reduced ICH and skeletal myopathy severities in Col4a1 mutant mice, which has significant clinical implications for patients with COL4A1 and COL4A2 mutations.

Citation:
Dis Model Mech. 2018 Jun 12. pii: dmm.034157. doi: 10.1242/dmm.034157. [Epub ahead of print]

http://dmm.biologists.org/content/early/2018/06/09/dmm.034157

EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury

Authors:
Hongyan Lu, Sarvesh Chelvanambi, Christophe Poirier, Jacob Saliba, Keith L. March, Matthias Clauss, Natalia V. Bogatcheva

Abstract:
Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

Citation:
Mol Ther. 2018 Jun 14. pii: S1525-0016(18)30220-X. doi: 10.1016/j.ymthe.2018.05.017. [Epub ahead of print]

https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(18)30220-X

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells

Authors:
Ma N, Zhang J, Itzhaki I, Zhang SL, Chen H, Haddad F, Kitani T, Wilson KD, Tian L, Shrestha R, Wu H, Lam CK, Sayed N, Wu JC

Abstract:
Background -The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. Methods -To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUSMYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). Results -The heterozygous VUSMYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. Conclusions -Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.

Citation:
Circulation. 2018 Jun 18. pii: CIRCULATIONAHA.117.032273. doi: 10.1161/CIRCULATIONAHA.117.032273. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29914921

Progress, obstacles, and limitations in the use of stem cells in organ-on-a-chip models

Authors:
Wnorowski A, Yang H, Wu JC

Abstract:
In recent years, drug development costs have soared, primarily due to the failure of preclinical animal and cell culture models, which do not directly translate to human physiology. Organ-on-a-chip (OOC) is a burgeoning technology with the potential to revolutionize disease modeling, drug discovery, and toxicology research by strengthening the relevance of culture-based models while reducing costly animal studies. Although OOC models can incorporate a variety of tissue sources, the most robust and relevant OOC models going forward will include stem cells. In this review, we will highlight the benefits of stem cells as a tissue source while considering current limitations to their complete and effective implementation into OOC models.

Citation:
Adv Drug Deliv Rev. 2018 Jun 6. pii: S0169-409X(18)30132-7. doi: 10.1016/j.addr.2018.06.001. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29885330

Clinical factors that influence the cellular responses of saphenous veins used for arterial bypass

Authors:
Sobel M, Kikuchi S, Chen L, Tang GL, Wight TN, Kenagy RD

Abstract:
OBJECTIVE: When an autogenous vein is harvested and used for arterial bypass, it suffers physical and biologic injuries that may set in motion the cellular processes that lead to wall thickening, fibrosis, stenosis, and ultimately graft failure. Whereas the injurious effects of surgical preparation of the vein conduit have been extensively studied, little is known about the influence of the clinical environment of the donor leg from which the vein is obtained. METHODS: We studied the cellular responses of fresh saphenous vein samples obtained before implantation in 46 patients undergoing elective lower extremity bypass surgery. Using an ex vivo model of response to injury, we quantified the outgrowth of cells from explants of the adventitial and medial layers of the vein. We correlated this cellular outgrowth with the clinical characteristics of the patients, including the Wound, Ischemia, and foot Infection classification of the donor leg for ischemia, wounds, and infection as well as smoking and diabetes. RESULTS: Cellular outgrowth was significantly faster and more robust from the adventitial layer than from the medial layer. The factors of leg ischemia (P < .001), smoking (P = .042), and leg infection (P = .045) were associated with impaired overall outgrowth from the adventitial tissue on multivariable analysis. Only ischemia (P = .046) was associated with impaired outgrowth of smooth muscle cells (SMCs) from the medial tissue. Co-culture of adventitial cells and SMCs propagated from vein explants revealed that adventitial cells significantly inhibited the growth of SMCs, whereas SMCs promoted the growth of adventitial cells. The AA genotype of the -838C>A p27 polymorphism (previously associated with superior graft patency) enhanced these effects, whereas the factor of smoking attenuated adventitial cell inhibition of SMC growth. Comparing gene expression, the cells cultured from the media overexpress Kyoto Encyclopedia of Genes and Genomes pathways associated with inflammation and infection, whereas those from the adventitia overexpress gene families associated with development and stem/progenitor cell maintenance. CONCLUSIONS: The adverse clinical environment of the leg may influence the biologic behavior of the cells in the vein wall, especially the adventitial cells. Chronic ischemia was the most significant factor that retards adventitial cell outgrowth. The cells arising from the vein adventitia may be key players in determining a healthy adaptive or a pathologic response to the injuries associated with vein grafting.

Citation:
J Vasc Surg. 2018 Jun 15. pii: S0741-5214(18)31029-2. doi: 10.1016/j.jvs.2018.03.436. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29914830

Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS

Authors:
Carole Y. Perrot, Junko Sawada and Masanobu Komatsu

Abstract:
The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element–binding protein (CREB) 3–dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.—Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cyclic AMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

Citation:
FASEB J. 2018 May 18:fj201700818RRR. doi: 10.1096/fj.201700818RRR. [Epub ahead of print]

https://www.fasebj.org/doi/abs/10.1096/fj.201700818RRR?journalCode=fasebj

Lack of Tone in Mouse Small Mesenteric Arteries Leads to Outward Remodeling, which can be Prevented by Prolonged Agonist-Induced Vasoconstriction

Authors:
Anika Klein, Philomeena Daphne Joseph, Vibeke Grøsfjeld Christensen, Lars Jørn Jensen, and Jens Christian Brings Jacobsen

Abstract:
Inward remodeling of resistance vessels is an independent risk factor for cardiovascular events. Thus far, the remodeling process remains incompletely elucidated, but the activation level of the vascular smooth muscle cell (VSMC) appears to play a central role. Accordingly, previous data suggest that an antagonistic and, supposedly beneficial, response - outward remodeling - may follow prolonged vasodilatation. This study aims to determine if 1) outward remodeling follows 3 days of vessel culture without tone, 2) a similar response can be elicited in a much shorter 4-hour time frame and finally 3) a 4-hour response can be prevented or reversed by the presence of vasoconstrictors in the medium. Cannulated mouse small mesenteric arteries (SMA) were organo-cultured for 3 days in the absence of tone, leading to outward remodeling that continued throughout the culture period. In more acute experiments in which cannulated SMAs were maintained in physiological saline without tone for 4 hours, we detected a similar outward remodeling that proceeded at a rate several times faster. In the 4-hour experimental setting, continuous vasoconstriction to approximately 50% tone by abluminal application of uridine 5'-triphosphate (UTP) or norepinephrine (NE) + neuropeptide Y (NPY) prevented outward remodeling but did not cause inward remodeling. Computational modelling was used to simulate and interpret these findings and to derive time constants of the remodeling processes. It is suggested that depriving resistance arteries of activation will lead to eutrophic outward remodeling, which can be prevented by VSMC activation induced by prolonged vasoconstrictor exposure.

Citation:
Am J Physiol Heart Circ Physiol. 2018 May 18. doi: 10.1152/ajpheart.00111.2018. [Epub ahead of print]

https://www.physiology.org/doi/abs/10.1152/ajpheart.00111.2018

Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown

Authors:
Guangliang Bi, Lei Wu, Shamima Islam, Daniel P. Heruth, Li Qin Zhang, Ding-You Li, Venkatesh Sampath, Weimin Huang, Brett A. Simon, Ronald Blaine Easley, Joe G.N. Garcia and Shui Qing Ye

Abstract:
Although a deficiency of surfactant protein B (SFTPB) has been associated with lung injury, SFTPB expression has not yet been linked with nicotinamide phosphoribosyltransferase (NAMPT), a potential biomarker of acute lung injury (ALI). The effects of Nampt in the pulmonary epithelial cell on both SFTPB expression and lung inflammation were investigated in a LPS-induced ALI mouse model. Pulmonary epithelial cell-specific knockdown of Nampt gene expression, achieved by the crossing of Nampt gene exon 2 floxed mice with mice expressing epithelial-specific transgene Cre or by the use of epithelial-specific expression of anti-Nampt antibody cDNA, significantly attenuated LPS-induced ALI. Knockdown of Nampt expression was accompanied by lower levels of bronchoalveolar lavage (BAL) neutrophil infiltrates, total protein and TNF-α levels, as well as lower lung injury scores. Notably, Nampt knockdown was also associated with significantly increased BAL SFTPB levels relative to the wild-type control mice. Down-regulation of NAMPT increased the expression of SFTPB and rescued TNF-α-induced inhibition of SFTPB, whereas overexpression of NAMPT inhibited SFTPB expression in both H441 and A549 cells. Inhibition of NAMPT up-regulated SFTPB expression by enhancing histone acetylation to increase its transcription. Additional data indicated that these effects were mainly mediated by NAMPT nonenzymatic function via the JNK pathway. This study shows that pulmonary epithelial cell-specific knockdown of NAMPT expression attenuated ALI, in part, via up-regulation of SFTPB expression. Thus, epithelial cell-specific knockdown of Nampt may be a potential new and viable therapeutic modality to ALI.-Bi, G., Wu, L., Huang, P., Islam, S., Heruth, D. P., Zhang, L. Q., Li, D.-Y., Sampath, V., Huang, W., Simon, B. A., Easley, R. B., Ye, S. Q. Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.

Citation:
FASEB J. 2018 Feb 8:fj201701059R. doi: 10.1096/fj.201701059R. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29452569

Suppression of TGFβ-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition

Authors:
Kim DJ, Dunleavey JM, Xiao L, Ollila DW, Troester MA, Otey CA, Li W, Barker TH, Dudley AC

Abstract:
BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFβ-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.

Citation:
Br J Cancer. 2018 Apr 26. doi: 10.1038/s41416-018-0072-3. [Epub ahead of print]

https://www.nature.com/articles/s41416-018-0072-3

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment

Authors:
Griveau A, Seano G, Shelton SJ, Kupp R, Jahangiri A, Obernier K, Krishnan S, Lindberg OR, Yuen TJ, Tien AC, Sabo JK, Wang N, Chen I, Kloepper J, Larrouquere L, Ghosh M, Tirosh I, Huillard E, Alvarez-Buylla A, Oldham MC, Persson AI, Weiss WA, Batchelor TT, Stemmer-Rachamimov A, Suvà ML, Phillips JJ, Aghi MK, Mehta S, Jain RK, Rowitch DH

Abstract:
Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Citation:
Cancer Cell. 2018 Apr 6. pii: S1535-6108(18)30125-9. doi: 10.1016/j.ccell.2018.03.020. [Epub ahead of print]

http://www.cell.com/cancer-cell/fulltext/S1535-6108(18)30125-9

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model

Authors:
Zhu W, Chen W, Zou D, Wang L, Bao C, Zhan L, Saw D, Wang S, Winkler E, Li Z, Zhang M, Shen F, Shaligram S, Lawton M, Su H

Abstract:
BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. METHODS: bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. RESULTS: Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. CONCLUSIONS: Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Citation:
Stroke. 2018 Mar 28. pii: STROKEAHA.117.020356. doi: 10.1161/STROKEAHA.117.020356. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=Thalidomide+Reduces+Hemorrhage+of+Brain++Arteriovenous+Malformations+in+a+Mouse+Model

Early Heterogenic Response of Renal Microvasculature to Hemorrhagic Shock/Resuscitation and the Influence of NF-κB Pathway Blockade

Authors:
Yan R, van Meurs M, Popa ER, Li R, Zwiers PJ, Zijlstra JG, Moser J, Molema G

Abstract:
Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with an NF-κB inhibitor on these responses. We found that HS/R disturbed the balance of the Angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, pro-inflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Citation:
Shock. 2018 Feb 21. doi: 10.1097/SHK.0000000000001126. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29470361

Mir-126 is a conserved modulator of lymphatic development

Authors:
Zacharias Kontarakis, Andrea Rossi, Sophie Ramas, Michael T. Dellinger, Didier Y.R. Stainier

Abstract:
Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4 +/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4 +/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.

Citation:
Developmental Biology, Available online 15 March 2018

https://www.sciencedirect.com/science/article/pii/S0012160618301386

Cross-talk between blood vessels and neural progenitors in the developing brain

Authors:
Mathew Tata, Christiana Ruhrberg

Abstract:
The formation of the central nervous system (CNS) involves multiple cellular and molecular interactions between neural progenitor cells (NPCs) and blood vessels to establish extensive and complex neural networks and attract a vascular supply that support their function. In this review, we discuss studies that have performed genetic manipulations of chick, fish and mouse embryos to define the spatiotemporal roles of molecules that mediate the reciprocal regulation of NPCs and blood vessels. These experiments have highlighted core functions of NPC-expressed ligands in initiating vascular growth into and within the neural tube as well as establishing the blood–brain barrier. More recent findings have also revealed indispensable roles of blood vessels in regulating NPC expansion and eventual differentiation, and specific regional differences in the effect of angiocrine signals. Accordingly, NPCs initially stimulate blood vessel growth and maturation to nourish the brain, but blood vessels subsequently also regulate NPC behaviour to promote the formation of a sufficient number and diversity of neural cells. A greater understanding of the molecular cross-talk between NPCs and blood vessels will improve our knowledge of how the vertebrate nervous system forms and likely help in the design of novel therapies aimed at regenerating neurons and neural vasculature following CNS disease or injury.

Citation:
Neuronal Signaling, Mar 30, 2018, 2(1)NS20170139; DOI: 10.1042/NS20170139

http://www.neuronalsignaling.org/content/2/1/NS20170139

Spatiotemporal Multi-omics Mapping Generates a Molecular Atlas of the Aortic Valve and Reveals Networks Driving Disease

Authors:
Schlotter F, Halu A, Goto S, Blaser MC, Body SC, Lee LH, Higashi H, DeLaughter DM, Hutcheson JD, Vyas P, Pham T, Rogers MA, Sharma A, Seidman CE, Loscalzo J, Seidman JG, Aikawa M, Singh SA, Aikawa E

Abstract:
Background -No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without invasive valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages. Moreover, it occurs in the context of a complex, multi-layered tissue architecture, a rich and abundant extracellular matrix phenotype, and a unique, highly plastic and multipotent resident cell population. Methods -A total of 25 human stenotic aortic valves obtained from valve replacement surgeries were analyzed by multiple modalities, including transcriptomics and global unlabeled and label-based tandem-mass-tagged proteomics. Segmentation of valves into disease-stage-specific samples was guided by near infrared molecular imaging, and anatomical layer-specificity was facilitated by laser capture microdissection. Side-specific cell cultures were subjected to multiple calcifying stimuli, and their calcification potential and basal/stimulated proteomes were evaluated. Molecular (protein-protein) interaction networks were built and their central proteins and disease associations were identified. Results -Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein (GFAP) as a specific marker of valvular interstitial cells (VICs) from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that in vitro, fibrosa-derived VICs demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured VICs, and that VICs exposed to ALPL-dependent and ALPL-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions -A spatially- and temporally-resolved multi-omics, and network and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a novel means of systematic disease ontology that is broadly applicable to comprehensive omics studies of cardiovascular diseases.

