Here is a listing of electronic publications authored by NAVBO members and of relevance to the entire Vascular Biology Community.
ePubs
Evidence for endothelial-to-mesenchymal transition in human brain arteriovenous malformations
Authors:
Lorelei D Shoemaker, Aaron K McCormick, Breanna M Allen, Steven D Chang
Abstract:
Background: Brain arteriovenous malformations (AVMs) are rare, potentially devastating cerebrovascular lesions that can occur in both children and adults. AVMs are largely sporadic and the basic disease biology remains unclear, limiting advances in both detection and treatment. This study aimed to investigate human brain AVMs for endothelial-to-mesenchymal transition (EndMT), a process recently implicated in cerebral cavernous malformations (CCMs).
Methods: We used 29 paraffin-embedded and 13 fresh/frozen human brain AVM samples to profile expression of panels of EndMT-associated proteins and RNAs. CCMs, a cerebrovascular disease also characterized by abnormal vasculature, were used as a primary comparison, given that EndMT specifically contributes to CCM disease biology. AVM-derived cell lines were isolated from three fresh, surgical AVM samples and characterized by protein expression.
Results: We observed high collagen deposition, high PAI-1 expression, and expression of EndMT-associated transcription factors such as KLF4, SNAI1, and SNAI2 and mesenchymal-associated markers such as VIM, ACTA2, and S100A4. SMAD-dependent TGF-β signaling was not strongly activated in AVMs and this pathway may be only partially involved in mediating EndMT. Using serum-free culture conditions, we isolated myofibroblast-like cell populations from AVMs that expressed a unique range of proteins associated with mature cell types and with EndMT. Conditioned medium from these cells led to increased proliferation of HUVECs and SMCs.
Conclusions: Collectively, our results suggest a role for EndMT in AVM disease. This may lead to new avenues for disease models to further our understanding of disease mechanisms, and to the development of improved diagnostics and therapeutics.
Citation:
Clin Transl Med. 2020 Jun 21. doi: 10.1002/ctm2.99. Online ahead of print.
N6-methyladenine in DNA antagonizes SATB1 in early development
Authors:
Li Z, Zhao S, Nelakanti RV, Lin K, Wu TP, Alderman MH 3rd, Guo C, Wang P, Zhang M, Min W, Jiang Z, Wang Y, Li H, Xiao AZ
Abstract:
The recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2,3,4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD–SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.
Citation:
Nature (2020). https://doi.org/10.1038/s41586-020-2500-9. Online ahead of print.
Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations
Authors:
Wang K, Zhang H, He Y, Jiang Q, Tanaka Y, Park IH, Pober JS, Min W, Zhou HJ
Abstract:
Objective: Cerebral cavernous malformations (CCM), consisting of dilated capillary channels formed by a single layer of endothelial cells lacking surrounding mural cells. It is unclear why CCM lesions are primarily confined to brain vasculature, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in brain mural cell in CCM pathogenesis. Approach and Results: SM22α-Cre was used to drive a specific deletion of Ccm3 in mural cells, including pericytes and smooth muscle cells (Ccm3smKO). Ccm3smKO mice developed CCM lesions in the brain with onset at neonatal stages. One-third of Ccm3smKO mice survived upto 6 weeks of age, exhibiting seizures, and severe brain hemorrhage. The early CCM lesions in Ccm3smKO neonates were loosely wrapped by mural cells, and adult Ccm3smKO mice had clustered and enlarged capillary channels (caverns) formed by a single layer of endothelium lacking mural cell coverage. Importantly, CCM lesions throughout the entire brain in Ccm3smKO mice, which more accurately mimicked human disease than the current endothelial cell-specific CCM3 deletion models. Mechanistically, CCM3 loss in brain pericytes dramatically increased paxillin stability and focal adhesion formation, enhancing ITG-β1 (integrin β1) activity and extracellular matrix adhesion but reducing cell migration and endothelial cell-pericyte associations. Moreover, CCM3-wild type, but not a paxillin-binding defective mutant, rescued the phenotypes in CCM3-deficient pericytes. Conclusions: Our data demonstrate for the first time that deletion of a CCM gene in the brain mural cell induces CCM pathogenesis.
Citation:
Arterioscler Thromb Vasc Biol. 2020 Jul 9;ATVBAHA120314586. doi: 10.1161/ATVBAHA.120.314586. Online ahead of print.
https://www.ahajournals.org/doi/abs/10.1161/ATVBAHA.120.314586
Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican
Authors:
Hong CC, Tang AT, Detter MR, Choi JP, Wang R, Yang X, Guerrero AA, Wittig CF, Hobson N, Girard R, Lightle R, Moore T, Shenkar R, Polster SP, Goddard LM, Ren AA, Leu NA, Sterling S, Yang J, Li L, Chen M, Mericko-Ishizuka P, Dow LE, Watanabe H, Schwaninger M, Min W, Marchuk DA, Zheng X, Awad IA, Kahn ML
Abstract:
Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.
Citation:
J Exp Med 2020 Oct;217(10).
Suppression of Endothelial AGO1 Promotes Adipose Tissue Browning and Improves Metabolic Dysfunction
Authors:
Xiaofang Tang, Yifei Miao, Yingjun Luo, Kiran Sriram, Zhijie Qi, Feng-Mao Lin, Yusu Gu, Chih-Hung Lai, Chien-Yi Hsu, Kirk L. Peterson, Kendall Van Keuren-Jensen, Patrick T. Fueger, Gene W. Yeo, Rama Natarajan, Sheng Zhong, and Zhen Bouman Chen
Abstract:
Background: Metabolic disorders such as obesity and diabetes can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than VEGF-VEGFR-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. Methods: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-KO) and their wild-type littermates (WT) to a fast-food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We employed crosslinking immunoprecipitation (iCLIP)- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy and obese/Type 2 diabetic donors. Results: We identified an anti-obesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an anti-angiogenic and pro-inflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from obese and/or type 2 diabetic donors, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. Conclusions: Our study suggests a novel mechanism by which ECs, through AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.
Citation:
Circulation. 2020 May 12. doi: 10.1161/CIRCULATIONAHA.119.041231. Online ahead of print.
https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.119.041231
Localization of TRPA1 Channels and Characterization of TRPA1 Mediated Responses in Dural and Pial Arteries in Vivo After Intracarotid Infusion of Na 2 S
Authors:
Anna Koldbro Hansted, Lars Jørn Jensen, Jes Olesen, Inger Jansen-Olesen
Abstract:
Background: The Transient Receptor Potential Ankyrin 1 (TRPA1) channel might play a role in migraine. However, different mechanisms for this have been suggested. The purpose of our study was to investigate the localization and significance of TRPA1 channels in rat pial and dural arteries. Methods: Immunofluorescence microscopy was used to localize TRPA1 channels in dural arteries, pial arteries, dura mater and trigeminal ganglion. The genuine closed cranial window model was used to examine the effect of Na2S, a donor of the TRPA1 channel opener H2S, on the diameter of pial and dural arteries. Further, we performed blocking experiments with TRPA1 antagonist HC-030031, calcitonin gene-related peptide (CGRP) receptor antagonist olcegepant and KCa3.1 channel blocker TRAM-34. Results: TRPA1 channels were localized to the endothelium of both dural and pial arteries and in nerve fibers in dura mater. Further, we found TRPA1 expression in the membrane of trigeminal ganglia neuronal cells, some of them also staining for CGRP. Na2S caused dilation of both dural and pial arteries. In dural arteries, this was inhibited by HC-030031 and olcegepant. In pial arteries, the dilation was inhibited by TRAM-34, suggesting involvement of the KCa3.1 channel. Conclusion: Na2S causes a TRPA1- and CGRP-dependent dilation of dural arteries and a KCa3.1 channel-dependent dilation of pial arteries in rats.
Citation:
Cephalalgia. 2020 Jul 1;333102420937724. doi: 10.1177/0333102420937724. Online ahead of print.