Citation:
Circulation. 2018 Mar 27. pii: CIRCULATIONAHA.117.032291. doi: 10.1161/CIRCULATIONAHA.117.032291. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2018/03/26/CIRCULATIONAHA.117.032291

Minoxidil improves vascular compliance, restores cerebral blood flow and alters extracellular matrix gene expression in a model of chronic vascular stiffness

Authors:
Knutsen R, Beeman SC, Broekelmann TJ, Liu D, Tsang KM, Kovacs A, Ye L, Danback J, Watson A, Wardlaw A, Wagenseil J, Garbow JR, Shoykhet M, Kozel BA

Abstract:
Increased vascular stiffness correlates with higher risk of cardiovascular complications in aging adults. Elastin insufficiency, as observed in patients with Williams-Beuren syndrome or with familial supravalvular aortic stenosis, also increases vascular stiffness and leads to arterial narrowing. We used Eln+/- mice to test the hypothesis that pathologically increased vascular stiffness with concomitant arterial narrowing leads to decreased blood flow to end organs such as the brain. We also hypothesized that drugs which remodel arteries and increase lumen diameter would improve flow. To test these hypotheses, we compared carotid blood flow using ultrasound and cerebral blood flow using MRI-based arterial spin labeling in WT and Eln+/- mice. We then studied how minoxidil, a KATP channel opener and vasodilator, affects vessel mechanics, blood flow and gene expression. Both carotid and cerebral blood flows were lower in Eln+/- than in WT mice. Treatment of Eln+/- mice with minoxidil lowered blood pressure and reduced functional arterial stiffness to WT levels. Minoxidil also improved arterial diameter and restored carotid and cerebral blood flows in Eln+/- mice. Beneficial effects persisted for weeks after drug removal. RNA-Seq analysis revealed differential expression of 127 extracellular matrix-related genes among the treatment groups. These results indicate that elastin insufficiency impairs end organ perfusion, which may contribute to increased cardiovascular risk. Minoxidil, despite lowering blood pressure, improves end organ perfusion. Changes in matrix gene expression and persistence of treatment effects after drug withdrawal suggest arterial remodeling. Such remodeling may benefit patients with genetic or age-dependent elastin insufficiency.

Citation:
Am J Physiol Heart Circ Physiol. 2018 Mar 2. doi: 10.1152/ajpheart.00683.2017. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29498532

Elastin-driven genetic diseases

Authors:
Lasio MLD, Kozel BA

Abstract:
Elastic fibers provide recoil to tissues that undergo repeated deformation, such as blood vessels, lungs and skin. Composed of elastin and its accessory proteins, the fibers are produced within a restricted developmental window and are stable for decades. Their eventual breakdown is associated with a loss of tissue resiliency and aging. Rare alteration of the elastin (ELN) gene produces disease by impacting protein dosage (supravalvar aortic stenosis, Williams Beuren syndrome and Williams Beuren region duplication syndrome) and protein function (autosomal dominant cutis laxa). This review highlights aspects of the elastin molecule and its assembly process that contribute to human disease and also discusses potential therapies aimed at treating diseases of elastin insufficiency.

Citation:
Matrix Biol. 2018 Feb 28. pii: S0945-053X(17)30369-4. doi: 10.1016/j.matbio.2018.02.021. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29501665

Extracellular bone morphogenetic protein modulator BMPER and twisted gastrulation homolog 1 preserve arterial-venous specification in zebrafish blood vessel development and regulate Notch signaling in endothelial cells

Authors:
Esser JS, Steiner RE, Deckler M, Schmitt H, Engert B, Link S, Charlet A, Patterson C, Bode C, Zhou Q, Moser M

Abstract:
The bone morphogenetic protein (BMP) signaling pathway plays a central role during vasculature development. Mutations or dysregulation of the BMP pathway members have been linked to arteriovenous malformations. In the present study, we investigated the effect of the BMP modulators bone morphogenetic protein endothelial precursor-derived regulator (BMPER) and twisted gastrulation protein homolog 1 (TWSG1) on arteriovenous specification during zebrafish development and analyzed downstream Notch signaling pathway in human endothelial cells. Silencing of bmper and twsg1b in zebrafish embryos by morpholinos resulted in a pronounced enhancement of venous ephrinB4a marker expression and concomitant dysregulated arterial ephrinb2a marker expression detected by in situ hybridization. As arteriovenous specification was disturbed, we assessed the impact of BMPER and TWSG1 protein stimulation on the Notch signaling pathway on endothelial cells from different origin. Quantitative real-time PCR (qRT-PCR) and western blot analysis showed increased expression of Notch target gene hairy and enhancer of split, HEY1/2 and EPHRINB2. Consistently, silencing of BMPER in endothelial cells by siRNAs decreased Notch signaling and downstream effectors. BMP receptor antagonist DMH1 abolished BMPER and BMP4 induced Notch signaling pathway activation. In conclusion, we found that in endothelial cells, BMPER and TWSG1 are necessary for regular Notch signaling activity and in zebrafish embryos BMPER and TWSG1 preserve arteriovenous specification to prevent malformations.

Citation:
FEBS J. 2018 Feb 23. doi: 10.1111/febs.14414. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=Extracellular+bone+morphogenetic+protein+modulator+BMPER+and+twisted+gastrulation+homolog+1+preserve+arterial-venous+specification+in+zebrafish+blood+vessel+development+and+regulate+Notch+signaling+in+endothelial+cells.

Peripheral post-ischemic vascular repair is impaired in a murine model of Alzheimer's disease

Authors:
Merkulova-Rainon T, Mantsounga CS, Broquères-You D, Pinto C, Vilar J, Cifuentes D, Bonnin P, Kubis N, Henrion D, Silvestre JS, Lévy BI

Abstract:
The pathophysiology of sporadic Alzheimer's disease (AD) remains uncertain. Along with brain amyloid-β (Aβ) deposits and neurofibrillary tangles, cerebrovascular dysfunction is increasingly recognized as fundamental to the pathogenesis of AD. Using an experimental model of limb ischemia in transgenic APPPS1 mice, a model of AD (AD mice), we showed that microvascular impairment also extends to the peripheral vasculature in AD. At D70 following femoral ligation, we evidenced a significant decrease in cutaneous blood flow (- 29%, P < 0.001), collateral recruitment (- 24%, P < 0.001), capillary density (- 22%; P < 0.01) and arteriole density (- 28%; P < 0.05) in hind limbs of AD mice compared to control WT littermates. The reactivity of large arteries was not affected in AD mice, as confirmed by unaltered size, and vasoactive responses to pharmacological stimuli of the femoral artery. We identified blood as the only source of Aβ in the hind limb; thus, circulating Aβ is likely responsible for the impairment of peripheral vasculature repair mechanisms. The levels of the majority of pro-angiogenic mediators were not significantly modified in AD mice compared to WT mice, except for TGF-β1 and PlGF-2, both of which are involved in vessel stabilization and decreased in AD mice (P = 0.025 and 0.019, respectively). Importantly, endothelin-1 levels were significantly increased, while those of nitric oxide were decreased in the hind limb of AD mice (P < 0.05). Our results suggest that vascular dysfunction is a systemic disorder in AD mice. Assessment of peripheral vascular function may therefore provide additional tools for early diagnosis and management of AD.

Citation:
Angiogenesis. 2018 Mar 7. doi: 10.1007/s10456-018-9608-7. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29516292

Postnatal development of lymphatic vasculature in the brain meninges

Authors:
Izen RM, Yamazaki T, Nishinaka-Arai Y, Hong YK, Mukouyama YS

Abstract:
BACKGROUND: Traditionally, the central nervous system (CNS) has been viewed as an immune-privileged environment with no lymphatic vessels. This view was partially overturned by the discovery of lymphatic vessels in the dural membrane that surrounds the brain, in contact with the interior surface of the skull. We here examine the distribution and developmental timing of these lymphatic vessels.

RESULTS: Using the Prox1-GFP BAC transgenic reporter and immunostaining with antibodies to lymphatic markers LYVE-1, Prox1, and Podoplanin, we have carried out whole-mount imaging of dural lymphatic vasculature at postnatal stages. We have found that between birth - postnatal day 13 (P)13, lymphatic vessels extend alongside dural blood vessels from the side of the skull towards the midline. Between P13 - P20, lymphatic vessels along the transverse sinuses (TS) reach the superior sagittal sinus (SSS) and extend along the SSS towards the olfactory bulb.CONCLUSION: Compared with the embryonic developmental timing of lymphatic vessels in other tissues, e.g. skin, dural lymphatic vessel development is dramatically delayed. This study provides useful anatomical data for continuing investigations of the fundamental mechanisms that underlie dural lymphatic vessel development. This article is protected by copyright. All rights reserved.

Citation:
Dev Dyn. 2018 Mar 1. doi: 10.1002/dvdy.24624. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29493038

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice

Authors:
Hsu JJ, Lu J, Umar S, Lee JT, Kulkarni R, Ding Y, Chang CC, Hsiai TK, Hokugo A, Gkouveris I, Tetradis S, Nishimura I, Demer LL, Tintut Y

Abstract:
Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit onset of aortic calcification. Whether teriparatide affects progression of pre-existing aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over one year to induce aortic calcification, treated for 4.5 weeks with daily injections of control vehicle (PBS), teriparatide 40 µg/kg (PTH40) or 400 µg/kg (PTH400), and assayed for aortic calcification by micro-computed tomography (microCT) before and after treatment. In a follow-up cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400, and assayed for aortic calcification by serial microCT and micro-positron emission tomography (microPET). In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial microPET, increased in the PBS group (+14{plus minus}5%). In contrast, 18F-NaF incorporation decreased in the PTH400 group (-33{plus minus}20%; p=0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400 group compared with the PBS group (p=0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice receiving PTH400 (2.1{plus minus}0.6% vs. controls, 0.5{plus minus}0.1%; p=0.02). In summary, while teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability.

Citation:
Am J Physiol Heart Circ Physiol. 2018 Feb 16. doi: 10.1152/ajpheart.00718.2017. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29451816

Endothelial immune activation programs cell-fate decisions and angiogenesis by inducing DLL4 through TLR4-ERK-FOXC2 signalling

Authors:
Xia S, Menden HL, Korfhagen TR, Kume T, Sampath V

Abstract:
Endothelial cells (EC) mediate a specific and robust immune response to bacteria in sepsis through the activation of Toll-Like Receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis program endothelial cell (EC) specification and altered angiogenesis remain unclear. We postulated that the Forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung EC, LPS induced ERK phosphorylation, FOXC2, and DLL4 expression in a TLR4-dependent manner. LPS mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung EC. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK-inhibition. FOXC2+/- mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2+/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programs EC specification and altered angiogenesis. This article is protected by copyright. All rights reserved.

Citation:
J Physiol. 2018 Jan 30. doi: 10.1113/JP275453. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29380370

RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia

Authors:
Herbert, LM, Resta TC, Jernigan NL

Abstract:
Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.

Citation:
Am J Physiol Cell Physiol. 2018 Feb 1;314(2):C166-C176. doi: 10.1152/ajpcell.00159.2017. Epub 2017 Oct 25.

http://www.physiology.org/doi/abs/10.1152/ajpcell.00159.2017?journalCode=ajpcell

Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca2+ entry

Authors:
Zhang B, Naik JS, Jernigan NL, Walker BR, Resta TC

Abstract:
Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca2+ entry (SOCE) and diminished Ca2+-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.

Citation:
Am J Physiol Heart Circ Physiol. 2018 Feb 1;314(2):H359-H369. doi: 10.1152/ajpheart.00540.2017. Epub 2017 Nov 3.

http://www.physiology.org/doi/abs/10.1152/ajpheart.00540.2017?journalCode=ajpheart

Endothelial-dependent dilation following chronic hypoxia involves TRPV4-mediated activation of endothelial BK channels

Authors:
Naik JS, Walker BR

Abstract:
Following chronic hypoxia (CH), the systemic vasculature exhibits blunted vasoconstriction due to endothelial-dependent hyperpolarization (EDH). Previous data demonstrate that subsequent to CH, EDH-mediated vasodilation switches from a reliance on SKca and IKca channels to activation of the endothelial BKca channels (eBK). The mechanism by which endothelial cell stimulation activates eBK channels following CH is not known. We hypothesized that following CH, EDH-dependent vasodilation involves a TRPV4-dependent activation of eBK channels. ACh induced concentration-dependent dilation in pressurized gracilis arteries from both normoxic and CH rats. Inhibition of TRPV4 (RN-1734) attenuated the ACh response in arteries from CH rats but had no effect in normoxic animals. In the presence of L-NNA and indomethacin, TRPV4 blockade attenuated ACh-induced vasodilation in arteries from CH rats. ACh elicited endothelial TRPV4-mediated Ca2+ events in arteries from both groups. GSK1016790A (GSK101, TRPV4 agonist) elicited vasodilation in arteries from normoxic and CH rats. In arteries from normoxic animals, TRAM-34/apamin abolished the dilation to TRPV4 activation, whereas luminal iberiotoxin had no effect. In CH rats, only administration of all three Kca channel inhibitors abolished the dilation to TRPV4 activation. Using Duolink®, we observed co-localization between Cav-1, TRPV4, and BK channels in gracilis arteries and in RAECs. Disruption of endothelial caveolae with methyl-β-cyclodextrin significantly decreased ACh-induced vasodilation in arteries from both groups. In gracilis arteries, endothelial membrane cholesterol was significantly decreased following 48 h of CH. In conclusion, CH results in a functional coupling between muscarinic receptors, TRPV4 and Kca channels in gracilis arteries.

Citation:
Pflugers Arch. 2018 Jan 29. doi: 10.1007/s00424-018-2112-5. [Epub ahead of print]

https://link.springer.com/article/10.1007%2Fs00424-018-2112-5

Cell Type-Specific Contributions of the Angiotensin II Type 1a Receptor to Aorta Homeostasis and Aneurysmal Disease

Authors:
Galatioto J, Caescu CI, Hansen J, Cook J, Miramontes I, Iyengar R, Ramirez F

Abstract:
OBJECTIVE: Two were the aims of this study: first, to translate whole-genome expression profiles into computational predictions of functional associations between signaling pathways that regulate aorta homeostasis and the activity of angiotensin II type 1a receptor (At1ar) in either vascular endothelial or smooth muscle cells and second, to characterize the impact of endothelial cell- or smooth muscle cell-specific At1ar disruption on the development of thoracic aortic aneurysm in fibrillin-1 hypomorphic mice, a validated animal model of early onset progressively severe Marfan syndrome. APPROACH AND RESULTS: Cdh5-Cre and Sm22-Cre transgenic mice were used to inactivate the At1ar-coding gene (Agt1ar) in either intimal or medial cells of both wild type and Marfan syndrome mice, respectively. Computational analyses of differentially expressed genes predicted dysregulated signaling pathways of cell survival and matrix remodeling in Agt1arCdh5-/- aortas and of cell adhesion and contractility in Agt1arSm22-/- aortas. Characterization of fibrillin-1 hypomorphic; Agt1arCdh5-/- mice revealed increased median survival associated with mitigated aneurysm growth and media degeneration, as well as reduced levels of phosphorylated (p-) Erk1/2 but not p-Smad2. By contrast, levels of both p-Erk1/2 and p-Smad2 proteins were normalized in fibrillin-1 hypomorphic; Agt1arSm22-/- aortas in spite of them showing no appreciable changes in thoracic aortic aneurysm pathology. CONCLUSIONS: Physiological At1ar signaling in the intimal and medial layers is associated with distinct regulatory processes of aorta homeostasis and function, and improper At1ar activity in the vascular endothelium is a significant determinant of thoracic aortic aneurysm development in Marfan syndrome mice.

Citation:
Arterioscler Thromb Vasc Biol. 2018 Jan 25. pii: ATVBAHA.117.310609. doi: 10.1161/ATVBAHA.117.310609. [Epub ahead of print]

http://atvb.ahajournals.org/content/early/2018/01/24/ATVBAHA.117.310609.long

Epithelial Na+ channel (ENaC) differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice

Authors:
Ashley Z, Mugloo S, McDonald FJ, Fronius M

Abstract:
A potential "new player" in arteries for mediating shear stress responses is the Epithelial Sodium Channel (ENaC). ENaC's contribution as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance) is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (3rd order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no-flow), in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and following inhibition of endothelial nitric oxide synthase (eNOS) by L-Name (100 µM). Under no-flow conditions, amiloride increased the internal and external diameter of carotid (13{plus minus}2%, p<0.05), but not mesenteric (0.5{plus minus}0.9%, p>0.05) arteries. In response to increased intraluminal pressure amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress/strain curves of mesenteric arteries. With increased shear stress ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2{plus minus}5.3%) compared with control (no amiloride, 6.2{plus minus}3.3%; p<0.05). In mesenteric arteries amiloride induced a flow-mediated constriction (-11.5{plus minus}6.6%) compared with control (-2.2{plus minus}4.5%; p<0.05). L-Name mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. The ENaC-mediated effects were associated with NO production although may involve different - artery dependent - downstream signalling pathways.

Citation:
Am J Physiol Heart Circ Physiol. 2018 Jan 26. doi: 10.1152/ajpheart.00506.2017. [Epub ahead of print]

http://www.physiology.org/doi/abs/10.1152/ajpheart.00506.2017?journalCode=ajpheart

Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease

Authors:
Foster AA, Dewi RE, Cai L, Hou L, Strassberg Z, Alcazar CA, Heilshorn SC

Abstract:
A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

Citation:
Biomater Sci. 2018 Feb 6. doi: 10.1039/c7bm00883j. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29406542

Long-term diet-induced hypertension in rats is associated with reduced expression and function of small artery SKCa, IKCa, and Kir2.1 channels

Authors:
Anna K.J. Gradel, Max Salomonsson, Charlotte M. Sørensen, Niels-Henrik Holstein-Rathlou, Lars J. Jensen

Abstract:
Rationale: Abdominal obesity and/or a high intake of fructose may cause hypertension. K+ channels, Na/K-ATPase, and voltage-gated Ca2+ channels are crucial determinants of resistance artery tone and thus the control of blood pressure. Limited information is available on the role of K+ transporters in long-term diet-induced hypertension in rats. Hypothesis: A 28-week diet rich in fat, fructose, or both, will lead to changes in K+ transporter expression and function, which is associated with increased blood pressure and decreased arterial function. Methods and Results: Male Sprague Dawley rats received a diet containing normal chow (Control), high-fat chow (High Fat), high-fructose in drinking water (High Fructose), or a combination of high-fat and high-fructose diet (High Fat/Fruc) for 28 weeks from age 4-weeks. Measurements included body weight (BW), systolic blood pressure (SBP), mRNA expression of vascular K+ transporters, and vessel myography in small mesenteric arteries. BW was increased in the High Fat and High Fat/Fruc groups, and SBP was increased in the High Fat/Fruc group. mRNA expression of SKCa, IKCa, and Kir2.1 K+ channels were reduced in the High Fat/Fruc group. Reduced EDH-type relaxation to acetylcholine was seen in the High Fat and High Fat/Fruc groups. Ba2+-sensitive dilatation to extracellular K+ was impaired in all experimental diet groups. Conclusions: Reduced expression and function of SKCa, IKCa and Kir2.1 channels is associated with elevated blood pressure in rats fed a long-term high fat/high fructose diet. Rats fed a 28-week high fat/high fructose diet provide a relevant model of diet-induced hypertension.

Citation:
Clinical Science Feb 07, 2018, CS20171408; DOI: 10.1042/CS20171408

http://www.clinsci.org/content/early/2018/02/07/CS20171408

Rapamycin activates TGF receptor independently of its ligand: implications for endothelial dysfunction

Authors:
Miyakawa AA, Girao-Silva T, Krieger JE, Edelman ER

Abstract:
Rapamycin, the macrolide immunosuppressant and active pharmaceutic in drug-eluting stents (DES), has a well-recognized anti-proliferative action that involves inhibition of the mTOR pathway after binding to the cytosolic protein FKBP12. TGF receptor-type I (TGFRI) spontaneous activation is inhibited by the association with FKBP12. We hypothesized that rapamycin, in addition to inhibition of mTOR signaling, activates TGFRI independent of TGFb. Human umbilical vein endothelial cells (HUVEC) were treated with rapamycin (10nmoL/L) and/or TGF-b RI kinase inhibitor (TGFRIi, 100nmoL/L) for 24 hours. Rapamycin induced SMAD phosphorylation (SMAD1, SMAD2 and SMAD5) and PAI-1 up-regulation, which was specifically abrogated by SMAD2 knockdown. TGFRIi efficiently blocked phosphorylation of SMAD2, but not SMAD1/5. Interestingly, the inhibitor did not alter cell proliferation arrest induced by rapamycin. Active TGFb secretion was not affected by the treatment. Neutralizing TGFβ experiments did not influence SMAD2 phosphorylation or PAI-1 expression indicating that activation of this pathway is independent of the ligand. In addition, rapamycin induction of endothelial-to-mesenchymal transition (EndMT) was potentiated by IL-1b, and efficiently blocked by TGFRIi. In vivo, the pro-thrombogenic effects of rapamycin and up-regulation of PAI-1 in murine carotid arteries were reduced by TGFRIi treatment. In conclusion, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGFb, in concert with promotion of PAI-1 expression and changes in endothelial phenotype. These undesirable effects, the prothrombogenic state and activation of EndMT, are SMAD2-dependent and independent of the therapeutic rapamycin-induced cell proliferation arrest.

Citation:
Clin Sci (Lond). 2018 Jan 17. pii: CS20171457. doi: 10.1042/CS20171457. [Epub ahead of print]

http://www.clinsci.org/content/early/2018/01/17/CS20171457

New pro-resolving n-3 mediators bridge resolution of infectious inflammation to tissue regeneration

Authors:
Serhan CN, Chiang N, Dalli J

Abstract:
While protective, the acute inflammatory response when uncontrolled can lead to further tissue damage and chronic inflammation that is now widely recognized to play important roles in many commonly occurring diseases, such as cardiovascular disease, neurodegenerative diseases, metabolic syndrome, and many other diseases of significant public health concern. The ideal response to initial challenges of the host is complete resolution of the acute inflammatory response, which is now recognized to be a biosynthetically active process governed by specialized pro-resolving mediators (SPM). These chemically distinct families include lipoxins, resolvins, protectins and maresins that are biosynthesized from essential fatty acids. The biosynthesis and complete stereochemical assignments of the major SPM are established, and new profiling procedures have recently been introduced to document the activation of these pathways in vivo with isolated cells and in human tissues. The active resolution phase leads to tissue regeneration, where we've recently identified new molecules that communicate during resolution of inflammation to activate tissue regeneration in model organisms. This review presents an update on the documentation of the roles of SPMs and the biosynthesis and structural elucidation of novel mediators that stimulate tissue regeneration, coined conjugates in tissue regeneration. The identification and actions of the three families, maresin conjugates in tissue regeneration (MCTR), protectin conjugates in tissue regeneration (PCTR), and resolvin conjugates in tissue regeneration (RCTR), are reviewed here. The identification, structural elucidation and the pathways and biosynthesis of these new mediators in tissue regeneration demonstrate the host capacity to protect from collateral tissue damage, stimulate clearance of bacteria and debris, and promote tissue regeneration via endogenous pathways and molecules in the resolution metabolome.

Citation:
Mol Aspects Med. 2017 Sep 1. pii: S0098-2997(17)30091-2. doi: 10.1016/j.mam.2017.08.002. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=New+pro-resolving+n-3+mediators+bridge+resolution+of+infectious+inflammation+to+tissue+regeneration.

Potent anti-inflammatory and pro-resolving effects of anabasum in a human model of self-resolving acute inflammation

Authors:
Motwani MP, Bennett F, Norris PC, Maini AA, George MJ, Newson J, Henderson A, Hobbs AJ, Tepper M, White B, Serhan CN, MacAllister R, Gilroy DW

Abstract:
Anabasum is a synthetic analogue of Δ8 - tetrahydrocannabinol (THC)-11-oic acid that in pre-clinical models of experimental inflammation exerts potent anti-inflammatory actions with minimal CNS cannabimimetic activity. Here we used a novel model of acute inflammation driven by i.d. UV-killed E. coli in healthy humans and found that Anabasum (5mg) exerted a potent anti-inflammatory effect equivalent to that of prednisolone in terms of inhibiting neutrophil infiltration, the hallmark of acute inflammation. These effects arose from the inhibition of the neutrophil chemoattractant LTB4 , while the inhibition of anti-phagocytic prostanoids (PGE2 , TxB2 and PGF2 α) resulted in enhanced clearance of inflammatory stimulus from the injected site. Anabasum at the higher dose of 20mg possessed the additional properties of triggering the biosynthesis of specialised pro-resolving lipid mediators including LXA4 , LXB4 , RvD1 and RvD3. Collectively, we demonstrate for the first-time striking anti-inflammatory and pro-resolution effects of a synthetic analogue of THC in healthy humans. This article is protected by copyright. All rights reserved.

Citation:
Clin Pharmacol Ther. 2017 Dec 14. doi: 10.1002/cpt.980. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=Potent+anti-inflammatory+and+pro-resolving+effects+of+anabasum+in+a+human+model+of+self-resolving+acute+inflammation

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

Authors:
Patrick A Murphy1, Vincent L Butty, Paul L Boutz, Shahinoor Begum1, Amy L Kimble, Phillip A Sharp, Christopher B Burge, Richard O Hynes

Abstract:
Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation.

Citation:
Elife. 2018 Jan 2;7. pii: e29494. doi: 10.7554/eLife.29494.

https://elifesciences.org/articles/29494

Nanoparticle Functionalization with Platelet Membrane Enables Multifactored Biological Targeting and Detection of Atherosclerosis

Authors:
Wei X, Ying M, Dehaini D, Su Y, Kroll AV, Zhou J, Gao W, Fang RH, Chien S, Zhang L

Abstract:
Cardiovascular disease represents one of the major causes of death across the global population. Atherosclerosis, one of its most common drivers, is characterized by the gradual buildup of arterial plaque over time, which can ultimately lead to life-threatening conditions. Given the impact of the disease on public health, there is a great need for effective and noninvasive imaging modalities that can provide valuable information on its biological underpinnings during development. Here, we leverage the role of platelets in atherogenesis to design nanocarriers capable of targeting multiple biological elements relevant to plaque development. Biomimetic nanoparticles are prepared by coating platelet membrane around a synthetic nanoparticulate core, the product of which is capable of interacting with activated endothelium, foam cells, and collagen. The effects are shown to be exclusive to platelet membrane-coated nanoparticles. These biomimetic nanocarriers are not only capable of efficiently localizing to well-developed atherosclerotic plaque, but can also target subclinical regions of arteries susceptible to plaque formation. Using a commonly employed magnetic resonance imaging contrast agent, live detection is demonstrated using an animal model of atherosclerosis. Ultimately, this strategy may be leveraged to better assess the development of atherosclerosis, offering additional information to help clinicians better manage the disease.

Citation:
ACS Nano. 2017 Dec 12. doi: 10.1021/acsnano.7b07720. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29216423

Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain

Authors:
Nikolaev SI, Vetiska S, Bonilla X, Boudreau E, Jauhiainen S, Rezai Jahromi B, Khyzha N, DiStefano PV, Suutarinen S, Kiehl TR, Mendes Pereira V, Herman AM, Krings T, Andrade-Barazarte H, Tung T, Valiante T, Zadeh G, Tymianski M, Rauramaa T, Ylä-Herttuala S, Wythe JD, Antonarakis SE, Frösen J, Fish JE, Radovanovic I

Abstract:
Background Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. Methods We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples. Results We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling. Conclusions We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).

Citation:
N Engl J Med. 2018 Jan 3. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29298116

Akt2 Stabilizes ATP7A, a Copper Transporter for SOD3 (Extracellular Superoxide Dismutase), in Vascular Smooth Muscles: Novel Mechanism to Limit Endothelial Dysfunction in Type 2 Diabetes Mellitus

Authors:
Varadarajan Sudhahar, Mustafa Nazir Okur, Zsolt Bagi, John P. O’Bryan, Nissim Hay, Ayako Makino, Vijay S. Patel, Shane A. Phillips, David Stepp, Masuko Ushio-Fukai, Tohru Fukai

Abstract:
Objective - Copper transporter ATP7A (copper-transporting/exporting ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM mice and human in which insulin–Akt pathway is selectively impaired. Approach and Results - Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet–induced or db/db T2DM mice. Akt2 activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B−/− T2DM mice, which enhance insulin–Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2−/− vessels or VSMCs, which is rescued by ATP7A overexpression. Conclusion - Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Citation:
Arterioscler Thromb Vasc Biol. 2018 Jan 4. pii: ATVBAHA.117.309819. doi: 10.1161/ATVBAHA.117.309819. [Epub ahead of print]

http://atvb.ahajournals.org/content/early/2018/01/03/ATVBAHA.117.309819?ijkey=q28IZtmlXl7zrCn&keytype=ref

Telomerase reverse transcriptase protects against Angiotensin II induced microvascular endothelial dysfunction

Authors:
Ait-Aissa K, Kadlec AO, Hockenberry J, Gutterman DD and Beyer AM

Abstract:
A rise in reactive oxygen species (ROS) may contribute to cardiovascular disease by reducing nitric oxide (NO) levels, leading to loss of NO's vasodilator and anti-inflammatory effects. Although primarily studied in the larger conduit arteries, excess ROS release and a corresponding loss of NO also occurs in the smaller resistance arteries of the microcirculation, but underlying mechanisms and therapeutic targets have not been fully characterized. We examined whether either of the two subunits of telomerase, telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC), affect microvascular ROS production and peak vasodilation at baseline and in response to in vivo administration to angiotensin II (ANG II). We report that genetic loss of TERT [max dilation (%): Vehicle 52.0{plus minus}6.1, L-NAME: 60.4{plus minus}12.9, Peg-CAT: 32.2{plus minus}12.2*; N = 9-19; *P<0.05] but not TERC [max dilation (%): Vehicle 79{plus minus}5; L-NAME 10.7{plus minus}9.8*; Peg-CAT 86.4{plus minus}8.4; N = 4-7; *P<0.05] promotes flow-induced ROS formation. Moreover, TERT KO exacerbates the microvascular dysfunction resulting from in vivo ANG II treatment, whereas TERT overexpression is protective [max dilation (%): vehicle: 88.22{plus minus}4.6 vs. ANG II (1000ng/kg/min) 74.0{plus minus}7.3; N = 4; P=NS]. Therefore, loss of TERT but not TERC may be a key contributor to the elevated microvascular ROS levels and reduced peak dilation observed in several cardiovascular disease pathologies.