Platelet factor 4 is a biomarker for lymphatic-promoted disorders
Authors:
Wanshu Ma, Hyea Jin Gil, Noelia Escobedo, Alberto Benito-Martín, Pilar Ximénez-Embún, Javier Muñoz, Héctor Peinado, Stanley G Rockson, Guillermo Oliver
Abstract:
Genetic or acquired defects of the lymphatic vasculature often result in disfiguring, disabling and, occasionally, life-threatening clinical consequences. Advanced forms of lymphedema are readily diagnosed clinically, but more subtle presentations often require invasive imaging or other technologies for a conclusive diagnosis. On the other hand, lipedema, a chronic lymphatic microvascular disease with pathological accumulation of subcutaneous adipose tissue is often misdiagnosed as obesity or lymphedema; currently there are no biomarkers or imaging criteria available for a conclusive diagnosis. Recent evidence suggests that otherwise asymptomatic defective lymphatic vasculature likely contributes to an array of other pathologies, including obesity, inflammatory bowel disease and neurological disorders, among others. Accordingly, identification of biomarkers of lymphatic malfunction will provide a valuable resource for the diagnosis and clinical discrimination of lymphedema, lipedema, obesity and other potential lymphatic-related pathologies. In this paper we profiled and compared blood plasma exosomes isolated from mouse models and from human subjects with and without symptomatic lymphatic pathologies. We identified platelet factor 4 (PF4/CXCL4) as a biomarker that could be used to diagnose lymphatic vasculature dysfunction. Furthermore, we determined that PF4 levels in circulating blood plasma exosomes were also elevated in lipedema patients, supporting current claims arguing that at least some of the underlying attributes of this disease are also the consequence of lymphatic defects.
Citation:
JCI Insight. 2020 Jun 11;135109. doi: 10.1172/jci.insight.135109. Online ahead of print.
Deficiency of MFSD7c results in microcephaly-associated vasculopathy in Fowler syndrome
Authors:
Pazhanichamy Kalailingam, Kai Qi Wang, Xiu Ru Toh, Toan Q Nguyen, Madhuvanthi Chandrakanthan, Zafrul Hasan, Clair Habib, Aharon Schif, Francesca Clementina Radio , Bruno Dallapiccola, Karin Weiss, Long N Nguyen
Abstract:
Several missense mutations in the orphan transporter FLVCR2 have been reported in Fowler syndrome. Affected subjects exhibit signs of severe neurological defects. We identified the mouse ortholog Mfsd7c as a gene, which is expressed in the blood brain barrier. Here, we report the characterizations of Mfsd7c knockout (KO) mice and compare it to phenotypic findings in humans with bi-allelic FLVCR2 mutations. Global KO of Mfsd7c in mice resulted in late gestation lethality, likely due to central nervous system (CNS) phenotypes. We found that the angiogenic growth of CNS blood vessels in the brain of Mfsd7c KO embryos was inhibited in cortical ventricular zones and ganglionic eminences. Vascular tips were dilated and fused resulting in glomeruloid vessels. Nonetheless, CNS blood vessels were intact without haemorrhage. Both embryos and humans with bi-allelic FLVCR2 mutations exhibited reduced cerebral cortical layers, enlargement of the cerebral ventricles, and microcephaly. Transcriptomic analysis of Mfsd7c knockout (KO) embryonic brains revealed upregulation of genes involved in glycolysis and angiogenesis. The Mfsd7c KO brain exhibited hypoxia and neuronal cell death. Our results indicate MFSD7c is required for the normal growth of CNS blood vessels and ablation of this gene results in microcephaly-associated vasculopathy in mice and humans.
Citation:
J Clin Invest. 2020 May 5;136727. doi: 10.1172/JCI136727. Online ahead of print.
Extracellular vesicles enhance remodeling of cell-free silk vascular scaffolds in rat aortae
Authors:
Eoghan M Cunnane, Katherine L Lorentz, Aneesh K Ramaswamy, Prerak Gupta, Biman B Mandal, Fergal J O'Brien, Justin S Weinbaum, David Vorp
Abstract:
Vascular tissue engineering (VTE) is aimed at developing regenerative vascular grafts to restore tissue function by bypassing or replacing defective arterial segments with tubular biodegradable scaffolds. Scaffolds are often combined with stem/progenitor cells to prevent acute thrombosis and initiate scaffold remodeling. However, there are limitations to cell-based technologies regarding safety and clinical translation. Extracellular vesicles (EVs) are nano-sized particles released by most cell types, including stem/progenitor cells, that serve to transmit protein/RNA cargo to target cells throughout the body. EVs have been shown to replicate the therapeutic effect of their parent cell, therefore EVs derived from stem/progenitor cells may serve as a more translatable, cell-free, therapeutic base for VTE. Our study aims to determine if EV incorporation provides a positive effect on graft patency and remodeling in vivo. We first assessed the effect of human adipose derived mesenchymal stem cell (hADMSC) EVs on vascular cells using in vitro bioassays. We then developed an EV-functionalized vascular graft by vacuum-seeding EVs into porous silk-based tubular scaffolds. These constructs were implanted as aortic interposition grafts in Lewis rats and their remodeling capacity was compared to that observed for hADMSC-seeded and blank (non-seeded) controls. The EV group demonstrated improved patency (100%) compared to the hADMSC (56%) and blank controls (82%) following 8 weeks in vivo. The EV group also produced significantly more elastin (126.46%) and collagen (44.59%) compared to the blank group, while the hADMSC group failed to produce significantly more elastin (57.64%) or collagen (11.21%) compared to the blank group. Qualitative staining of the explanted neo-tissue revealed improved endothelium formation, increased smooth muscle cell infiltration, and reduced macrophage numbers in the EV group compared to the controls which aids in explaining this group's favorable preclinical outcomes.
Citation:
ACS Appl Mater Interfaces. 2020 May 22. doi: 10.1021/acsami.0c06609. Online ahead of print.
Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier
Authors:
Santander N, Lizama CO, Meky E, McKinsey GL, Jung B, Sheppard D, Betsholtz C, Arnold TD
Abstract:
Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler Syndrome by inactivating Flvcr2 in mice. We show that Flvcr2 is necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood brain barrier. Endothelial cells lacking Flvcr2 have altered expression of angiogenic factors, fail to adopt tip-cell properties and display reduced sprouting leading to vascular malformations similar to those seen in humans with Fowler Syndrome. Brain hypo-vascularization is associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier is maintained in Flvcr2 mutant mice. Our new Fowler syndrome models therefore define the pathobiology of this disease, and provide new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
Citation:
J Clin Invest. 2020 May 5. pii: 136578. doi: 10.1172/JCI136578. [Epub ahead of print]
Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation
Authors:
Le Cras TD, Goines J, Lakes N, Pastura P, Hammill AM, Adams DM, Boscolo E
Abstract:
Capillary lymphatic venous malformations (CLVM) are complex vascular anomalies characterized by aberrant and enlarged lymphatic and blood vessels. CLVM appear during fetal development and enlarge after birth, causing life-long complications such as coagulopathy, pulmonary embolism, chronic pain, and disfigurement. Treatment includes surgical debulking, amputation, and recurrent sclerotherapy. Somatic, mosaic mutations in the 110-kD catalytic α-subunit of phosphoinositide-3-kinase (PIK3CA) gene have been previously identified in affected tissues from CLVM patients; however, the cell population harboring the mutation is still unknown. In this study, we hypothesized that endothelial cells (EC) carry the PIK3CA mutations and play a major role in the cellular origin of CLVM. We isolated EC from the lesions of seven patients with CLVM and identified PIK3CA hotspot mutations. The CLVM EC exhibited constitutive phosphorylation of the PI3K effector AKT as well as hyperproliferation and increased resistance to cell death compared to normal EC. Inhibitors of PIK3CA (BYL719) and AKT (ARQ092) attenuated the proliferation of CLVM EC in a dose-dependent manner. A xenograft model of CLVM was developed by injecting patient-derived EC into the flanks of immunocompromised mice. CLVM EC formed lesions with enlarged lymphatic and vascular channels, recapitulating the patient histology. EC subpopulations were further obtained by both immunomagnetic separation into lymphatic EC (LEC) and vascular EC (VEC) and generation of clonal populations. By sequencing these subpopulations, we determined that both LEC and VEC from the same patient express the PIK3CA mutation, exhibit increased AKT activation and can form lymphatic or vascular lesions in mouse.
Citation:
Angiogenesis. 2020 Apr 30. doi: 10.1007/s10456-020-09722-0. [Epub ahead of print]
Neutrophil extracellular traps in COVID-19
Authors:
Zuo Y, Yalavarthi S, Shi H, Gockman K, Zuo M, Madison JA, Blair CN, Weber A, Barnes BJ, Egeblad M, Woods RJ, Kanthi Y, Knight JS
Abstract:
In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have potential to propagate inflammation and microvascular thrombosis - including in the lungs of patients with acute respiratory distress syndrome. While elevated levels of blood neutrophils predict worse outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera from patients with COVID-19 (n = 50 patients, n = 84 samples) have elevated levels of cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter two are highly specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute phase reactants including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. In summary, these data reveal high levels of NETs in many patients with COVID-19, where they may contribute to cytokine release and respiratory failure. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts, and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.