Citation:
American Journal of Physiology-Heart and Circulatory Physiology, 22 Dec 2017, doi.org/10.1152/ajpheart.00472.2017

http://www.physiology.org/doi/abs/10.1152/ajpheart.00472.2017

R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature

Authors:
Fangfei Li, Junko Sawada, Masanobu Komatsu

Abstract:
The formation of endothelial lumen is fundamental to angiogenesis and essential to the oxygenation of hypoxic tissues. The molecular mechanism underlying this important process remains obscure. Here, we show that Akt activation by a Ras homolog, R-Ras, stabilizes the microtubule cytoskeleton in endothelial cells leading to endothelial lumenogenesis. The activation of Akt by the potent angiogenic factor VEGF-A does not strongly stabilize microtubules or sufficiently promote lumen formation, hence demonstrating a distinct role for the R-Ras-Akt axis. We show in mice that this pathway is important for the lumenization of new capillaries and microvessels developing in ischemic muscles to allow sufficient tissue reperfusion after ischemic injury. Our work identifies a role for Akt in lumenogenesis and the significance of the R-Ras-Akt signaling for the patency of regenerating blood vessels.

Citation:
Nature Communications 8, Article number: 1720 (2017) doi:10.1038/s41467-017-01865-x

https://www.nature.com/articles/s41467-017-01865-x

Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion

Authors:
Fitzpatrick E, Han X, Liu W, Corcoran E, Burtenshaw D, Morrow D, Helt JC, Cahill PA, Redmond EM

Abstract:
BACKGROUND: Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1+ ) progenitor stem cell involvement during pathologic arterial remodeling. METHODS: Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH. RESULTS: The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1+ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1+ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1+ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1+ adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. CONCLUSIONS: EtOH reduces SHh-responsive Sca1+ progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1+ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.

Citation:
Alcohol Clin Exp Res. 2017 Sep 18. doi: 10.1111/acer.13499. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28921619

Mechanisms of angiogenesis in microbe-regulated inflammatory and neoplastic conditions

Authors:
Sajib S, Zahra FT, Lionakis MS, German NA, Mikelis CM

Abstract:
Commensal microbiota inhabit all the mucosal surfaces of the human body. It plays significant roles during homeostatic conditions, and perturbations in numbers and/or products are associated with several pathological disorders. Angiogenesis, the process of new vessel formation, promotes embryonic development and critically modulates several biological processes during adulthood. Indeed, deregulated angiogenesis can induce or augment several pathological conditions. Accumulating evidence has implicated the angiogenic process in various microbiota-associated human diseases. Herein, we critically review diseases that are regulated by microbiota and are affected by angiogenesis, aiming to provide a broad understanding of how angiogenesis is involved and how microbiota regulate angiogenesis in microbiota-associated human conditions.

Citation:
Angiogenesis. 2017 Nov 6. doi: 10.1007/s10456-017-9583-4. [Epub ahead of print]

https://link.springer.com/article/10.1007/s10456-017-9583-4

The infarcted myocardium solicits GM-CSF for the detrimental oversupply of inflammatory leukocytes

Authors:
Anzai A, Choi J, He S, Fenn A, Nairz M, Rattik S, McAlpione C, Mindur J, Chan C, Iwamoto Y, Tricot B, Wojtkiewicz G, Weissleder R, Libby P, Nahrendorf M, Stone J, Becher B, and Swirski F

Abstract:
Myocardial infarction (MI) elicits massive inflammatory leukocyte recruitment to the heart. Here, we hypothesized that excessive leukocyte invasion leads to heart failure and death during acute myocardial ischemia. We found that shortly and transiently after onset of ischemia, human and mouse cardiac fibroblasts produce granulocyte/macrophage colony-stimulating factor (GM-CSF) that acts locally and distally to generate and recruit inflammatory and proteolytic cells. In the heart, fibroblast-derived GM-CSF alerts its neighboring myeloid cells to attract neutrophils and monocytes. The growth factor also reaches the bone marrow, where it stimulates a distinct myeloid-biased progenitor subset. Consequently, hearts of mice deficient in either GM-CSF or its receptor recruit fewer leukocytes and function relatively well, whereas mice producing GM-CSF can succumb from left ventricular rupture, a complication mitigated by anti–GM-CSF therapy. These results identify GM-CSF as both a key contributor to the pathogenesis of MI and a potential therapeutic target, bolstering the idea that GM-CSF is a major orchestrator of the leukocyte supply chain during inflammation.

Citation:
J Exp Med. 2017 Oct 4. pii: jem.20170689. doi: 10.1084/jem.20170689. [Epub ahead of print]

http://jem.rupress.org/content/early/2017/10/03/jem.20170689

Epigenetics and precision medicine in cardiovascular patients: from basic concepts to the clinical arena

Authors:
Costantino S, Libby P, Kishore R, Tardif JC, El-Osta A, Paneni F

Abstract:
Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide and also inflict major burdens on morbidity, quality of life, and societal costs. Considering that CVD preventive medications improve vascular outcomes in less than half of patients (often relative risk reductions range from 12% to 20% compared with placebo), precision medicine offers an attractive approach to refine the targeting of CVD medications to responsive individuals in a population and thus allocate resources more wisely and effectively. New tools furnished by advances in basic science and translational medicine could help achieve this goal. This approach could reach beyond the practitioners 'eyeball' assessment or venerable markers derived from the physical examination and standard laboratory evaluation. Advances in genetics have identified novel pathways and targets that operate in numerous diseases, paving the way for 'precision medicine'. Yet the inherited genome determines only part of an individual's risk profile. Indeed, standard genomic approaches do not take into account the world of regulation of gene expression by modifications of the 'epi'genome. Epigenetic modifications defined as 'heritable changes to the genome that do not involve changes in DNA sequence' have emerged as a new layer of biological regulation in CVD and could advance individualized risk assessment as well as devising and deploying tailored therapies. This review, therefore, aims to acquaint the cardiovascular community with the rapidly advancing and evolving field of epigenetics and its implications in cardiovascular precision medicine.

Citation:
Eur Heart J. 2017 Oct 23. doi: 10.1093/eurheartj/ehx568. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29069341

Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

Authors:
Lopez-Ramirez MA, Fonseca G, Zeineddine HA, Girard R, Moore T, Pham A, Cao Y, Shenkar R, de Kreuk BJ, Lagarrigue F, Lawler J, Glass CK, Awad IA, Ginsberg MH

Abstract:
KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1 In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1ECKO ) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.

Citation:
J Exp Med. 2017 Sep 28. pii: jem.20171178. doi: 10.1084/jem.20171178. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28970240

Identification of miR-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 and PKM2

Authors:
Caruso P, Dunmore BJ, Schlosser K, Schoors S, Dos Santos CC, Perez-Iratxeta C, Lavoie JR, Zhang H, Long L, Flockton AR, Frid MG, Upton PD, D'Alessandro A, Hadinnapola C, Kiskin FN, Taha M, Hurst LA, Ormiston ML, Hata A, Stenmark KR, Carmeliet P, Stewart DJ, Morrell NW

Abstract:
Background -Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells (PAECs). However, the mechanisms underlying alterations in energy production have not been identified. Methods -Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH (HPAH) due to mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH (IPAH) to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from PAH patients, the downregulation of miR-124, determined using a tiered systems biology approach, is responsible for increased expression of the splicing factor polypyrimidine-tract-binding protein (PTBP1), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently, increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Results -HPAH and IPAH BOECs recapitulated the metabolic abnormalities observed in PAECs from IPAH patients, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124, or siRNA silencing of PTPB1, restored normal proliferation and glycolysis in HPAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivoConclusions -Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124 leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA, or its targets, could represent a novel therapeutic approach for the treatment of PAH.

Citation:
Circulation. 2017 Sep 26. pii: CIRCULATIONAHA.117.028034. doi: 10.1161/CIRCULATIONAHA.117.028034. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2017/09/22/CIRCULATIONAHA.117.028034

The Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension is Regulated through a MiR-124/PTBP1/PKM Axis

Authors:
Zhang H, Wang D, Li M, Plecitá-Hlavatá L, D'Alessandro A, Tauber J, Riddle S, Kumar S, Flockton AR, McKeon BA, Frid MG, Reisz JA, Caruso P, El Kasmi KC, Ježek P, Morrell NW, Hu CJ, Stenmark KR

Abstract:
Background -An emerging "metabolic theory" of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyper-proliferative, apoptosis-resistant, pro-inflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the TCA cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that, in PH-Fibs, miR-124 regulates polypyrimidine tract binding protein 1 (PTBP1) expression to control alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and PKM2) resulting in an increased PKM2/PKM1 ratio which promotes glycolysis and proliferation even in aerobic environments. Methods -Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects (CO-Fibs). PTBP1 gene knockdown was achieved via PTBP1-siRNA, restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and Shikonin were utilized to manipulate PKM2 glycolytic function. HDACi were used to treat cells. Metabolic products were determined by Mass spectrometry-based metabolomics analyses (UHPLC-MS), and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. Results -We detected an increased PKM2/PKM1 ratio in PH-Fibs compared to CO-Fibs. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation and attenuated macrophage IL-1β expression. Further, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, i.e. lactate), rescued mitochondrial reprogramming and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and Shikonin, or treatment with histone deacetylase inhibitors (HDACi), produced similar results. Conclusions -In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Citation:
Circulation. 2017 Sep 26. pii: CIRCULATIONAHA.117.028069. doi: 10.1161/CIRCULATIONAHA.117.028069. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2017/09/22/CIRCULATIONAHA.117.028069

Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities

Authors:
Fung KY, Fairn GD, Lee WL

Abstract:
Vesicle-mediated transcellular transport or simply "transcytosis" is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between two neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells the process often involves receptor-mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm, and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.

Citation:
Traffic. 2017 Oct 6. doi: 10.1111/tra.12533. [Epub ahead of print]

http://onlinelibrary.wiley.com/doi/10.1111/tra.12533/abstract

Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth

Authors:
Mathivet T, Bouleti C, Van Woensel M, Stanchi F, Verschuere T, Phng LK, Dejaegher J, Balcer M, Matsumoto K, Georgieva PB, Belmans J, Sciot R, Stockmann C, Mazzone M, De Vleeschouwer S, Gerhardt H

Abstract:
Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by in situ repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences.

Citation:
EMBO Mol Med. 2017 Oct 16. pii: e201607445. doi: 10.15252/emmm.201607445. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29038312

Somatic second hit mutation of RASA1 in vascular endothelial cells in capillary malformation-arteriovenous malformation

Authors:
Lapinski PE, Doosti A, Salato V, North P, Burrows PE, King PD

Abstract:
Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant vascular disorder that is associated with inherited inactivating mutations of the RASA1 gene in the majority of cases. Characteristically, patients exhibit one or more focal cutaneous CM that may occur alone or together with AVM, arteriovenous fistulas or lymphatic vessel abnormalities. The focal nature and varying presentation of lesions has led to the hypothesis that somatic "second hit" inactivating mutations of RASA1 are necessary for disease development. In this study, we examined CM from four different CM-AVM patients for the presence of somatically acquired RASA1 mutations. All four patients were shown to possess inactivating heterozygous germline RASA1 mutations. In one of the patients, a somatic inactivating RASA1 mutation (c.1534C > T, p.Arg512*) was additionally identified in CM lesion tissue. The somatic RASA1 mutation was detected within endothelial cells specifically and was in trans with the germline RASA1 mutation. Together with the germline RASA1 mutation (c.2125C > T, p.Arg709*) in the same patient, the endothelial cell somatic RASA1 mutation likely contributed to lesion development. These studies provide the first clear evidence of the second hit model of CM-AVM pathogenesis.

Citation:
Eur J Med Genet. 2017 Oct 9. pii: S1769-7212(17)30256-2. doi: 10.1016/j.ejmg.2017.10.004. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/29024832

Hdac3 regulates lymphovenous and lymphatic valve formation

Authors:
Janardhan HP, Milstone ZJ, Shin M, Lawson ND, Keaney JF Jr, Trivedi CM

Abstract:
Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box-GATA-ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Citation:
J Clin Invest. 2017 Oct 16. pii: 92852. doi: 10.1172/JCI92852. [Epub ahead of print]

https://www.jci.org/articles/view/92852

YAP/TAZ-CDC42 signaling regulates vascular tip cell migration

Authors:
Sakabe M, Fan J, Odaka Y, Liu N, Hassan A, Duan X, Stump P, Byerly L, Donaldson M, Hao J, Fruttiger M, Lu QR, Zheng Y, Lang RA, Xin M

Abstract:
Angiogenesis and vascular remodeling are essential for the establishment of vascular networks during organogenesis. Here we show that the Hippo signaling pathway effectors YAP and TAZ are required, in a gene dosage-dependent manner, for the proliferation and migration of vascular endothelial cells (ECs) during retinal angiogenesis. Intriguingly, nuclear translocation of YAP and TAZ induced by Lats1/2-deletion blocked endothelial migration and phenocopied Yap/Taz-deficient mutants. Furthermore, overexpression of a cytoplasmic form of YAP (YAPS127D) partially rescued the migration defects caused by loss of YAP and TAZ function. Finally, we found that cytoplasmic YAP positively regulated the activity of the small GTPase CDC42, deletion of which caused severe defects in endothelial migration. These findings uncover a previously unrecognized role of cytoplasmic YAP/TAZ in promoting cell migration by activating CDC42 and provide insight into how Hippo signaling in ECs regulates angiogenesis.

Citation:
Proc Natl Acad Sci U S A. 2017 Sep 25. pii: 201704030. doi: 10.1073/pnas.1704030114. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28973878

Identifying early pathogenic events during vascular calcification in uremic rats

Authors:
Hortells L, Sosa C, Guillén N, Lucea S, Millán Á, Sorribas V

Abstract:
Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

Citation:
Kidney Int. 2017 Aug 23. pii: S0085-2538(17)30479-9. doi: 10.1016/j.kint.2017.06.019. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28844316

A Proinflammatory Function of Toll-Like Receptor 2 in the Retinal Pigment Epithelium as a Novel Target for Reducing Choroidal Neovascularization in Age-Related Macular Degeneration

Authors:
Feng L, Ju M, Lee KYV, Mackey A, Evangelista M, Iwata D, Adamson P, Lashkari K, Foxton R, Shima D, Ng YS

Abstract:
Current treatments for choroidal neovascularization, a major cause of blindness for patients with age-related macular degeneration, treat symptoms but not the underlying causes of the disease. Inflammation has been strongly implicated in the pathogenesis of choroidal neovascularization. We examined the inflammatory role of Toll-like receptor 2 (TLR2) in age-related macular degeneration. TLR2 was robustly expressed by the retinal pigment epithelium in mouse and human eyes, both normal and with macular degeneration/choroidal neovascularization. Nuclear localization of NF-κB, a major downstream target of TLR2 signaling, was detected in the retinal pigment epithelium of human eyes, particularly in eyes with advanced stages of age-related macular degeneration. TLR2 antagonism effectively suppressed initiation and growth of spontaneous choroidal neovascularization in a mouse model, and the combination of anti-TLR2 and antivascular endothelial growth factor receptor 2 yielded an additive therapeutic effect on both area and number of spontaneous choroidal neovascularization lesions. Finally, in primary human fetal retinal pigment epithelium cells, ligand binding to TLR2 induced robust expression of proinflammatory cytokines, and end products of lipid oxidation had a synergistic effect on TLR2 activation. Our data illustrate a functional role for TLR2 in the pathogenesis of choroidal neovascularization, likely by promoting inflammation of the retinal pigment epithelium, and validate TLR2 as a novel therapeutic target for reducing choroidal neovascularization.

Citation:
Am J Pathol. 2017 Oct;187(10):2208-2221. doi: 10.1016/j.ajpath.2017.06.015. Epub 2017 Jul 21.

http://www.sciencedirect.com/science/article/pii/S0002944017302043

Polyester vascular patches acquire arterial or venous identity depending on their environment

Authors:
Bai H, Hu H, Guo J, Ige M, Wang T, Isaji T, Kudze T, Liu H, Yatsula B, Takuya H, Xing Y, Dardik A

Abstract:
Polyester is commonly used in vascular surgery for patch angioplasty and grafts. We hypothesized that polyester patches heal by infiltration of arterial or venous progenitor cells depending on the site of implantation. Polyester patches were implanted to the Wistar rat aorta or inferior vena cava and explanted on days 7 or 30. Neointima that formed on polyester patches was thicker in the venous environment compared to the amount that formed on patches in the arterial environment. Venous patches had more cell proliferation and greater numbers of VCAM-positive and CD68-positive cells, whereas arterial patches had greater numbers of vimentin-positive and alpha-actin-positive cells. Although there were similar numbers of endothelial progenitor cells in the neointimal endothelium, cells in the arterial patch were Ephrin-B2- and notch-4-positive while those in the venous patch were Eph-B4- and COUP-TFII-positive. Venous patches treated with an arteriovenous fistula had decreased neointimal thickness; however, neointimal endothelial cells expressed Ephrin-B2 and notch-4 in addition to Eph-B4 and COUP-TFII. Polyester patches in the venous environment acquire venous identity, whereas patches in the arterial environment acquire arterial identity; patches in the fistula environment acquire dual arterial-venous identity. This data suggests that synthetic patches heal by acquisition of identity of their environment.

Citation:
J Biomed Mater Res A. 2017 Sep 6. doi: 10.1002/jbm.a.36193. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28877393

The Lymphatic Vasculature: Its Role in Adipose Metabolism and Obesity

Authors:
Noelia Escobedo, Guillermo Oliver

Abstract:
Obesity is a key risk factor for metabolic and cardiovascular diseases, and although we understand the mechanisms regulating weight and energy balance, the causes of some forms of obesity remain enigmatic. Despite the well-established connections between lymphatics and lipids, and the fact that intestinal lacteals play key roles in dietary fat absorption, the function of the lymphatic vasculature in adipose metabolism has only recently been recognized. It is well established that angiogenesis is tightly associated with the outgrowth of adipose tissue, as expanding adipose tissue requires increased nutrient supply from blood vessels. Results supporting a crosstalk between lymphatic vessels and adipose tissue, and linking lymphatic function with metabolic diseases, obesity, and adipose tissue, also started to accumulate in the last years. Here we review our current knowledge of the mechanisms by which defective lymphatics contribute to obesity and fat accumulation in mouse models, as well as our understanding of the lymphatic-adipose tissue relationship.

Citation:
Cell Metab. 2017 Aug 22. pii: S1550-4131(17)30485-0. doi: 10.1016/j.cmet.2017.07.020. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28844882

ASK1-dependent endothelial cell activation is critical in ovarian cancer growth and metastasis

Authors:
Yin M, Zhou HJ, Zhang J, Lin C, Li H, Li X, Li Y, Zhang H, Breckenridge DG, Ji W, Min W

Abstract:
We have recently reported that tumor-associated macrophages (TAMs) promote early transcoelomic metastasis of ovarian cancer by facilitating TAM-ovarian cancer cell spheroid formation. ASK1 is known to be important for macrophage activation and inflammation-mediated tumorigenesis. In the present study, we show that ASK1 deficiency attenuates TAM-spheroid formation and ovarian cancer progression in an orthotopic ovarian cancer model. Interestingly, ASK1 in stroma, but not in TAMs, is critical for peritoneal tumor growth of ovarian cancer. Moreover, overexpression of an ASK1 inhibitory protein (suppressor of cytokine signaling-1; SOCS1) in vascular endothelium attenuates vascular permeability, TAM infiltration, and ovarian cancer growth. Mechanistically, we show that ASK1 mediates degradation of endothelial junction protein VE-cadherin via a lysosomal pathway to promote macrophage transmigration. Importantly, a pharmacological ASK1 inhibitor prevents tumor-induced vascular leakage, macrophage infiltration, and tumor growth in two mouse models. Since transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our study provides ASK1 as a therapeutic target for the treatment of ovarian cancer and other transcoelomic metastasis cancers.

Citation:
JCI Insight. 2017 Sep 21;2(18). pii: 91828. doi: 10.1172/jci.insight.91828. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28931753

Glioblastoma stem cells exploit the αvβ8 integrin-TGFβ1 signaling axis to drive tumor initiation and progression

Authors:
Guerrero PA, Tchaicha JH, Chen Z, Morales JE, McCarty N, Wang Q, Sulman EP, Fuller G, Lang FF, Rao G, McCarty JH

Abstract:
Glioblastoma (GBM) is a primary brain cancer that contains populations of stem-like cancer cells (GSCs) that home to specialized perivascular niches. GSC interactions with their niche influence self-renewal, differentiation and drug resistance, although the pathways underlying these events remain largely unknown. Here, we report that the integrin αvβ8 and its latent transforming growth factor β1 (TGFβ1) protein ligand have central roles in promoting niche co-option and GBM initiation. αvβ8 integrin is highly expressed in GSCs and is essential for self-renewal and lineage commitment in vitro. Fractionation of β8high cells from freshly resected human GBM samples also reveals a requirement for this integrin in tumorigenesis in vivo. Whole-transcriptome sequencing reveals that αvβ8 integrin regulates tumor development, in part, by driving TGFβ1-induced DNA replication and mitotic checkpoint progression. Collectively, these data identify the αvβ8 integrin-TGFβ1 signaling axis as crucial for exploitation of the perivascular niche and identify potential therapeutic targets for inhibiting tumor growth and progression in patients with GBM.Oncogene advance online publication, 7 August 2017; doi:10.1038/onc.2017.248.

Citation:
Oncogene. 2017 Aug 7. doi: 10.1038/onc.2017.248. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28783169

Effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial

Authors:
Ridker PM, MacFadyen JG, Thuren T, Everett BM, Libby P, Glynn RJ, CANTOS Trial Group

Abstract:
BACKGROUND: Inflammation in the tumour microenvironment mediated by interleukin 1β is hypothesised to have a major role in cancer invasiveness, progression, and metastases. We did an additional analysis in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), a randomised trial of the role of interleukin-1β inhibition in atherosclerosis, with the aim of establishing whether inhibition of a major product of the Nod-like receptor protein 3 (NLRP3) inflammasome with canakinumab might alter cancer incidence. METHODS: We did a randomised, double-blind, placebo-controlled trial of canakinumab in 10 061 patients with atherosclerosis who had had a myocardial infarction, were free of previously diagnosed cancer, and had concentrations of high-sensitivity C-reactive protein (hsCRP) of 2 mg/L or greater. To assess dose-response effects, patients were randomly assigned by computer-generated codes to three canakinumab doses (50 mg, 150 mg, and 300 mg, subcutaneously every 3 months) or placebo. Participants were followed up for incident cancer diagnoses, which were adjudicated by an oncology endpoint committee masked to drug or dose allocation. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, NCT01327846. The trial is closed (the last patient visit was in June, 2017). FINDINGS: Baseline concentrations of hsCRP (median 6·0 mg/L vs 4·2 mg/L; p<0·0001) and interleukin 6 (3·2 vs 2·6 ng/L; p<0·0001) were significantly higher among participants subsequently diagnosed with lung cancer than among those not diagnosed with cancer. During median follow-up of 3·7 years, compared with placebo, canakinumab was associated with dose-dependent reductions in concentrations of hsCRP of 26-41% and of interleukin 6 of 25-43% (p<0·0001 for all comparisons). Total cancer mortality (n=196) was significantly lower in the pooled canakinumab group than in the placebo group (p=0·0007 for trend across groups), but was significantly lower than placebo only in the 300 mg group individually (hazard ratio [HR] 0·49 [95% CI 0·31-0·75]; p=0·0009). Incident lung cancer (n=129) was significantly less frequent in the 150 mg (HR 0·61 [95% CI 0·39-0·97]; p=0·034) and 300 mg groups (HR 0·33 [95% CI 0·18-0·59]; p<0·0001; p<0·0001 for trend across groups). Lung cancer mortality was significantly less common in the canakinumab 300 mg group than in the placebo group (HR 0·23 [95% CI 0·10-0·54]; p=0·0002) and in the pooled canakinumab population than in the placebo group (p=0·0002 for trend across groups). Fatal infections or sepsis were significantly more common in the canakinumab groups than in the placebo group. All-cause mortality did not differ significantly between the canakinumab and placebo groups (HR 0·94 [95% CI 0·83-1·06]; p=0·31). INTERPRETATION: Our hypothesis-generating data suggest the possibility that anti-inflammatory therapy with canakinumab targeting the interleukin-1β innate immunity pathway could significantly reduce incident lung cancer and lung cancer mortality. Replication of these data in formal settings of cancer screening and treatment is required.

Citation:
Lancet. 2017 Aug 25. pii: S0140-6736(17)32247-X. doi: 10.1016/S0140-6736(17)32247-X. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=Effect+of+interleukin-1%CE%B2+inhibition+with+canakinumab+on+incident+lung+cancer+in+patients+with+atherosclerosis%3A+exploratory+results+from+a+randomised%2C+double-blind%2C+placebo-controlled+trial

Annexin A2 supports pulmonary microvascular integrity by linking vascular endothelial cadherin and protein tyrosine phosphatases

Authors:
Luo M, Flood EC, Almeida D, Yan L, Berlin DA, Heerdt PM, Hajjar KA

Abstract:
Relative or absolute hypoxia activates signaling pathways that alter gene expression and stabilize the pulmonary microvasculature. Alveolar hypoxia occurs in disorders ranging from altitude sickness to airway obstruction, apnea, and atelectasis. Here, we report that the phospholipid-binding protein, annexin A2 (ANXA2) functions to maintain vascular integrity in the face of alveolar hypoxia. We demonstrate that microvascular endothelial cells (ECs) from Anxa2-/- mice display reduced barrier function and excessive Src-related tyrosine phosphorylation of the adherens junction protein vascular endothelial cadherin (VEC). Moreover, unlike Anxa2+/+ controls, Anxa2-/- mice develop pulmonary edema and neutrophil infiltration in the lung parenchyma in response to subacute alveolar hypoxia. Mice deficient in the ANXA2-binding partner, S100A10, failed to demonstrate hypoxia-induced pulmonary edema under the same conditions. Further analyses reveal that ANXA2 forms a complex with VEC and its phosphatases, EC-specific protein tyrosine phosphatase (VE-PTP) and Src homology phosphatase 2 (SHP2), both of which are implicated in vascular integrity. In the absence of ANXA2, VEC is hyperphosphorylated at tyrosine 731 in response to vascular endothelial growth factor, which likely contributes to hypoxia-induced extravasation of fluid and leukocytes. We conclude that ANXA2 contributes to pulmonary microvascular integrity by enabling VEC-related phosphatase activity, thereby preventing vascular leak during alveolar hypoxia.

Citation:
J Exp Med. 2017 Jul 10. pii: jem.20160652. doi: 10.1084/jem.20160652. [Epub ahead of print]

http://jem.rupress.org/content/early/2017/07/07/jem.20160652

A possible Fourier transform infrared-based plasma fingerprint of angiotensin-converting enzyme inhibitor-induced reversal of endothelial dysfunction in diabetic mice

Authors:
Staniszewska-Slezak E, Wiercigroch E, Fedorowicz A, Buczek E, Mateuszuk L, Baranska M, Chlopicki S, Malek K

Abstract:
Angiotensin-converting enzyme inhibitors (ACE-I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)-based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased β-sheet formation. Spectral markers at 3010, 1520/1238 cm-1 , representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE-I.

Citation:
J Biophotonics. 2017 Jul 12. doi: 10.1002/jbio.201700044. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28700133

Rapamycin reversal of VEGF-C–driven lymphatic anomalies in the respiratory tract

Authors:
Peter Baluk, Li-Chin Yao, Julio C. Flores, Dongwon Choi, Young-Kwon Hong, and Donald M. McDonald

Abstract:
Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C–driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

Citation:
JCI Insight. 2017 Aug 17;2(16). pii: 90103. doi: 10.1172/jci.insight.90103. [Epub ahead of print]

https://insight.jci.org/articles/view/90103

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation

Authors:
Bailey KA, Haj FG, Simon SI, Passerini AG

Abstract:
Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.

Citation:
Sci Rep. 2017 Aug 15;7(1):8196. doi: 10.1038/s41598-017-08417-9.

https://www.ncbi.nlm.nih.gov/pubmed/?term=shear+stress+activates+endoplasmic+reticulum+stress+to+promote+endothelial+inflammation

Endothelial cells respond to the direction of mechanical stimuli through SMAD signaling to regulate coronary artery size

Authors:
Poduri A, Raftrey B, Chang AH, Rhee S, Van M, Red-Horse K

Abstract:
How mechanotransduction intersects with chemical and transcriptional factors to shape organogenesis is an important question in developmental biology. This is particularly relevant to the cardiovascular system, which uses mechanical signals from flowing blood to stimulate cytoskeletal and transcriptional responses that form a highly efficient vascular network. Using this system, artery size and structure are tightly regulated, but the underlying mechanisms are poorly understood. Here, we demonstrate that deletion of Smad4 increased the diameter of coronary arteries during embryonic development, a phenotype that followed the initiation of blood flow. At the same time, the BMP signal transducers SMAD1/5/8 were activated in developing coronary arteries. In a culture model of blood flow-induced shear stress, coronary artery endothelial cells failed to align when either BMPs were inhibited or Smad4 was depleted. In contrast to control cells, Smad4-deficient cells did not migrate against the direction of shear stress and increased proliferation rates specifically under flow. Similar alterations were seen in coronary arteries in vivo Thus, endothelial cells perceive the direction of blood flow and respond through SMAD signaling to regulate artery size.

Citation:
Development. 2017 Jul 31. pii: dev.150904. doi: 10.1242/dev.150904. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28760815

DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis

Authors:
Chang AH, Raftrey BC, D'Amato G, Surya VN, Poduri A, Chen HI, Goldstone AB, Woo J, Fuller GG, Dunn AR, Red-Horse K

Abstract:
Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronary arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 had small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12 Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis but was down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically down-regulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model in which DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that down-regulates DACH1. Thus, we identified a mechanism by which blood flow quality balances artery growth and maturation.