Citation:
JCI Insight. 2020 Apr 24. pii: 138999. doi: 10.1172/jci.insight.138999. [Epub ahead of print]
https://insight.jci.org/articles/view/138999#version_history
Beyond a Passive Conduit: Implications of Lymphatic Biology for Kidney Diseases
Authors:
Jafree DJ, Long DA
Abstract:
The kidney contains a network of lymphatic vessels that clear fluid, small molecules, and cells from the renal interstitium. Through modulating immune responses and via crosstalk with surrounding renal cells, lymphatic vessels have been implicated in the progression and maintenance of kidney disease. In this Review, we provide an overview of the development, structure, and function of lymphatic vessels in the healthy adult kidney. We then highlight the contributions of lymphatic vessels to multiple forms of renal pathology, emphasizing CKD, transplant rejection, and polycystic kidney disease and discuss strategies to target renal lymphatics using genetic and pharmacologic approaches. Overall, we argue the case for lymphatics playing a fundamental role in renal physiology and pathology and treatments modulating these vessels having therapeutic potential across the spectrum of kidney disease.
Citation:
J Am Soc Nephrol. 2020 Apr 15. pii: ASN.2019121320. doi: 10.1681/ASN.2019121320. [Epub ahead of print]
https://jasn.asnjournals.org/content/early/2020/04/15/ASN.2019121320
Podosomes in endothelial cell-microenvironment interactions
Authors:
Alonso F, Spuul P, Génot E
Abstract:
PURPOSE OF REVIEW: The discovery of podosomes in endothelial cells during the process of angiogenesis in vivo opens a new era in vascular biology. Podosomes are actin-based microdomains located at the plasma membrane that have been extensively described but in vitro and in other cells. This review focuses on podosomes in endothelial cells and aims to rise hypotheses about when and how these structures mediate cell--microenvironment interactions.
RECENT FINDINGS: A wealth of new information regarding podosome organization and functioning has been collected in simple 2D models. Characterization of their modular architecture has unravelled their mechanics. However, context matters and podosome characteristics and functioning are shaped by the microenvironment. Although matrix degradation was seen as the typical function of podosomes, mechanosensing now appears equally prominent and involved in setting of the proteolytic machinery. Endothelial podosomes breach the basement membrane, and are thus, involved in vascular remodelling.
SUMMARY: In endothelial cells, podosomes are involved in breaking up the basement membrane, giving the cells the opportunity to invade adjacent tissues and to engage in new cell--cell interactions. Such functions are particularly relevant to vascular biology and the exploration of podosomes in in vivo settings should bring clues to many unanswered questions.
Citation:
Curr Opin Hematol. 2020 Feb 27. doi: 10.1097/MOH.0000000000000575. [Epub ahead of print]
Annexin A1 drives macrophage skewing to accelerate muscle regeneration through AMPK activation
Authors:
McArthur S, Juban G, Gobbetti T, Desgeorges T, Theret M, Gondin J, Toller-Kawahisa JE, Reutelingsperger CP, Chazaud B, Perretti M, Mounier R
Abstract:
Understanding the circuits that promote an efficient resolution of inflammation is crucial to deciphering the molecular and cellular processes required to promote tissue repair. Macrophages play a central role in the regulation of inflammation, resolution, and repair/regeneration. Using a model of skeletal muscle injury and repair, herein we identified annexin A1 (AnxA1) as the extracellular trigger of macrophage skewing toward a pro-reparative phenotype. Brought into the injured tissue initially by migrated neutrophils, and then overexpressed in infiltrating macrophages, AnxA1 activated FPR2/ALX receptors and the downstream AMPK signaling cascade, leading to macrophage skewing, dampening of inflammation, and regeneration of muscle fibers. Mice lacking AnxA1 in all cells or only in myeloid cells displayed a defect in this reparative process. In vitro experiments recapitulated these properties, with AMPK-null macrophages lacking AnxA1-mediated polarization. Collectively, these data identified the AnxA1/FPR2/AMPK axis as an important pathway in skeletal muscle injury regeneration.
Citation:
J Clin Invest. 2020 Feb 4. pii: 124635. doi: 10.1172/JCI124635. [Epub ahead of print]
LDL induces cholesterol loading, inhibits endothelial proliferation, and angiogenesis in Matrigels: Correlation with impaired angiogenesis during wound healing
Authors:
Bogachkov YY, Chen L, Le Master E, Fancher IS, Zhao Y, Oh MJ, Wary KK, DiPietro LA, Levitan I
Abstract:
Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and Laurdan two-photon imaging, we show that elevated levels of LDL, like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxLDL. The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase whereas oxLDL enhances endothelial proliferation in S-phase of the cycle. LDL but not oxLDL suppresses the expression of VEGFR2 while enhancing the expression of VEGF. Furthermore, we show that aged (8-10 months) hypercholesterolemic ApoE-/- mice display delayed wound closure as compared to age-matched C57/BL6 wild type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of CD31/PECAM endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors, VEGF and bFGF is observed in ApoE-/- mice in Matrigel plugs, as well as in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is anti-angiogenic due to elevated levels of LDL.
Citation:
Am J Physiol Cell Physiol. 2020 Jan 29. doi: 10.1152/ajpcell.00495.2018. [Epub ahead of print]
https://journals.physiology.org/doi/abs/10.1152/ajpcell.00495.2018
Altered Lipid Domains Facilitate Enhanced Pulmonary Vasoconstriction Following Chronic Hypoxia
Authors:
Norton CE, Weise-Cross L, Ahmadian R, Yan S, Jernigan NL, Paffett ML, Naik JS, Walker BR, Resta TC
Abstract:
Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. NADPH oxidase and epidermal growth factor receptor (EGFR) signaling contribute to this response. Caveolin-1 regulates the activity of a variety of proteins including EGFR and NADPH oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction following CH. Although exposure of rats to CH (4 wk, ∼380 mmHg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted, pulmonary arteries (∼150 µm i.d.) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH than control rats, and increased superoxide production assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide AP-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction following CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction following CH.
Citation:
Am J Respir Cell Mol Biol. 2020 Jan 16. doi: 10.1165/rcmb.2018-0318OC. [Epub ahead of print]
PKCβ and Reactive Oxygen Species Mediate Enhanced Pulmonary Vasoconstrictor Reactivity following Chronic Hypoxia in Neonatal Rats
Authors:
Sheak JR, Yan S, Weise-Cross L, Ahmadian R, Walker BR, Jernigan NL, Resta TC
Abstract:
Reactive oxygen species (ROS), mitochondrial dysfunction and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. Based on evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial-derived ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor, LY-333,531, the ROS scavenger, TEMPOL, or the mitochondrial antioxidants, MitoQ and mitoTEMPO, on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12 day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to U-46619. LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared to controls, which was inhibited by both LY-333,531 and mitochondrial-targeted antioxidants. Additional experiments assessing mitoROS generation using the mitochondrial-targeted ROS indicator, MitoSOX, revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate (PMA) in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrial-localized PKCβ in pulmonary arteries as assessed by western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.
Citation:
Am J Physiol Heart Circ Physiol. 2020 Jan 10. doi: 10.1152/ajpheart.00629.2019. [Epub ahead of print]
VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours
Authors:
Eric Song, Tianyang Mao, Huiping Dong, Ligia Simoes Braga Boisserand, Salli Antila, Marcus Bosenberg , Kari Alitalo, Jean-Leon Thomas, Akiko Iwasaki
Abstract:
Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However, the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1,2,3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system, the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system, resulting in uncontrolled tumour growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a new therapeutic approach to treat brain tumours.