Citation:
Genes Dev. 2017 Aug 4. doi: 10.1101/gad.301549.117. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28779009

SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling

Authors:
Zhu X, Ding S, Qiu C, Shi Y, Song L, Wang Y, Wang Y, Li J, Wang Y, Sun Y, Qin L, Chen J, Simons M, Min W, Yu L

Abstract:
Rationale: The highly conserved NOTCH signaling pathway functions as a key cell-cell interaction mechanism controlling cell-fate and tissue patterning, while its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls it signal transduction. Our previous study indicated the potential role of post-translational small ubiquitin-like modifier (SUMO) modification (SUMOylation) in vascular disorders. Objective: To investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Methods and Results: Endothelial SENP1 deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system) deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with NOTCH1 intracellular domain (N1ICD) it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a co-transcriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 stimulation. This in turn suppressed VEGF receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. Conclusions: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in non-vascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

Citation:
Circ. Res. 2017 Jul 31. pii: CIRCRESAHA.117.310696. doi: 10.1161/CIRCRESAHA.117.310696. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28760777

Annexin A2 supports pulmonary microvascular integrity by linking vascular endothelial cadherin and protein tyrosine phosphatases

Authors:
Luo M, Flood EC, Almeida D, Yan L, Berlin DA, Heerdt PM, Hajjar KA

Abstract:
Relative or absolute hypoxia activates signaling pathways that alter gene expression and stabilize the pulmonary microvasculature. Alveolar hypoxia occurs in disorders ranging from altitude sickness to airway obstruction, apnea, and atelectasis. Here, we report that the phospholipid-binding protein, annexin A2 (ANXA2) functions to maintain vascular integrity in the face of alveolar hypoxia. We demonstrate that microvascular endothelial cells (ECs) from Anxa2−/− mice display reduced barrier function and excessive Src-related tyrosine phosphorylation of the adherens junction protein vascular endothelial cadherin (VEC). Moreover, unlike Anxa2+/+ controls, Anxa2−/− mice develop pulmonary edema and neutrophil infiltration in the lung parenchyma in response to subacute alveolar hypoxia. Mice deficient in the ANXA2-binding partner, S100A10, failed to demonstrate hypoxia-induced pulmonary edema under the same conditions. Further analyses reveal that ANXA2 forms a complex with VEC and its phosphatases, EC-specific protein tyrosine phosphatase (VE-PTP) and Src homology phosphatase 2 (SHP2), both of which are implicated in vascular integrity. In the absence of ANXA2, VEC is hyperphosphorylated at tyrosine 731 in response to vascular endothelial growth factor, which likely contributes to hypoxia-induced extravasation of fluid and leukocytes. We conclude that ANXA2 contributes to pulmonary microvascular integrity by enabling VEC-related phosphatase activity, thereby preventing vascular leak during alveolar hypoxia.

Citation:
J Exp Med. 2017 Jul 10. pii: jem.20160652. doi: 10.1084/jem.20160652. [Epub ahead of print]

http://jem.rupress.org/content/early/2017/07/07/jem.20160652

Tubulogenesis of co-cultured human iPS-derived endothelial cells and human mesenchymal stem cells in fibrin and gelatin methacrylate gels

Authors:
Calderon GA, Thai P, Hsu CW, Grigoryan B, Gibson SM, Dickinson ME, Miller JS

Abstract:
Here, we investigate the tubulogenic potential of commercially-sourced iPS-ECs with and without supporting commercially-sourced hMSCs within 3D natural fibrin or semi-synthetic gelatin methacrylate (GelMA) hydrogels. We developed a selectable dual color third generation lentiviral reporter (hEF1α-H2B-mOrange2-IRES-EGFP PGK-Puro) to differentially label the nucleus and cytoplasm of iPS-ECs which allowed real-time tracking of key steps of vascular morphogenesis such as vacuole formation and coalescence to form shared multicellular lumens. We implement 3D quantification of the network character and validate that transduced and untransduced iPS-ECs can form tubules in fibrin with or without supporting hMSCs. In addition to natural fibrin gels, we also investigated tubulogenesis in GelMA, a semi-synthetic material that has received increased interest due to its ability to be photopatterned and 3D printed, and which may thus boost development of complex 3D models for regenerative medicine studies. We find that iPS-ECs alone have a muted tubulogenic response within GelMA, but that their tubulogenic response is enhanced when they are co-cultured with a small fraction of hMSCs (2% of total cells). Our work bolsters previous findings by validating established tubulogenic mechanisms with commercially available iPS-ECs, and we expect our findings will benefit biologic studies of vasculogenesis and will have applications in tissue engineering to pre-vascularize tissue constructs which are fabricated with advanced photopatterning and three-dimensional printing.

Citation:
Biomater Sci. 2017 Jun 29. doi: 10.1039/c7bm00223h. [Epub ahead of print]

http://pubs.rsc.org/en/content/articlelanding/2017/bm/c7bm00223h#!divAbstract

RASA1 regulates the function of lymphatic vessel valves in mice

Authors:
Lapinski PE, Lubeck BA, Chen D, Doosti A, Zawieja SD, Davis MJ, King PD

Abstract:
Capillary malformation-arteriovenous malformation (CM-AVM) is a blood and lymphatic vessel (LV) disorder that is caused by inherited inactivating mutations of the RASA1 gene, which encodes p120 RasGAP (RASA1), a negative regulator of the Ras small GTP-binding protein. How RASA1 mutations lead to the LV leakage defects that occur in CM-AVM is not understood. Here, we report that disruption of the Rasa1 gene in adult mice resulted in loss of LV endothelial cells (LECs) specifically from the leaflets of intraluminal valves in collecting LVs. As a result, valves were unable to prevent fluid backflow and the vessels were ineffective pumps. Furthermore, disruption of Rasa1 in midgestation resulted in LEC apoptosis in developing LV valves and consequently failed LV valvulogenesis. Similar phenotypes were observed in induced RASA1-deficient adult mice and embryos expressing a catalytically inactive RASA1R780Q mutation. Thus, RASA1 catalytic activity is essential for the function and development of LV valves. These data provide a partial explanation for LV leakage defects and potentially other LV abnormalities observed in CM-AVM.

Citation:
J Clin Invest. 2017 May 22. pii: 89607. doi: 10.1172/JCI89607. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28530642

Cell adhesion controlled by adhesion G-protein coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising Intersectins and Elmo/Dock

Authors:
Hernández-Vásquez MN, Adame-García SR, Hamoud N, Chidiac R, Reyes-Cruz G, Gratton JP, Côté JF, Vázquez-Prado J

Abstract:
Developmental angiogenesis and the maintenance of blood brain barrier involve endothelial cell adhesion, which are linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an Adhesion GPCR-family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the GPCR at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the RhoGEFs ELMO/Dock and Intersectin (ITSN). Small fragments of either ELMO or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124/ELMO complex. Furthermore, GPR124 also promotes the activation of the ELMO/Dock complex as measured by ELMO phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, ELMO and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-ELMO and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via ELMO/Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional RhoGEFs for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

Citation:
J Biol Chem. 2017 Jun 9. pii: jbc.M117.780304. doi: 10.1074/jbc.M117.780304. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28600358

Host non-inflammatory neutrophils mediate the engraftment of bioengineered vascular networks

Authors:
Ruei-Zeng Lin, Chin Nien Lee, Rafael Moreno-Luna, Joseph Neumeyer, Breanna Piekarski, Pingzhu Zhou, Marsha A. Moses, Monisha Sachdev, William T. Pu, Sitaram Emani, & Juan M. Melero-Martin

Abstract:
Notwithstanding the remarkable progress in vascular network engineering, implanted bioengineered microvessels mostly fail to form anastomoses with the host vasculature. Here we demonstrate that implants containing assembled human vascular networks (A-grafts) fail to engraft owing to their inability to engage non-inflammatory host neutrophils upon implantation into mice. By contrast, unassembled vascular cells (U-grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-grafts re-engaged neutrophils and enhanced revascularization in A-grafts, a process that was recapitulated by blocking Notch signalling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues.

Citation:
Nature Biomedical Engineering 1, Article number: 0081 (2017) doi:10.1038/s41551-017-0081

http://www.nature.com/articles/s41551-017-0081

Altered feto-placental vascularization, feto-placental malperfusion, and fetal growth restriction in mice with Egfl7 loss-of-function

Authors:
Lauretta A. Lacko, Romulo Hurtado, Samantha Hinds, Michael G. Poulos, Jason M. Butler, Heidi Stuhlmann

Abstract:
EGFL7 is a secreted, angiogenic factor produced by embryonic endothelial cells. To understand its role in placental development, we established a novel Egfl7 knockout mouse. The mutant mice have gross defects in chorioallantoic branching morphogenesis and placental vascular patterning. Microangiography and 3D imaging revealed patchy perfusion of Egfl7−/− placentas marked by impeded blood conductance through sites of narrowed vessels. Consistent with poor feto-placental perfusion, Egfl7 knockout resulted in reduced placental weight and fetal growth restriction. The placentas also showed abnormal fetal vessel patterning and >50% reduction in fetal blood space. In vitro, placental endothelial cells were deficient in migration, cord formation, and sprouting. Expression of genes involved in branching morphogenesis, Gcm1, SynA, and SynB, and patterning of the extracellular matrix, Mmrn1, were temporally dysregulated in the placentas. Egfl7 knockout did not affect expression of the microRNA embedded within intron 7. Collectively, these data reveal that Egfl7 is critical for placental vascularization and embryonic growth, and may provide insight into etiological factors underlying placental pathologies associated with intrauterine growth restriction, a significant cause of infant morbidity and mortality.

Citation:
Development 2017 : doi: 10.1242/dev.147025

http://dev.biologists.org/content/early/2017/05/18/dev.147025

Endothelial Myocyte Enhancer Factor 2c Inhibits Migration of Smooth Muscle Cells Through Fenestrations in the Internal Elastic Lamina

Authors:
Lu YW, Lowery AM, Sun LY, Singer HA, Dai G, Adam AP, Vincent PA, Schwarz JJ

Abstract:
OBJECTIVE: Laminar flow activates myocyte enhancer factor 2 (MEF2) transcription factors in vitro to induce expression of atheroprotective genes in the endothelium. Here we sought to establish the role of Mef2c in the vascular endothelium in vivo. APPROACH AND RESULTS: To study endothelial Mef2c, we generated endothelial-specific deletion of Mef2c using Tie2-Cre or Cdh5-Cre-ERT2 and examined aortas and carotid arteries by en face immunofluorescence. We observed enhanced actin stress fiber formation in the Mef2c-deleted thoracic aortic endothelium (laminar flow region), similar to those observed in normal aortic inner curvature (disturbed flow region). Furthermore, Mef2c deletion resulted in the de novo formation of subendothelial intimal cells expressing markers of differentiated smooth muscle in the thoracic aortas and carotids. Lineage tracing showed that these cells were not of endothelial origin. To define early events in intimal development, we induced endothelial deletion of Mef2c and examined aortas at 4 and 12 weeks postinduction. The number of intimal cell clusters increased from 4 to 12 weeks, but the number of cells within a cluster peaked at 2 cells in both cases, suggesting ongoing migration but minimal proliferation. Moreover, we identified cells extending from the media through fenestrations in the internal elastic lamina into the intima, indicating transfenestral smooth muscle migration. Similar transfenestral migration was observed in wild-type carotid arteries ligated to induce neointimal formation. CONCLUSIONS: These results indicate that endothelial Mef2c regulates the endothelial actin cytoskeleton and inhibits smooth muscle cell migration into the intima.

Citation:
Arterioscler Thromb Vasc Biol. 2017 May 4. pii: ATVBAHA.117.309180. doi: 10.1161/ATVBAHA.117.309180. [Epub ahead of print]

http://atvb.ahajournals.org/content/early/2017/05/04/ATVBAHA.117.309180

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells

Authors:
Kenagy RD, Kikuchi S, Chen L, Wijelath ES, Stergachis AB, Stamatoyannopoulos J, Tang GL, Clowes AW, Sobel M

Abstract:
BACKGROUND: Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. METHODS: Human saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs. RESULTS: p27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response. CONCLUSIONS: These results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

Citation:
J Vasc Surg. 2017 May 16. pii: S0741-5214(17)30104-0. doi: 10.1016/j.jvs.2016.12.113. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28526559

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution

Authors:
Kroon J, Heemskerk N, Kalsbeek MJT, de Waard V, van Rijssel J, van Buul JD

Abstract:
Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, if sustained and local activation of Rac1 is required for long-term flow-induced cell alignment is not clear. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 hours upon long-term flow. Silencing studies revealed that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and as a result for long-term flow-induced cell alignment. Surprisingly, Trio appeared to be not involved in flow-induced activation of Rac1. In conclusion, our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner. Our data further show that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions.

Citation:
Mol Biol Cell. 2017 May 17. pii: mbc.E16-06-0389. doi: 10.1091/mbc.E16-06-0389. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28515142

Engineering Vascularized Flaps Using Adipose-Derived Microvascular EC and MSC

Authors:
Freiman A, Shandalov Y, Rosenfeld D, Shor E, Ben-David D, Meretzki S, Levenberg S, Egozi D

Abstract:
Human adipose-derived microvascular endothelial cells (HAMEC) and mesenchymal stem cells (MSC) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co-culturing HAMEC:MSC on a porous biodegradable scaffold in vitro, later implanted as a graft around femoral blood vessels in a rat, will result in its vascularization by host vessels, creating a functional vascular flap that can effectively treat a range of large full-thickness soft tissue defects. HAMEC were co-cultured with MSC on polymeric 3D porous constructs. Grafts were then implanted around the femoral vessels of a rat. To ensure vessel-sprouting from the main femoral vessels, grafts were pre-isolated from the surrounding tissue. Graft vascularization was monitored to confirm full vascularization before flap transfer. Flaps were then transferred to treat both abdominal wall and exposed bone and tendon of an ankle defects. Flaps were analyzed to determine vascular properties in terms of maturity, functionality and survival of implanted cells. Findings show that pre-isolated grafts bearing the HAMEC:MSC combination, promoted formation of highly vascularized flaps, which were better integrated in both defect-models. The results of this study show the essentiality of specific adipose-derived cell-combination in successful graft vascularization and integration, two processes crucial for flap survival.

Citation:
J Tissue Eng Regen Med. 2017 Apr 6. doi: 10.1002/term.2436. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28382732

Integrative meta-modeling identifies endocytic vesicles, late endosome and the nucleus as the cellular compartments primarily directing RTK signaling

Authors:
Weddell JC, Imoukhuede PI

Abstract:
Recently, intracellular receptor signaling has been identified as a key component mediating cell responses for various receptor tyrosine kinases (RTKs). However, the extent each endocytic compartment (endocytic vesicle, early endosome, recycling endosome, late endosome, lysosome and nucleus) contributes to receptor signaling has not been quantified. Furthermore, our understanding of endocytosis and receptor signaling is complicated by cell- or receptor-specific endocytosis mechanisms. Therefore, towards understanding the differential endocytic compartment signaling roles, and identifying how to achieve signal transduction control for RTKs, we delineate how endocytosis regulates RTK signaling. We achieve this via a meta-analysis across eight RTKs, integrating computational modeling with experimentally derived cell (compartment volume, trafficking kinetics and pH) and ligand–receptor (ligand/receptor concentration and interaction kinetics) physiology. Our simulations predict the abundance of signaling from eight RTKs, identifying the following hierarchy in RTK signaling: PDGFRβ > IGFR1 > EGFR > PDGFRα > VEGFR1 > VEGFR2 > Tie2 > FGFR1. We find that endocytic vesicles are the primary cell signaling compartment; over 43% of total receptor signaling occurs within the endocytic vesicle compartment for these eight RTKs. Mechanistically, we found that high RTK signaling within endocytic vesicles may be attributed to their low volume (5.3 × 10−19 L) which facilitates an enriched ligand concentration (3.2 μM per ligand molecule within the endocytic vesicle). Under the analyzed physiological conditions, we identified extracellular ligand concentration as the most sensitive parameter to change; hence the most significant one to modify when regulating absolute compartment signaling. We also found that the late endosome and nucleus compartments are important contributors to receptor signaling, where 26% and 18%, respectively, of average receptor signaling occurs across the eight RTKs. Conversely, we found very low membrane-based receptor signaling, exhibiting <1% of the total receptor signaling for these eight RTKs. Moreover, we found that nuclear translocation, mechanistically, requires late endosomal transport; when we blocked receptor trafficking from late endosomes to the nucleus we found a 57% reduction in nuclear translocation. In summary, our research has elucidated the significance of endocytic vesicles, late endosomes and the nucleus in RTK signal propagation.