Citation:
Nature (2020) doi:10.1038/s41586-019-1912-x
Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis after Carotid Artery Ligation
Authors:
Becher T, Riascos-Bernal DF, Kramer DJ, Almonte V, Chi J, Tong T, Oliveira-Paula GH, Koleilat I, Chen W, Cohen P, Sibinga NE
Abstract:
Rationale: Remodeling of the vessel wall and the formation of vascular networks are dynamic processes that occur during mammalian embryonic development and in adulthood. Plaque development and excessive neointima formation are hallmarks of atherosclerosis and vascular injury. As our understanding of these complex processes evolves, there is a need to develop new imaging techniques to study underlying mechanisms. Objective: We used tissue clearing and light-sheet microscopy for three-dimensional (3D) profiling of the vascular response to carotid artery ligation and induction of atherosclerosis in mouse models.Methods and Results: Adipo-Clear and immunolabeling in combination with light-sheet microscopy were applied to image carotid arteries (CAs) and brachiocephalic arteries (BCAs), allowing for 3D reconstruction of vessel architecture. Entire 3D neointima formations with different geometries were observed within the CA and scored by volumetric analysis. Additionally, we identified a CD31-positive adventitial plexus after ligation of the CA that evolved and matured over time. We also used this method to characterize plaque extent and composition in the BCA of ApoE-deficient mice on high-fat diet. The plaques exhibited inter-animal differences in terms of plaque volume, geometry, and ratio of acellular core to plaque volume. A 3D reconstruction of the endothelium overlying the plaque was also generated.Conclusions: We present a novel approach to characterize vascular remodeling in adult mice using Adipo-Clear in combination with light-sheet microscopy. Our method reconstructs 3D neointima formation after arterial injury and allows for volumetric analysis of remodeling, in addition to revealing angiogenesis and maturation of a plexus surrounding the CA. This method generates complete 3D reconstructions of atherosclerotic plaques and uncovers their volume, geometry, acellular component, surface, and spatial position within the BCA. Our approach may be used in a number of mouse models of cardiovascular disease to assess vessel geometry and volume.
Citation:
Circ Res. 2020 Jan 1. doi: 10.1161/CIRCRESAHA.119.315804. [Epub ahead of print]
BMX Represses Thrombin-PAR1-Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis
Authors:
Li Z, Yin M, Zhang H, Ni W, Pierce R, Zhou HJ, Min W
Abstract:
Rationale: Bone marrow kinase on the X chromosome (BMX) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. Objective: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture (CLP) models.Methods and Results: The CLP model was applied to wild-type and BMX knockout mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect trans-endothelial electrical resistance in vitro and the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early CLP-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells (ECs) by 2-3-fold. The expression of BMX in macrophages, neutrophils, platelets and lung epithelial cells was undetectable compared with that in ECs, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-protease-activated receptor 1 (PAR1) signaling in ECs by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pre-treatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early CLP-induced sepsis. Conclusions: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.
Citation:
Circ Res. 2020 Jan 8. doi: 10.1161/CIRCRESAHA.119.315769. [Epub ahead of print]
Congenital Fibular Dystrophisms Conform to Embryonic Arterial Dysgenesis
Authors:
Hootnick DR
Abstract:
The congenital short limb (CSL) with fibular deficiency has traditionally been graded by plain radiography. The most popular orthopedic classification sorts the fibular dysmorphologies into three radiographic groupings: IA (thinned), IB (proximally truncated) or II (absent). In contrast, the soft tissues have been relatively neglected. Since bone formation of the fibula progresses from the anlage, a scaffolding cartilage mold intermediate, cartilage transformation to bone is dependent upon timely embryonic arterial invasion. Absences of the requisite arteries predicate specific skeletal dysmorphologies. The usual arterial supply of the fibula is comprised primarily of the Anterior Tibialis Artery (ATA) which uniquely supplies the proximal portion of the fibula, but also joins the Peroneal Artery (PA) in supplying the mid to distal fibular shaft. Combinations of the two nutrient arteries allow four potential variations of fibular vascular supply, among which the ATA and PA conjoin to supply the normal fibula and variably supply the three dysmorphic fibular models. The IA and IB deformities conform respectively to absences of the PA and the ATA. Combined ATA and PA absences present in the radiographically "absent" fibula. Thus, each of the four fibular (dys)morphologies conforms to a specific embryonic pattern of arterial development. The term "dystrophism" most accurately characterizes such malformed long bones. This article is protected by copyright. All rights reserved.
Citation:
Anat Rec (Hoboken). 2019 Dec 24. doi: 10.1002/ar.24348. [Epub ahead of print]
Hyperglycemia-induced transcriptional regulation of ROCK1 and TGM2 expression is involved in small artery remodeling in obese diabetic Göttingen Minipigs
Authors:
T.P. Ludvigsen, L.H. Olsen, H.D. Pedersen, B.O. Christoffersen, L.J. Jensen
Abstract:
Obesity and diabetes in humans are associated with hypertrophic remodeling and increased media:lumen ratio of small resistance arteries, which is an independent predictor of cardiovascular events. In order to minimize increases in media:lumen ratio, hypertrophic remodeling should be accompanied by outward remodeling. We aimed to investigate the mechanisms of structural remodeling in small pial arteries (PA) and terminal mesenteric arteries (TMA) from obese Göttingen Minipigs with or without diabetes. Göttingen Minipigs received either control diet (LC), high fat/high fructose/high cholesterol diet (FFC), or FFC diet with streptozotocin-induced diabetes (FFC/STZ) for 13 months. At study end (20 months), we assessed body weight, fasting plasma biochemistry, passive vessel dimensions, mRNA expression (MMP2, MMP9, TIMP1, TGM2, ROCK1, TGFBR2, and IGFR1 genes), and immunofluorescence in PAs and TMAs. We performed multiple linear correlation analyses using plasma values, structural data, and gene expression data. We detected outward hypertrophic remodeling in TMAs and hypertrophic remodeling in PAs from FFC/STZ animals. ROCK1 and TGM2 genes were up-regulated in PAs and TMAs from the FFC/STZ group. Passive lumen diameter of TMAs was correlated with plasma values of glucose, fructosamine, total cholesterol, and triglycerides. ROCK1 and TGM2 expressions in TMAs were correlated with passive lumen diameter, plasma glucose, fructosamine, and total cholesterol. ROCK1 and TGM2 proteins were immunolocalized in the media of PAs and TMAs, and their fluorescence levels were increased in the FFC/STZ group. Hyperglycemia/hyperlipidemia is involved in regulation of ROCK1 and TGM2 expression leading to outward remodeling of small resistance arteries in obese diabetic Göttingen Minipigs.
Citation:
Clin Sci (Lond) (2019) CS20191066., https://doi.org/10.1042/CS20191066
The potassium channel Kcne3 is a VEGFA-inducible gene selectively expressed by vascular endothelial tip cells
Authors:
Deckelbaum RA, Lobov IB, Cheung E, Halasz G, Rajamani S, Lerner J, Tong C, Li Z, Boland P, Dominguez M, Hughes V, Yancopoulos GD, Murphy AJ, Thurston G, Cao J, Romano C, Gale NW
Abstract:
Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and a lacZ reporter allele to identify Kcne3 as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development, Kcne3 expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently, Kcne3 activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity of Kcne3 in several injury and tumor models. Together, our data show that Kcne3 is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.
Citation:
Angiogenesis. 2019 Nov 21. doi: 10.1007/s10456-019-09696-8. [Epub ahead of print]
Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease
Authors:
Jafree DJ, Moulding D, Kolatsi-Joannou M, Perretta Tejedor N, Price KL, Milmoe NJ, Walsh CL, Correra RM, Winyard PJ, Harris PC, Ruhrberg C, Walker-Samuel S, Riley PR, Woolf AS, Scambler P, Long DA
Abstract:
Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis, a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.
Citation:
Elife. 2019 Dec 6;8. pii: e48183. doi: 10.7554/eLife.48183. [Epub ahead of print]
Immunoglobulin-Driven Complement Activation Regulates Pro-Inflammatory Remodeling in Pulmonary Hypertension
Authors:
Frid MG, McKeon BA, Thurman JM, Maron BA, Li M, Zhang H, Kumar S, Sullivan T, Laskowsky J, Fini MA, Hu S, Tuder RM, Gandjeva A, Wilkins MR, Rhodes CJ, Ghataorhe P, Leopold JA, Wang RS, Holers VM, Stenmark KR
Abstract:
RATIONALE: Pulmonary (arterial) hypertension (PH/PAH) is a life-threatening cardiopulmonary disorder where inflammation and immunity have emerged as critical early pathogenic elements. Although pro-inflammatory processes in PH/PAH are the focus of extensive investigation, the initiating mechanisms remain elusive. OBJECTIVES: We tested whether activation of the complement cascade is critical in regulating pro-inflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH, and can serve as a prognostic biomarker of outcome in human PAH. METHODS: We employed immunostaining of lung tissues from experimental PH models and PAH patients; analyses of genetic murine models lacking specific complement components or circulating immunoglobulins; cultured human pulmonary adventitial fibroblasts; and network medicine analysis of a biomarker risk panel from plasma of PAH patients. MEASUREMENTS AND MAIN RESULTS: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH/PAH in experimental animal models and humans. In experimental hypoxic PH, pro-inflammatory and pro-proliferative responses were complement (Alternative pathway and C5)-dependent, and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identify Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of PAH patients, we demonstrate that complement signaling can serve as a prognostic factor for clinical outcome in PAH. CONCLUSIONS: The present study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating pro-inflammatory/pro-proliferative processes in the initiation of experimental hypoxic PH, and demonstrates complement signaling as a critical determinant of clinical outcome of in PAH. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Citation:
Am J Respir Crit Care Med. 2019 Sep 23. doi: 10.1164/rccm.201903-0591OC. [Epub ahead of print]
Combining laser microdissection and microRNA expression profiling to unmask microRNA signatures in complex tissues
Authors:
Skalicky S, Zwiers PJ, Kuiper T, Schraml E, Hackl M, Molema G.