Citation:
Integr Biol (Camb). 2017 Apr 24. doi: 10.1039/c7ib00011a. [Epub ahead of print]

http://pubs.rsc.org/en/content/articlelanding/2017/ib/c7ib00011a#!divAbstract

Roles of NADPH oxidase and mitochondria in flow-induced vasodilation of human adipose arterioles: ROS induced ROS release in coronary artery disease

Authors:
Zinkevich NS, Fancher IS, Gutterman DD, Phillips SA

Abstract:
OBJECTIVES: Hydrogen peroxide (H2 O2 ) contributes to flow-induced dilation (FID) of human arterioles. This study is designed to examine the roles of mitochondria and NADPH oxidase in modulating the release of ROS and in mediating FID. We tested whether NADPH oxidase contributes to mitochondrial ROS generation in arterioles during coronary artery disease (CAD). METHODS: Visceral adipose arterioles obtained from patients with or without CAD were cannulated and pressurized for videomicroscopic measurement of arteriolar diameters. Dilator responses and ROS production during flow were determined in the presence and absence of the NADPH oxidase inhibitor gp91ds-tat and the mitochondrial electron transport inhibitor rotenone. RESULTS: Both dilation and H2 O2 generation during flow were reduced in the presence of rotenone (13.5±8% vs. 97±17% without rotenone) or gp91ds-tat in patients with CAD, while patients without CAD exhibited H2 O2 -independent dilations. Mitochondrial superoxide production during flow was attenuated by gp91ds-tat in arterioles from CAD patients. CONCLUSIONS: These findings indicate that ROS produced by NADPH oxidase are an upstream component of the mitochondria-dependent pathway contributing to flow-dependent H2 O2 generation and dilation in peripheral microvessels from patients with CAD. We conclude that in CAD, both mitochondria and NADPH oxidase contribute to FID through a redox mechanism in visceral arterioles. This article is protected by copyright. All rights reserved.

Citation:
Microcirculation. 2017 May 8. doi: 10.1111/micc.12380. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28480622

Development of the larval lymphatic system in the zebrafish

Authors:
Hyun Min Jung, Daniel Castranova, Matthew R. Swift, Van N. Pham, Marina Venero Galanternik, Sumio Isogai, Matthew G. Butler, Timothy S. Mulligan, Brant M. Weinstein

Abstract:
The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking, and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)y251 transgenic zebrafish using a Mannose receptor C type 1 (MRC1) promoter that drives strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal lymphatics, spinal lymphatic, superficial lateral lymphatics, and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

Citation:
Development 2017 : doi: 10.1242/dev.145755

http://dev.biologists.org/content/early/2017/05/11/dev.145755

Mechanotransmission in endothelial cells subjected to oscillatory and multi-directional shear flow

Authors:
Mahsa Dabagh, Payman Jalali, Peter J. Butler, Amanda Randles, John M. Tarbell

Abstract:
Local haemodynamics are linked to the non-uniform distribution of atherosclerosic lesions in arteries. Low and oscillatory (reversing in the axial flow direction) wall shear stress (WSS) induce inflammatory responses in endothelial cells (ECs) mediating disease localization. The objective of this study is to investigate computationally how the flow direction (reflected in WSS variation on the EC surface over time) influences the forces experienced by structural components of ECs that are believed to play important roles in mechanotransduction. A three-dimensional, multi-scale, multi-component, viscoelastic model of focally adhered ECs is developed, in which oscillatory WSS (reversing or non-reversing) parallel to the principal flow direction, or multi-directional oscillatory WSS with reversing axial and transverse components are applied over the EC surface. The computational model includes the glycocalyx layer, actin cortical layer, nucleus, cytoskeleton, focal adhesions (FAs), stress fibres and adherens junctions (ADJs). We show the distinct effects of atherogenic flow profiles (reversing unidirectional flow and reversing multi-directional flow) on subcellular structures relative to non-atherogenic flow (non-reversing flow). Reversing flow lowers stresses and strains due to viscoelastic effects, and multi-directional flow alters stress on the ADJs perpendicular to the axial flow direction. The simulations predict forces on integrins, ADJ filaments and other substructures in the range that activate mechanotransduction.

Citation:
Journal of the Royal Society Interface, Published 17 May 2017. DOI: 10.1098/rsif.2017.0185

http://rsif.royalsocietypublishing.org/content/14/130/20170185

Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis

Authors:
Lam JD, Oh DJ, Wong LL, Amarnani D, Park-Windhol C, Sanchez AV, Cardona-Velez J, McGuone D, Stemmer-Rachamimov AO, Eliott D, Bielenberg DR, van Zyl T, Shen L, Gai X, D'Amore PA, Kim LA, Arboleda-Velasquez JF

Abstract:
Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population, and affects those with type 1 and type 2 diabetes mellitus. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31+ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVM) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVM and angiogenic tufts in the retina of mice with oxygen-induced retinopathy (OIR), suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in OIR, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.

Citation:
Diabetes. 2017 Apr 11. pii: db161035. doi: 10.2337/db16-1035. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28400392

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries

Authors:
Bloksgaard M, Leurgans TM, Spronck B, Heusinkveld MH, Thorsted B, Rosenstand K, Nissen I, Hansen UM, Brewer JR, Bagatolli LA, Rasmussen LM, Irmukhamedov A, Reesink KD, De Mey JGR

Abstract:
The impact of disease related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared to frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as model system, integrating vital imaging, pressure myography and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the loadbearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2≥0.99), while there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (mean±SEM) of 1.1±0.2 circumferentially and 0.20±0.01 longitudinally, corresponding to a pressure of ~40mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8±0.1 circumferentially and 0.06±0.02 longitudinally, reached at a distending pressure below 20mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show for the first time in resistance arteries a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.

Citation:
Am J Physiol Heart Circ Physiol. 2017 Apr 21:ajpheart.00110.2017. doi: 10.1152/ajpheart.00110.2017. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28432057

Functional and Biochemical Endothelial Profiling In Vivo in a Murine Model of Endothelial Dysfunction; Comparison of Effects of 1-Methylnicotinamide and Angiotensin-converting Enzyme Inhibitor

Authors:
Bar A, Olkowicz M, Tyrankiewicz U, Kus E, Jasinski K, Smolenski RT, Skorka T, Chlopicki S.

Abstract:
Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR-/-) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1-7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR-/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate®FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T1) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1-7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR-/- mice, Ach induced a vasoconstriction in BCA that amounted to -7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1-7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1-9) and Ang-(1-7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR-/- mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1-7) balance and in L-arginine/ADMA ratio in plasma.

Citation:
Front Pharmacol. 2017 Apr 10;8:183. doi: 10.3389/fphar.2017.00183. eCollection 2017.

https://www.ncbi.nlm.nih.gov/pubmed/28443021

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling

Authors:
Heinolainen K, Karaman S, D'Amico G, Tammela T, Sormunen R, Eklund L, Alitalo K, Zarkada G.

Abstract:
Rationale: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), while lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. Objective: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. Methods and Results: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated Vegfr2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3Conclusions: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

Citation:
Circ Res. 2017 Mar 15. pii: CIRCRESAHA.116.310477. doi: 10.1161/CIRCRESAHA.116.310477. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28298294

Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

Authors:
Leppänen VM, Saharinen P, Alitalo K

Abstract:
The endothelial cell (EC)-specific receptor tyrosine kinases Tie1 and Tie2 are necessary for the remodeling and maturation of blood and lymphatic vessels. Angiopoietin-1 (Ang1) growth factor is a Tie2 agonist, whereas Ang2 functions as a context-dependent agonist/antagonist. The orphan receptor Tie1 modulates Tie2 activation, which is induced by association of angiopoietins with Tie2 in cis and across EC-EC junctions in trans Except for the binding of the C-terminal angiopoietin domains to the Tie2 ligand-binding domain, the mechanisms for Tie2 activation are poorly understood. We report here the structural basis of Ang1-induced Tie2 dimerization in cis and provide mechanistic insights on Ang2 antagonism, Tie1/Tie2 heterodimerization, and Tie2 clustering. We find that Ang1-induced Tie2 dimerization and activation occurs via the formation of an intermolecular β-sheet between the membrane-proximal (third) Fibronectin type III domains (Fn3) of Tie2. The structures of Tie2 and Tie1 Fn3 domains are similar and compatible with Tie2/Tie1 heterodimerization by the same mechanism. Mutagenesis of the key interaction residues of Tie2 and Tie1 Fn3 domains decreased Ang1-induced Tie2 phosphorylation and increased the basal phosphorylation of Tie1, respectively. Furthermore, the Tie2 structures revealed additional interactions between the Fn 2 (Fn2) domains that coincide with a mutation of Tie2 in primary congenital glaucoma that leads to defective Tie2 clustering and junctional localization. Mutagenesis of the Fn2-Fn2 interface increased the basal phosphorylation of Tie2, suggesting that the Fn2 interactions are essential in preformed Tie2 oligomerization. The interactions of the membrane-proximal domains could provide new targets for modulation of Tie receptor activity.

Citation:
Proc Natl Acad Sci U S A. 2017 Apr 10. pii: 201616166. doi: 10.1073/pnas.1616166114. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=Structural+basis+of+Tie2+activation+and+Tie2%2FTie1+heterodimerization

Development of a novel strategy to target CD39 antithrombotic activity to the endothelial-platelet microenvironment in kidney ischemia-reperfusion injury

Authors:
Sashindranath M, Dwyer KM, Dezfouli S, Selan C, Crikis S, Lu B, Yuan Y, Hickey MJ, Peter K, Robson SC, Cowan PJ, Nandurkar HH.

Abstract:
Kidney ischemia-reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.

Citation:
Purinergic Signal. 2017 Mar 25. doi: 10.1007/s11302-017-9558-3. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28343356

4D Microvascular Analysis Reveals that Regenerative Angiogenesis in Ischemic Muscle Produces a Flawed Microcirculation

Authors:
John-Michael Arpino, Zengxuan Nong, Fuyan Li, Hao Yin, Nour W Ghonaim, Stephanie Milkovich, Brittany Balint, Caroline O'Neil, Graham M Fraser, Daniel Goldman, Christopher G Ellis, J G Pickering

Abstract:
Rationale: Angiogenesis occurs following ischemic injury to skeletal muscle and enhancing this response has been a therapeutic goal. However, to appropriately deliver oxygen, a precisely organized and exquisitely responsive microcirculation must form. Whether these network attributes exist in a regenerated microcirculation is unknown and methodologies for answering this have been lacking. Objective: To develop 4D methodologies for elucidating microarchitecture and function of the reconstructed microcirculation in skeletal muscle. Methods and Results: We established a model of complete microcirculatory regeneration following ischemia-induced obliteration in the mouse extensor digitorum longus muscle. Dynamic imaging of red blood cells revealed the regeneration of an extensive network of flowing neo-microvessels, which after 14 days structurally resembled that of uninjured muscle. However, the skeletal muscle remained hypoxic. Red blood cell transit analysis revealed slow and stalled flow in the regenerated capillaries and extensive arteriolar-venular shunting. Furthermore, spatial heterogeneity in capillary red cell transit was highly constrained. Moreover, red blood cell oxygen saturation was both low and profoundly variable. These abnormalities persisted to 120 days after injury. To determine if the regenerated microcirculation could regulate flow, the muscle was subjected to local hypoxia using an oxygen-permeable membrane. Hypoxia promptly increased red cell velocity and flux in control capillaries but in neo-capillaries the response was blunted. Three-dimensional confocal imaging revealed that neo-arterioles were aberrantly covered by smooth muscle cells, with increased inter-process spacing and haphazard actin microfilament bundles. Conclusions: Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed in both structure and function, with no evidence for normalizing over time. This network-level dysfunction must be recognized and overcome in order to advance regenerative approaches for ischemic disease.

Citation:
Circulation Research. 2017;CIRCRESAHA.116.310535

http://circres.ahajournals.org/content/early/2017/02/07/CIRCRESAHA.116.310535

Lymphatic deletion of calcitonin receptor–like receptor exacerbates intestinal inflammation

Authors:
Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, and Kathleen M. Caron

Abstract:
Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor–like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrlfl/fl/Prox1-CreERT2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrlfl/fl/Prox1-CreERT2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.

Citation:
JCI Insight. 2017;2(6):e92465. doi:10.1172/jci.insight.92465.

https://insight.jci.org/articles/view/92465

Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

Authors:
Brittany G. Durgin, Olga A. Cherepanova, Delphine Gomez, Themistoclis Karaoli, Gabriel F. Alencar, Joshua T. Butcher, Yu-Qing Zhou, Michelle P. Bendeck, Brant E. Isakson, Gary K. Owens, Jessica J. Connelly

Abstract:
Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMC) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMC are a crucial source of collagens and that this impacts lesion development or fibrous cap formation. We sought to determine how conditional SMC specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice affects advanced lesion formation given: 1) we previously identified a Col15a1 sequence variant associated with age related atherosclerosis; 2) COL15A1 is a matrix organizer enhancing tissue structural integrity; and 3) siRNA mediated Col15a1 knockdown increased migration and decreased proliferation of cultured human SMC. We hypothesized that SMC-derived COL15A1 is critical in advanced lesions, specifically in fibrous cap formation. Surprisingly, we demonstrate that SMC specific Col15a1 knockout mice fed a Western diet for 18 weeks failed to form advanced lesions. SMC specific Col15a1 knockout resulted in lesions reduced in size by 78%, with marked reductions in number and proliferating SMC, and lacked a SMC and ECM-rich lesion or fibrous cap. In vivo RNA-seq analyses on SMC Col15a1 knockout and wild type lesions suggest that a mechanism for these effects is through global repression of multiple pro-atherogenic inflammatory pathways involved in lesion development. These results provide the first direct evidence that a SMC-derived collagen, COL15A1, is critical during lesion pathogenesis but contrary to expectations, its loss resulted in marked attenuation rather than exacerbation of lesion pathogenesis.

Citation:
Am J Physiol Heart Circ Physiol. 2017 Mar 10:ajpheart.00029.2017. doi: 10.1152/ajpheart.00029.2017. [Epub ahead of print]

http://ajpheart.physiology.org/content/early/2017/03/10/ajpheart.00029.2017

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity

Authors:
Wan Zhu, Fanxia Shen, Lei Mao, Lei Zhan, Shuai Kang, Zhengda Sun, Jeffrey Nelson, Rui Zhang, Dingquan Zou, Cameron M. McDougall, Michael T. Lawton, Thiennu H. Vu, Zhijian Wu, Abraham Scaria, Peter Colosi, John Forsayeth, Hua Su

Abstract:
BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. METHODS: Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22αCre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. RESULTS: AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated model 1 mice. CONCLUSIONS: By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

Citation:
Stroke. 2017 Mar 21. pii: STROKEAHA.116.015713. doi: 10.1161/STROKEAHA.116.015713. [Epub ahead of print]

http://stroke.ahajournals.org/content/early/2017/03/21/STROKEAHA.116.015713

Pyk2 Phosphorylation of VE-PTP Downstream of STIM1 induced Ca2+ entry Regulates Disassembly of Adherens Junctions

Authors:
Soni D, Regmi SC, Wang DM, DebRoy A, Zhao YY, Vogel SM, Malik AB, Tiruppathi C

Abstract:
VE-PTP stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of STIM1 activation of store-operated Ca2+ entry (SOCE) in regulating adherens junction assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation, and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated the permeability response. Thus, Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP in turn binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin, and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.