Abstract:
Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression information from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment-specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopathology. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates.
Citation:
Biotechniques. 2019 Oct 17. doi: 10.2144/btn-2019-0032. [Epub ahead of print]
VEGF signalling enhances lesion burden in KRIT1 deficient mice
Authors:
DiStefano PV, Glading AJ
Abstract:
The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial-specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.
Citation:
J Cell Mol Med. 2019 Nov 20. doi: 10.1111/jcmm.14773. [Epub ahead of print]
The isolation and molecular characterization of cerebral microvessels
Authors:
Lee YK, Uchida H, Smith H, Ito A, Sanchez T
Abstract:
The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels with consistent cellular compositions and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood-brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions.
Citation:
Nat Protoc. 2019 Oct 4. doi: 10.1038/s41596-019-0212-0. [Epub ahead of print]
Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b
Authors:
Sudhahar V, Das A, Horimatsu T, Ash D, Leanhart S, Antipova O, Vogt S, Singla B, Csanyi G, White J, Kaplan JH, Fulton D, Weintraub NL, Won Kim H, Ushio-Fukai M, Fukai T
Abstract:
OBJECTIVE: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE/ mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.
Citation:
Arterioscler Thromb Vasc Biol. 2019 Sep 26:ATVBAHA119313374. doi: 10.1161/ATVBAHA.119.313374. [Epub ahead of print]
Novel Interaction of Antioxidant-1 with TRAF4: Role in Inflammatory Responses in Endothelial Cells
Authors:
Das A, Sudhahar V, Ushio-Fukai M, Fukai T
Abstract:
NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, have been implicated in vascular inflammatory diseases. We reported that in proinflammatory cytokine TNFa-stimulated endothelial cells (ECs), cytosolic Cu chaperone Atox1 functions as a Cu-dependent transcription factor for NOX organizer p47phox, thereby increasing ROS-dependent inflammatory gene expression. However, the role and mechanism of Atox1 nuclear translocation in inflamed ECs remain unclear. Using enface staining and nuclear fractionation, here we show that Atox1 was localized in the nucleus in inflamed aortas from ApoE-/- mice with Angiotensin II infusion on a high fat diet, while it was found in cytosol in those from control mice. In cultured human ECs, TNFα stimulation promoted Atox1 nuclear translocation within 15 min, which was associated with Atox1 binding to TNFα receptor associated factor (TRAF)4 in a Cu-dependent manner. TRAF4 depletion by siRNA significantly inhibited Atox1 nuclear translocation, p47phox expression and ROS production as well as its downstream VCAM1/ICAM1 expression and monocyte adhesion to inflamed ECs, which were rescued by overexpression of nuclear targeted Atox1. Furthermore, Atox1 colocalized with TRAF4 at nucleus in TNFα-stimulated inflamed ECs and vessels. In summary, Cu-dependent Atox1 binding to TRAF4 plays an important role in Atox1 nuclear translocation and ROS-dependent inflammatory responses in TNFα-stimulated ECs. Thus, the Atox1-TRAF4 axis is a novel therapeutic target for vascular inflammatory disease such as atherosclerosis.
Citation:
Am J Physiol Cell Physiol. 2019 Sep 25. doi: 10.1152/ajpcell.00264.2019. [Epub ahead of print]
Application of Transmural Flow Across In Vitro Microvasculature Enables Direct Sampling of Interstitial Therapeutic Molecule Distribution
Authors:
Offeddu GS, Possenti L, Loessberg-Zahl JT, Zunino P, Roberts J, Han X, Hickman D, Knutson CG, Kamm RD
Abstract:
In vitro prediction of physiologically relevant transport of therapeutic molecules across the microcirculation represents an intriguing opportunity to predict efficacy in human populations. On-chip microvascular networks (MVNs) show physiologically relevant values of molecular permeability, yet like most systems, they lack an important contribution to transport: the ever-present fluid convection through the endothelium. Quantification of transport through the MVNs by current methods also requires confocal imaging and advanced analytical techniques, which can be a bottleneck in industry and academic laboratories. Here, it is shown that by recapitulating physiological transmural flow across the MVNs, the concentration of small and large molecule therapeutics can be directly sampled in the interstitial fluid and analyzed using standard analytical techniques. The magnitudes of transport measured in MVNs reveal trends with molecular size and type (protein versus nonprotein) that are expected in vivo, supporting the use of the MVNs platform as an in vitro tool to predict distribution of therapeutics in vivo.
Citation:
Small. 2019 Sep 9:e1902393. doi: 10.1002/smll.201902393. [Epub ahead of print]
HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients
Authors:
Chelvanambi S, Gupta SK, Chen X, Ellis BW, Maier BF, Colbert TM, Kuriakose J, Zorlutuna P, Jolicoeur P, Obukhov AG, Clauss M
Abstract:
RATIONALE: Even in antiretroviral therapy (ART) treated patients, HIV continues to play a pathogenic role in cardiovascular diseases. A possible cofactor may be persistence of the early HIV response gene Nef, which we have demonstrated recently to persist in the lungs of HIV+ patients on ART. Previously, we have reported that HIV strains with Nef, but not Nef-deleted HIV strains, cause endothelial proinflammatory activation and apoptosis. OBJECTIVE: To characterize mechanisms through which HIV-Nef leads to the development of cardiovascular diseases using ex vivo tissue culture approaches as well as interventional experiments in transgenic murine models. METHODS AND RESULTS: EV (extracellular vesicles) derived from both peripheral blood mononuclear cells (PBMC) and plasma from HIV+ patient blood samples induced human coronary artery endothelial cells dysfunction. Plasma derived EV from ART+ patients that were HIV-Nef+ induced significantly greater endothelial apoptosis compared to HIV-Nef- plasma EV. Both HIV-Nef expressing T cells and HIV-Nef-induced EV increased transfer of cytosol and Nef protein to endothelial monolayers in a Rac1-dependent manner, consequently leading to endothelial adhesion protein upregulation and apoptosis. HIV-Nef induced Rac1 activation also led to dsDNA breaks in endothelial colony forming cells (ECFC), thereby resulting in ECFC premature senescence and eNOS downregulation. These Rac1 dependent activities were characterized by NOX2-mediated ROS production. Statin treatment equally inhibited Rac1 inhibition in preventing or reversing all HIV-Nef-induction abnormalities assessed. This was likely due to the ability of statins to block Rac1 prenylation as geranylgeranyl transferase inhibitors were effective in inhibiting HIV-Nef-induced ROS formation. Finally, transgenic expression of HIV-Nef in endothelial cells in a murine model impaired endothelium-mediated aortic ring dilation, which was then reversed by 3-week treatment with 5mg/kg atorvastatin. CONCLUSION: Conclusions: These studies establish a mechanism by which HIV-Nef persistence despite ART could contribute to ongoing HIV related vascular dysfunction which may then be ameliorated by statin treatment.
Citation:
Circ Res. 2019 Aug 27. doi: 10.1161/CIRCRESAHA.119.315082. [Epub ahead of print]
Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium
Authors:
Bailey KA, Moreno E, Haj FG, Simon SI, Passerini AG
Abstract:
Endothelial up-regulation of VCAM-1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x-box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF-α–stimulated VCAM-1 expression. High-resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM-1)–dependent, outside-in signaling as a function of SS magnitude. Differential expression of VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM-1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM-1 at low SS (2 dynes/cm2), whereas inhibition of ERK1/2 attenuated peak ICAM-1 at high SS (12 dynes/cm2). A shear stress–regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM-1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine-induced ER stress response to spatially regulate inflammation promoting atherosclerosis.—Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress–mediated inflammation by arterial endothelium.