Citation:
Am J Physiol Lung Cell Mol Physiol. 2017 Apr 6:ajplung.00008.2017. doi: 10.1152/ajplung.00008.2017. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28385807

Inhibition of Smooth Muscle β-Catenin Hinders Neointima Formation After Vascular Injury

Authors:
Riascos-Bernal DF, Chinnasamy P, Gross JN, Almonte V, Egaña-Gorroño L, Parikh D, Jayakumar S, Guo L, Sibinga NE

Abstract:
OBJECTIVE: Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although β-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of β-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC β-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of β-catenin inhibitors on vascular SMC growth. APPROACH AND RESULTS: We used an inducible, conditional genetic deletion of β-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC β-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC β-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking β-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, β-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. CONCLUSIONS: SMC β-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological β-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting β-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

Citation:
Arterioscler Thromb Vasc Biol. 2017 Mar 16. pii: ATVBAHA.116.308643. doi: 10.1161/ATVBAHA.116.308643. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28302627

VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

Authors:
Alessandro Fantin, Anastasia Lampropoulou, Valentina Senatore, James T. Brash, Claudia Prahst, Clemens A. Lange, Sidath E. Liyanage, Claudio Raimondi, James W. Bainbridge, Hellmut G. Augustin, and Christiana Ruhrberg

Abstract:
The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.

Citation:
J Exp Med. 2017 Mar 13. pii: jem.20160311. doi: 10.1084/jem.20160311. [Epub ahead of print]

http://jem.rupress.org/content/early/2017/03/10/jem.20160311

Role of Genetic Variation in Collateral Circulation in the Evolution of Acute Stroke: A Multimodal Magnetic Resonance Imaging Study

Authors:
Kao YJ, Oyarzabal EA, Zhang H, Faber JE, Shih YI

Abstract:
BACKGROUND AND PURPOSE: No studies have determined the effect of differences in pial collateral extent (number and diameter), independent of differences in environmental factors and unknown genetic factors, on severity of stroke. We examined ischemic tissue evolution during acute stroke, as measured by magnetic resonance imaging and histology, by comparing 2 congenic mouse strains with otherwise identical genetic backgrounds but with different alleles of the Determinant of collateral extent-1 (Dce1) genetic locus. We also optimized magnetic resonance perfusion and diffusion-deficit thresholds by using histological measures of ischemic tissue. METHODS: Perfusion, diffusion, and T2-weighted magnetic resonance imaging were performed on collateral-poor (congenic-Bc) and collateral-rich (congenic-B6) mice at 1, 5, and 24 hours after permanent middle cerebral artery occlusion. Magnetic resonance imaging-derived penumbra and ischemic core volumes were confirmed by histology in a subset of mice at 5 and 24 hours after permanent middle cerebral artery occlusion. RESULTS: Although perfusion-deficit volumes were similar between strains 1 hour after permanent middle cerebral artery occlusion, diffusion-deficit volumes were 32% smaller in collateral-rich mice. At 5 hours, collateral-rich mice had markedly restored perfusion patterns showing reduced perfusion-deficit volumes, smaller infarct volumes, and smaller perfusion-diffusion mismatch volumes compared with the collateral-poor mice (P<0.05). At 24 hours, collateral-rich mice had 45% smaller T2-weighted lesion volumes (P<0.005) than collateral-poor mice, with no difference in perfusion-diffusion mismatch volumes because of penumbral death occurring 5 to 24 hours after permanent middle cerebral artery occlusion in collateral-poor mice. CONCLUSIONS: Variation in collateral extent significantly alters infarct volume expansion, transiently affects perfusion and diffusion magnetic resonance imaging signatures, and impacts salvage of ischemic penumbra after stroke onset.

Citation:
Stroke. 2017 Feb 10. pii: STROKEAHA.116.015878. doi: 10.1161/STROKEAHA.116.015878. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28188261

Fluid shear stress induces upregulation of COX-2 and PGI2 release in endothelial cells via a pathway involving PECAM-1 , PI3K , FAK, and p38

Authors:
Russell-Puleri S, Dela Paz NG, Adams D, Chattopadhyay M, Cancel LM, Ebong E, Orr AW, Frangos JA, Tarbell JM.

Abstract:
Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important anti-atherogenic prostanoid and vasodilator produced in endothelial cells through the action of cyclooxygenase isoenzymes COX-1 and COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated, but are determined in the present study. We used cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5-hrs to examine shear stress induced induction of COX-2/PGI2Our results demonstrate the relationship between mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and intracellular mechano-responsive molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38, in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1 integrin, upregulation of COX-2 and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002)substantially inhibited FSS activation of α5β1 integrin, upregulation of COX-2 gene and protein expression and release of PGI2 in BAECs.Inhibition of integrin associated FAK (PF573228), and MAPK p38 (SB203580) also inhibited the shear induced up-regulation of COX-2. Finally, a PECAM-1 -/- mouse model was characterized by reduced COX-2 immunostaining in the aorta and reduced plasma PGI2 levels compared to wild type mice, as well as complete inhibition of acute flow-induced PGI2 release compared to wild type animals.

Citation:
Am J Physiol Heart Circ Physiol. 2016 Dec 23:ajpheart.00035.2016. doi: 10.1152/ajpheart.00035.2016. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28011582

Bone morphogenetic protein 4 regulates microRNAs miR-494 and miR-126-5p in control of endothelial cell function in angiogenesis

Authors:
Esser JS, Saretzki E, Pankratz F, Engert B, Grundmann S, Bode C, Moser M, Zhou Q

Abstract:
MicroRNAs are small non-coding RNAs that negatively regulate posttranscriptional gene expression. Several microRNAs have been described to regulate the process of angiogenesis. Previously, we have shown that bone morphogenetic protein 4 (BMP4) increased the pro-angiogenic activity of endothelial cells. In this project, we now investigated how the pro-angiogenic BMP4 effect is mediated by microRNAs. First, we performed a microRNA array with BMP4-stimulated human umbilical vein endothelial cells (HUVECs). Among the top-regulated microRNAs, we detected a decreased expression of miR-494 and increased expression of miR-126-5p. Next, we analysed the canonical Smad and alternative signalling pathways, through which BMP4 would regulate miR-126-5p and miR-494 expression. Furthermore, the functional effect of miR-494 and miR-126-5p on endothelial cells was investigated. MicroRNA-494 overexpression decreased endothelial cell proliferation, migration and sprout formation. Consistently, miR-494 inhibition increased endothelial cell function. As potential miR-494 targets, bFGF and BMP endothelial cell precursor-derived regulator (BMPER) were identified and confirmed by western blot. Luciferase assays showed direct miR-494 binding in BMPER 3'UTR. In contrast, miR-126-5p overexpression increased pro-angiogenic endothelial cell behaviour and, accordingly, miR-126-5p inhibition decreased endothelial cell function. As a direct miR-126-5p target we identified the anti-angiogenic thrombospondin-1 which was confirmed by western blot analysis and luciferase assays. In the Matrigel plug assay application of antagomiR-494 increased endothelial cell ingrowth, whereas antagomiR-126-5p treatment decreased cell ingrowth in vivo. Taken together, through differential regulation of the anti-angiomiR-494 and the angiomiR-126-5p by BMP4 both microRNAs contribute to the pro-angiogenic BMP4 effect on endothelial cells.

Citation:
Thromb Haemost. 2017 Jan 26. doi: 10.1160/TH16-08-0643. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28124060

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis

Authors:
Cyrill Géraud, Philipp-Sebastian Koch, Johanna Zierow, Kay Klapproth, Katrin Busch, Victor Olsavszky, Thomas Leibing, Alexandra Demory, Friederike Ulbrich, Miriam Diett, Sandhya Singh, Carsten Sticht, Katja Breitkopf-Heinlein, Karsten Richter, Sanna-Maria Karppinen, Taina Pihlajaniemi, Bernd Arnold, Hans-Reimer Rodewald, Hellmut G. Augustin, Kai Schledzewski, and Sergij Goerdt

Abstract:
Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.

Citation:
J Clin Invest. doi:10.1172/JCI90086

http://www.jci.org/articles/view/90086

Reduced nitric oxide bioavailability mediates cerebroarterial dysfunction independent of cerebral amyloid angiopathy in a mouse model of Alzheimer's disease

Authors:
Merlini M, Shi Y, Keller S, Savarese G, Akhmedov A, Derungs R, Spescha RD, Kulic L, Nitsch RM, Lüscher TF, Camici GG

Abstract:
In Alzheimer's disease (AD), cerebral arteries, in contrast to cerebral microvessels, show both cerebral amyloid angiopathy- (CAA) dependent and -independent vessel wall pathology. However, it remains unclear whether CAA-independent vessel wall pathology affects arterial function thereby chronically reducing cerebral perfusion, and if so which mechanisms mediate this effect. To this end, we assessed the ex vivo vascular function of the basilar artery and a similar-sized peripheral artery (femoral artery) in the Swedish-Arctic (SweArc) transgenic AD mouse model at different disease stages. Further, we used quantitative immunohistochemistry to analyze CAA, endothelial morphology, and molecular pathways pertinent to vascular relaxation. We found that endothelium-dependent, but not smooth muscle-dependent vasorelaxation was significantly impaired in basilar and femoral arteries of 15-month-old SweArc mice compared to that of age-matched wildtype (WT) and 6-month-old SweArc mice. This impairment was accompanied by significantly reduced levels of cyclic GMP (cGMP), indicating a reduced nitric oxide (NO) bioavailability. However, no age- and genotype-related differences in oxidative stress as measured by lipid peroxidation were observed. Although parenchymal capillaries, arterioles, and arteries showed abundant CAA in the 15-month-old SweArc mice, no CAA or changes in endothelial morphology were detected histologically in the basilar and femoral artery. Thus, our results suggest that in this AD mouse model dysfunction of large intracranial, extracerebral arteries important for brain perfusion is mediated by reduced NO bioavailability rather than by CAA. This finding supports the growing body of evidence highlighting the therapeutic importance of targeting the cerebrovasculature in AD.

Citation:
American Journal of Physiology - Heart and Circulatory Physiology Published 11 November 2016 Vol. no. , DOI: 10.1152/ajpheart.00607.2016

http://ajpheart.physiology.org/content/early/2016/11/09/ajpheart.00607.2016

Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1β Production

Authors:
Ceneri N, Zhao L, Young BD, Healy A, Coskun S, Vasavada H, Yarovinsky TO, Ike K, Pardi R, Qin L, Qin L, Tellides G, Hirschi K, Meadows J, Soufer R, Chun HJ, Sadeghi M, Bender JR, Morrison AR

Abstract:
OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1β (IL-1β) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1β expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1β levels. Moreover, we found that elevated IL-1β was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1β expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

Citation:
Arterioscler Thromb Vasc Biol. 2016 Nov 10. pii: ATVBAHA.116.308507. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/27834690

β-arrestin is critical for early shear stress-induced Akt/eNOS activation in human vascular endothelial cells

Authors:
Carneiro AP, Fonseca-Alaniz MH, Dallan LA, Miyakawa AA, Krieger JE

Abstract:
Recent evidence suggests that β-arrestins, which are involved in G protein-coupled receptors desensitization, may influence mechanotransduction. Here, we observed that nitric oxide (NO) production was abrogated in human saphenous vein endothelial cells (SVECs) transfected with siRNA against β-arrestin 1 and 2 subjected to shear stress (SS, 15 dynes/cm2, 10 min). The downregulation of β-arrestins 1/2 in SVECs cells also prevented the SS-induced rise in levels of phosphorylation of Akt and endothelial nitric oxide synthase (eNOS, Serine 1177). Interestingly, immunoprecipitation revealed that β-arrestin interacts with Akt, eNOS and caveolin-1 and these interactions are not influenced by SS. Our data indicate that β-arrestins and Akt/eNOS downstream signaling are required for early SS-induced NO production in SVECs, which is consistent with the idea that β-arrestins and caveolin-1 are part of a pre-assembled complex associated with the cellular mechanotransduction machinery.

Citation:
Biochem Biophys Res Commun. 2017 Jan 4. pii: S0006-291X(17)30003-7. doi: 10.1016/j.bbrc.2017.01.003. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/28062183

Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli

Authors:
Margaret F. Bennewitz, Maritza A. Jimenez, Ravi Vats, Egemen Tutuncuoglu, Jude Jonassaint, Gregory J. Kato, Mark T. Gladwin, and Prithu Sundd

Abstract:
In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.

Citation:
Citation Information: JCI Insight. 2017;2(1):e89761. doi:10.1172/jci.insight.89761.

https://insight.jci.org/articles/view/89761

T-type Ca2+ channels and Autoregulation of Local Blood Flow

Authors:
Jensen LJ, Nielsen MS, Salomonsson M, Sørensen CM

Abstract:
L-type voltage gated Ca2+ channels are considered to be the primary source of calcium influx during the myogenic response. However, many vascular beds also express T-type voltage gated Ca2+ channels. Recent studies suggest that these channels may also play a role in autoregulation. At low pressures (40–80 mm Hg) T-type channels affect myogenic responses in cerebral and mesenteric vascular beds. T-type channels also seem to be involved in skeletal muscle autoregulation. This review discusses the expression and role of T-type voltage gated Ca2+ channels in the autoregulation of several different vascular beds. Lack of specific pharmacological inhibitors has been a huge challenge in the field. Now the research has been strengthened by genetically modified models such as mice lacking expression of T-type voltage gated Ca2+ channels (CaV3.1 and CaV3.2). Hopefully, these new tools will help further elucidate the role of voltage gated T-type Ca2+ channels in autoregulation and vascular function.

Citation:
Channels (Austin). 2017 Jan 5:0. doi: 10.1080/19336950.2016.1273997. [Epub ahead of print]

http://www.tandfonline.com/doi/full/10.1080/19336950.2016.1273997

Vagal Regulation of Group 3 Innate Lymphoid Cells and the Immunoresolvent PCTR1 Controls Infection Resolution

Authors:
Dalli J, Colas RA, Arnardottir H, Serhan CN

Abstract:
Uncovering mechanisms that control immune responses in the resolution of bacterial infections is critical for the development of new therapeutic strategies that resolve infectious inflammation without unwanted side effects. We found that disruption of the vagal system in mice delayed resolution of Escherichia coli infection. Dissection of the right vagus decreased peritoneal group 3 innate lymphoid cell (ILC3) numbers and altered peritoneal macrophage responses. Vagotomy resulted in an inflammatory peritoneal lipid mediator profile characterized by reduced concentrations of pro-resolving mediators, including the protective immunoresolvent PCTR1, along with elevated inflammation-initiating eicosanoids. We found that acetylcholine upregulated the PCTR biosynthetic pathway in ILC3s. Administration of PCTR1 or ILC3s to vagotomized mice restored tissue resolution tone and host responses to E. coli infections. Together these findings elucidate a host protective mechanism mediated by ILC3-derived pro-resolving circuit, including PCTR1, that is controlled by local neuronal output to regulate tissue resolution tone and myeloid cell responses.

Citation:
Immunity DOI: http://dx.doi.org/10.1016/j.immuni.2016.12.009

http://www.cell.com/immunity/abstract/S1074-7613(16)30514-3