Citation:
FASEB J. 2019 Sep 7:fj201900236R. doi: 10.1096/fj.201900236R. [Epub ahead of print]
Carbon monoxide-releasing molecule-3 (CORM-3) offers protection in an in vitro model of compartment syndrome
Authors:
Bihari A, Chung KA, Cepinskas G, Sanders D, Schemitsch E, Lawendy AR
Abstract:
OBJECTIVE: Limb compartment syndrome (CS), a complication of trauma, results in muscle necrosis and cell death; ischemia and inflammation contribute to microvascular dysfunction and parenchymal injury. Carbon monoxide-releasing molecule-3 (CORM-3) has been shown to protect microvascular perfusion and reduce inflammation in animal models of CS. The purpose of the study was to test the effect of CORM-3 in human in vitro CS model, allowing exploration of the mechanism(s) of CO protection and potential development of pharmacologic treatment. METHODS: Confluent human vascular endothelial cells (HUVECs) were stimulated for 6 h with serum isolated from patients with CS. Intracellular oxidative stress (production of reactive oxygen species (ROS)) apoptosis, transendothelial resistance (TEER), polymorphonuclear leukocyte (PMN) activation and transmigration across the monolayer in response to the CS stimulus were assessed. All experiments were performed in the presence of CORM-3 (100 μM) or its inactive form, iCORM-3. RESULTS: CS serum induced a significant increase in ROS, apoptosis and endothelial monolayer breakdown; it also increased PMN superoxide production, leukocyte rolling and adhesion/transmigration. CORM-3 completely prevented CS-induced ROS production, apoptosis, PMN adhesion, rolling and transmigration, while improving monolayer integrity. CONCLUSION: CORM-3 offers potent anti-oxidant and anti-inflammatory effects, and may have a potential application to patients at risk of developing CS.
Citation:
Microcirculation. 2019 Jun 22:e12577. doi: 10.1111/micc.12577. [Epub ahead of print]
Regulation of CCL2 expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA
Authors:
Khyzha N, Khor M, DiStefano PV, Wang L, Matic L, Hedin U, Wilson MD, Maegdefessel L, Fish JE
Abstract:
Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)-regulated messenger RNA (mRNA)-lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2, which is transcribed divergently to the gene, CCL2, encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA-lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2, which may contribute to atherogenesis in humans.
Citation:
Proc Natl Acad Sci U S A. 2019 Jul 26. pii: 201904108. doi: 10.1073/pnas.1904108116. [Epub ahead of print]
Augmented pulmonary vasoconstrictor reactivity following chronic hypoxia requires Src kinase and EGFR signaling
Authors:
Norton CE, Sheak JR, Yan S, Weise-Cross L, Jernigan NL, Walker BR, Resta TC
Abstract:
RATIONALE: Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. OBJECTIVES: We tested the hypothesis that Src/epidermal growth factor receptor signaling mediates enhanced vasoconstrictor sensitivity following chronic hypoxia through NADPH oxidase-derived superoxide generation. METHODS: Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone following chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the epidermal growth factor receptor inhibitor gefitinib. MEASUREMENTS AND RESULTS: Inhibition of NADPH oxidase, Rac1, and epidermal growth factor receptor abolished pressure-induced pulmonary arterial tone and endothelin-1-dependent calcium sensitization and vasoconstriction following chronic hypoxia. Consistently, chronic hypoxia augmented endothelin-1-induced superoxide production through epidermal growth factor receptor signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were additionally activated by ET-1 following chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction to ET-1. A role for matrix metalloproteinase 2 to mediate Src dependent-epidermal growth factor receptor activation is further supported by our findings. CONCLUSIONS: Our studies support a novel role for a Src kinase, epidermal growth factor receptor, NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension following chronic hypoxia.
Citation:
Am J Respir Cell Mol Biol. 2019 Jul 2. doi: 10.1165/rcmb.2018-0106OC. [Epub ahead of print]
High‐Throughput Scaffold System for Studying the Effect of Local Geometry and Topology on the Development and Orientation of Sprouting Blood Vessels
Authors:
Szklanny, A. A., Debbi, L., Merdler, U., Neale, D., Muñiz, A., Kaplan, B., Guo, S., Lahann J. & Levenberg, S.
Abstract:
Live tissues require vascular networks for cell nourishing. Mimicking the complex structure of native vascular networks in vitro requires understanding the governing factors of early tubulogenesis. Current vascularization protocols allow for spontaneous formation of vascular networks; however, there is still a need to provide control over the defined network structure. Moreover, there is little understanding on sprouting decision and migration, especially within 3D environments. Here, tessellated polymer scaffolds with various compartment geometries and a novel two‐step seeding protocol are used to study vessel sprouting decisions. Endothelial cells first organize into hollow vessels tracing the shape contour with high fidelity. Subsequent sprouts emerge in specific directions, responding to compartment geometry. Time‐lapse imaging is used to track vessel migration, evidencing that sprouts frequently emerge from the side centers, mainly migrating toward opposing corners, where the density of support cells (SCs) is the highest, providing the highest levels of angiogenic factors. SCs distribution is quantified by smooth muscle actin expression, confirming the cells preference for curved compartment surfaces and corners. Displacements within the hydrogel correlate with SCs distribution during the initial tubulogenesis phase. This work provides new insight regarding vessel sprouting decisions that should be considered when designing scaffolds for vascularized engineered tissues.
Citation:
First published: 07 June 2019|https://doi.org/10.1002/adfm.201901335
https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201901335
Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction
Authors:
Li Z, Solomonidis EG, Meloni M, Taylor RS, Duffin R, Dobie R, Magalhaes MS, Henderson BEP, Louwe PA, D'Amico G, Hodivala-Dilke KM, Shah AM, Mills NL, Simons BD, Gray GA, Henderson NC, Baker AH, Brittan M
Abstract:
AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.
Citation:
Eur Heart J. 2019 Jun 4. pii: ehz305. doi: 10.1093/eurheartj/ehz305. [Epub ahead of print]
https://academic.oup.com/eurheartj/advance-article/doi/10.1093/eurheartj/ehz305/5510786
The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing
Authors:
Ishihara J, Ishihara A, Starke RD, Peghaire CR, Smith KE, McKinnon TA, Tabata Y, Sasaki K, White MJ, Fukunaga K, Laffan MA, Lutolf MP, Randi AM, Hubbell JA.
Abstract:
During wound healing, the distribution, availability and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GFs binding. The hemostatic protein von Willebrand factor (VWF), released by endothelial cells (ECs) in plasma and in the subendothelial matrix, has been shown to regulate angiogenesis; this function is relevant to patients where VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing, accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor (VEGF)-A isoforms and platelet derived growth factor (PDGF)-BB, mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and FGF-2 in human plasma and co-localizes with VEGF-A in EC. Incorporation of the VWF A1-HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1-HBD can function as a GFs reservoir, leading to effective angiogenesis and tissue regeneration.
Citation:
Blood. 2019 Apr 11. pii: blood.2019000510. doi: 10.1182/blood.2019000510. [Epub ahead of print]
Standardization of methods to quantify and culture endothelial colony-forming cells derived from peripheral blood: Position paper from the International Society on Thrombosis and Haemostasis SSC
Authors:
Smadja DM, Melero-Martin JM, Eikenboom J, Bowman M, Sabatier F, Randi AM
Citation:
J Thromb Haemost. 2019 Apr 25. doi: 10.1111/jth.14462. [Epub ahead of print]
A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesisand turnover
Authors:
Wang LW, Nandadasa S, Annis DS, Dubail J, Mosher DF, Willard BB, Apte SS
Abstract:
The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary ciliumduring mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few knowninteracting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminalGon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase bindingassays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact.ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lackingfibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining afterblockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in bothmolecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networksformed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike wildtype cells. Targeted LC–MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semi-tryptic peptidearising from cleavage at Gly-2196–Leu-2197. We noted that this scissile bond is in the linker between fibronectin modulesIII17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previouslyobserved in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.
Citation:
Journal of Biological Chemistry, First Published on May 13, 2019, doi: 10.1074/jbc.RA118.006479, jbc.RA118.006479
http://www.jbc.org/content/early/2019/05/13/jbc.RA118.006479.abstract
Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation
Authors:
Wafa Altalhi, Rupal Hatkar, James B. Hoying, Yasaman Aghazadeh, Sara S. Nunes
Abstract:
Introduction: The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects. Methods: We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs. Results: We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu. Conclusion: We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.
Citation:
Cellular and Molecular Bioengineering, First Online: 21 May 2019
https://link.springer.com/article/10.1007%2Fs12195-019-00574-3
FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation
Authors:
James M. Murphy, Kyuho Jeong, Yelitza A. R. Rodriguez, Jung-Hyun Kim, Eun-Young Erin Ahn & Ssang-Taek Steve Lim
Abstract:
Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE−/− mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.
Citation:
Scientific Reports 9, Article number: 7617 (2019)
Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-mediated Cyclin D1 Transcription
Authors:
Kyuho Jeong, Jung-Hyun Kim, James M Murphy, Hyeonsoo Park, Su-Jeong Kim, Yelitza Rodriguez, Hyunkyung Kong, Chungsik Choi,Jun-Lin Guan,Joan M Taylor, William T Gerthoffer , Jun-Sub Kim, Eun-Young Erin Ahn, David D Schlaepfer, Ssang-Taek Steve Lim
Abstract:
Rationale: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via focal adhesion kinase (FAK). Objective: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. Methods and Results: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (KD, Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4-cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitantly increased GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control, but not in VS-4718-treated and SMC-specific FAK-KD mice. Finally, lentiviral shGATA4 knockdown in the femoral artery wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared to control mice. Conclusions: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.
Citation:
Originally published, https://doi.org/10.1161/CIRCRESAHA.118.314344Circulation Research. ;0
https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.118.314344
Innate Immune Cells' Contribution to Systemic Lupus Erythematosus
Authors:
Andrés A. Herrada, Noelia Escobedo, Mirentxu Iruretagoyena, Rodrigo A. Valenzuela, Paula I. Burgos, Loreto Cuitino and Carolina Llanos
Abstract:
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies against nuclear antigens, immune complex deposition, and tissue damage in the kidneys, skin, heart and lung. Because of the pathogenic role of antinuclear antibodies and autoreactive T cells in SLE, extensive efforts have been made to demonstrate how B cells act as antibody-producing or as antigen-presenting cells that can prime autoreactive T cell activation. With the discovery of new innate immune cells and inflammatory mediators, innate immunity is emerging as a key player in disease pathologies. Recent work over the last decade has highlighted the importance of innate immune cells and molecules in promoting and potentiating SLE. In this review, we discuss recent evidence of the involvement of different innate immune cells and pathways in the pathogenesis of SLE. We also discuss new therapeutics targets directed against innate immune components as potential novel therapies in SLE.
Citation:
Front. Immunol., 15 April 2019| https://doi.org/10.3389/fimmu.2019.00772
https://www.frontiersin.org/articles/10.3389/fimmu.2019.00772/full
Disrupted inflammasome activation by CD39 in venous thrombosis
Authors:
Yadav V, Chi L, Zhao R, Tourdot BE, Yalavarthi S, Banka A, Jacobs BN, Liao H, Koonse S, Anyanwu AC, Visovatti SH, Holinstat M, Kahlenberg JM, Knight JS, Pinsky DJ, Kanthi Y
Abstract:
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1β (IL-1β) release in CD39-deficient mice. Substantiating these findings, an IL-1β-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1β is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.
Citation:
J Clin Invest. https://doi.org/10.1172/JCI124804
Tuning the Thromboinflammatory Response to Venous Flow Interruption by the Ectonucleotidase CD39
Authors:
Anuli C. Anyanwu , Yogendra Kanthi , Keigo Fukase , Hui Liao , Tekashi Mimura , Karl C. Desch , Martin Gruca , Saabir Kaskar , Hussein Sheikh-Aden , Liguo Chi , Raymond Zhao , Vinita Yadav , Thomas W. Wakefield , Matthew C. Hyman , David J. Pinsky
Abstract:
Objective — Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 (ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results — CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions — These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.
Citation:
Originally published28 Feb 2019https://doi.org/10.1161/ATVBAHA.119.312407Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39:e118–e129
Genetic compensation triggered by mutant mRNA degradation
Authors:
Mohamed A. El-Brolosy, Zacharias Kontarakis, Andrea Rossi, Carsten Kuenne, Stefan Günther, Nana Fukuda, Khrievono Kikhi, Giulia L. M. Boezio, Carter M. Takacs, Shih-Lei Lai, Ryuichi Fukuda, Claudia Gerri, Antonio J. Giraldez & Didier Y. R. Stainier
Abstract:
Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.
Citation:
Nature(2019)
Loss of Endothelial Derived WNT5A is Associated with Reduced Pericyte Recruitment and Small Vessel Loss in Pulmonary Arterial Hypertension
Authors:
Yuan K, Shamskhou EA, Orcholski ME, Nathan A, Reddy S, Honda H, Mani V, Zeng Y, Ozen MO, Wang L, Demirci U, Tian W, Nicolls MR, de Jesus Perez VA
Abstract:
Background: Pulmonary arterial hypertension (PAH) is a life-threatening disorder of the pulmonary circulation associated with loss and impaired regeneration of microvessels. Reduced pericyte coverage of pulmonary microvessels is a pathological feature of PAH and is partly due to the inability of pericytes to respond to signaling cues from neighboring pulmonary microvascular endothelial cells (PMVECs). We have shown that activation of the Wnt/PCP pathway is required for pericyte recruitment but whether production and release of specific Wnt ligands by PMVECs is responsible for Wnt/PCP activation in pericytes is unknown. Methods: Isolation of pericytes and PMVECs from healthy donor and PAH lungs was carried out using 3G5 or CD31 antibody conjugated magnetic beads. Wnt expression profile of PMVECs was documented via qPCR using a Wnt primer library. Exosome purification from PMVEC media was carried out using the ExoTIC device. Hemodynamic profile, right ventricular function and pulmonary vascular morphometry were obtained in a conditional endothelial specific Wnt5a knockout (Wnt5aECKO) mouse model under normoxia, chronic hypoxia and hypoxia recovery. Results: Quantification of Wnt ligand expression in healthy PMVECs co-cultured with pericytes demonstrated a 35-fold increase in Wnt5a, a known Wnt/PCP ligand. This Wnt5a spike was not seen in PAH PMVECs, which correlated with inability to recruit pericytes in matrigel co-culture assays. Exosomes purified from media demonstrated an increase in Wnt5a content when healthy PMVECs were co-cultured with pericytes, a finding that was not observed in exosomes of PAH PMVECs. Furthermore, the addition of either recombinant Wnt5a or purified healthy PMVEC exosomes increased pericyte recruitment to PAH PMVECs in co-culture studies. While no differences were noted in normoxia and chronic hypoxia, Wnt5aECKO mice demonstrated persistent pulmonary hypertension and right ventricular failure four weeks after recovery from chronic hypoxia, which correlated with significant reduction, muscularization and decreased pericyte coverage of microvessels. Conclusions: We identify Wnt5a as a key mediator for the establishment of pulmonary endothelial-pericyte interactions and its loss could contribute to PAH by reducing the viability of newly formed vessels. We speculate that therapies that mimic or restore Wnt5a production could help prevent loss of small vessels in PAH.
Citation:
Originally published 11 Dec 2018 https://doi.org/10.1161/CIRCULATIONAHA.118.037642 Circulation. 2018
https://www.ahajournals.org/doi/abs/10.1161/CIRCULATIONAHA.118.037642
Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β-induced Serpine1
Authors:
McCann JV, Xiao L, Kim DJ, Khan OF, Kowalski PS, Anderson DG, Pecot CV, Azam SH, Parker JS, Tsai YS, Wolberg AS, Turner SD, Tatsumi K, Mackman N, Dudley AC
Abstract:
In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β–mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1–dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.
Citation:
J Clin Invest. https://doi.org/10.1172/JCI123106
Effect of TRPA1 activator allyl isothiocyanate (AITC) on rat dural and pial arteries
Authors:
Anna Koldbro Hansted, Deepak Kumar Bhatt, Jes Olesen, Lars Jørn Jensen, Inger Jansen-Olesen
Abstract:
Background: Transient receptor potential ankyrin 1 (TRPA1) channels may have a role in migraine as some substances known to cause headache activate the channel. In the craniovascular system such activation causes a calcitonin gene-related peptide (CGRP)-dependent increase in meningeal blood flow. TRPA1 channels in the endothelium of cerebral arteries cause vasodilation when activated. The headache preventive substance feverfew inhibits activation of TRPA1 channels. In this study we aim to compare and characterize the effect of the TRPA1 agonist allyl isothiocyanate (AITC) on the diameter of rat dural and pial arteries in vivo. Material and methods: The genuine closed-cranial window technique in rats was used to examine changes in dural and pial artery diameter and mean arterial blood pressure (MABP) after intracarotid infusion of AITC. Blockade experiments were performed by intravenous infusion of olcegepant, HC-030031, sumatriptan or capsazepine immediately after infusion of AITC, in four different groups of rats. Results: AITC caused a significant dilation of dural arteries, which was inhibited by HC-030031, olcegepant and sumatriptan, but not by capsazepine. In pial arteries AITC caused a significant dilation, which was not inhibited by any of the pre-treatments, suggesting a poor penetration of the blood-brain barrier or autoregulation due to dimethyl sulfoxide (DMSO) mediated decrease in MABP during HC-030031 infusion. AITC did not cause a significant change in MABP. Conclusion: AITC causes dilation of dural arteries via a mechanism dependent on CGRP and TRPA1 that is sensitive to sumatriptan. AITC causes a small but significant dilation of pial arteries.
Citation:
Pharmacological Reports, Available online 21 February 2019
https://www.sciencedirect.com/science/article/abs/pii/S1734114018303463
Cardosin A Endocytosis Mediated by Integrin Leads to Lysosome Leakage and Apoptosis of Epithelial Cells
Authors:
Wu H, Castenheira P, Faro C, Tang J.
Abstract:
Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans.(1) Such a process, which has been called the Trojan Horse mechanism, is thought to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores. This article is protected by copyright. All rights reserved.
Citation:
Proteins. 2019 Feb 20. doi: 10.1002/prot.25672. [Epub ahead of print]
Structural and functional analysis of single-nucleotide polymorphic variants of purine-rich element-binding protein B
Authors:
Lauren A. Ferris & Robert J. Kelm Jr.
Abstract:
Purine-rich element-binding protein B (Purβ) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α-actin gene (Acta2). Several reports have identified the structural domains in Purβ that enable its characteristic interaction with purine-rich single-stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single-nucleotide polymorphisms that alter individual amino acid residues in Purβ. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purβ protein. Mapping the location of variant residues on a homology model of the Purβ homodimer suggested that most of the altered residues are remote from the predicted ssDNA-binding regions of the protein. The repressor activity of each Purβ variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter-reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5-fold greater to ~2-fold less than wild-type Purβ. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild-type Purβ in terms of quaternary structure and thermostability. Results of DNA and protein-binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine-rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purβ by altering its interaction with other transcription factors but not with ssDNA.
Citation:
J Cell Biochem. 2019 Apr;120(4):5835-5851. doi: 10.1002/jcb.27869. Epub 2018 Nov 1.
Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells
Authors:
Esmerats JF, Villa-Roel N, Kumar S, Gu L, Salim MT, Ohh M, Taylor WR, Nerem RM, Yoganathan AP, Jo H
Abstract:
Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.
Citation:
Arterioscler Thromb Vasc Biol. 2019 Jan 3:ATVBAHA118312233. doi: 10.1161/ATVBAHA.118.312233. [Epub ahead of print]
miR-214 is Stretch-Sensitive in Aortic Valve and Inhibits Aortic Valve Calcification
Authors:
Salim MT, Esmerats JF, Arjunon S, Villa-Roel N, Nerem RM, Jo H, Yoganathan AP
Abstract:
miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48 h in regular medium and for 1 week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2 weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.
Citation:
Ann Biomed Eng. 2019 Jan 22. doi: 10.1007/s10439-019-02206-3. [Epub ahead of print]
Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice
Authors:
Lubkin A., Lee W.L., Alonzo F. 3rd, Wang C., Aligo J., Keller M., Girgis N. M., Reyes-Robles T., Chan R., O’Malley A., Buckley P., Vozhilla N., Vasquez M., Su J., Sugiyama M., Yeung S.T., Coffre M., Bajwa S., Chen E., Martin P., Kim S.Y., Loomis C., Worthen G.S., Shopsin B., Khanna K.M., Weinstock D., Lynch A.S., Koralov S.B., Loke P., Cadwell K., and Torres V.J.
Abstract:
The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytesand lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB),are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstratethat LukED and HlgAB cause vascular congestion and derangements in vascular fluiddistribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines(DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the criticaltarget for lethality. Consistent with this, LukED and HlgAB injure primary human endothelialcells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells areresistant to toxin-mediated lethality. During bloodstream infection in mice, DARCtargeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potentialfor S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulencefactors.
Citation:
Cell Host and Microbe, Published: February 21, 2019•DOI:https://doi.org/10.1016/j.chom.2019.01.015
https://www.cell.com/cell-host-microbe/fulltext/S1931-3128(19)30053-8
MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis
Authors:
Moro A, Driscoll TP, Boraas LC, Armero W, Kasper DM, Baeyens N, Jouy C, Mallikarjun V, Swift J, Ahn SJ, Lee D, Zhang J, Gu M, Gerstein M, Schwartz M, Nicoli S
Abstract:
Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.
Citation:
Nat Cell Biol. 2019 Feb 11. doi: 10.1038/s41556-019-0272-y. [Epub ahead of print]
Evolution of hemodynamic forces in the pulmonary tree with progressively worsening pulmonary arterial hypertension in pediatric patients
Authors:
Yang W, Dong M, Rabinovitch M, Chan FP, Marsden AL, Feinstein JA
Abstract:
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling resulting in right ventricular (RV) dysfunction and ultimately RV failure. Mechanical stimuli acting on the vessel walls of the full pulmonary tree have not previously been comprehensively characterized. The goal of this study is to characterize wall shear stress (WSS) and strain in pediatric PAH patients at different stages of disease severity using computational patient-specific modeling. Computed tomography, magnetic resonance imaging and right heart catheterization data were collected and assimilated into pulmonary artery (PA) models for patients with and without PAH. Patients were grouped in three disease severity groups (control, moderate and severe) based on clinical evaluations. A finite element solver was employed to quantify hemodynamics and wall strains. To estimate WSS in the distal small PAs with diameters ranging from 50 to 500 [Formula: see text], a morphometric tree model was created, with inputs coming from outlets of the 3D model. WSS in the proximal PAs decreased with disease severity (control 20.5 vs. moderate 15.8 vs. severe 6.3 [Formula: see text], [Formula: see text]). Oscillatory shear index increased in the main pulmonary artery (MPA) with disease severity (0.13 vs. 0.13 vs. 0.2, [Formula: see text]). Wall strains measured by the first invariant of Green strain tensor decreased with disease severity (0.16 vs. 0.12 vs. 0.11, [Formula: see text]). Mean WSS for the distal PAs between 100 and 500 [Formula: see text] significantly increased with disease severity (20 vs. 52 vs. 116 [Formula: see text], [Formula: see text]). In conclusion, 3D flow simulations showed that WSS is significantly decreased in the MPA with disease while the mathematical morphometric model suggested increased WSS in the distal small vessels. Computational models can reveal mechanical stimuli acting on vessel walls that may inform patient risk stratification and flow shear experiments.
Citation:
Biomech Model Mechanobiol. 2019 Jan 12. doi: 10.1007/s10237-018-01114-0. [Epub ahead of print]
https://link.springer.com/article/10.1007/s10237-018-01114-0
Solid stress in brain tumours causes neuronal loss and neurological dysfunction and can be reversed by lithium
Authors:
Seano G, Nia HT, Emblem KE, Datta M, Ren J, Krishnan S, Kloepper J, Pinho M, Ho WW, Ghosh M, Askoxylakis V, Ferraro GB, Riedemann L, Gerstner ER, Batchelor TT, Wen PY, Lin NU, Grodzinsky AJ, Fukumura D, Huang P, Baish JW, Padera TP, Munn LL, Jain RK
Abstract:
The compression of brain tissue by a tumour mass is believed to be a major cause of the clinical symptoms seen in patients with brain cancer. However, the biological consequences of these physical stresses on brain tissue are unknown. Here, via imaging studies in patients and by using mouse models of human brain tumours, we show that a subgroup of primary and metastatic brain tumours, classified as nodular on the basis of their growth pattern, exert solid stress on the surrounding brain tissue, causing a decrease in local vascular perfusion as well as neuronal death and impaired function. We demonstrate a causal link between solid stress and neurological dysfunction by applying and removing cerebral compression, which respectively mimic the mechanics of tumour growth and of surgical resection. We also show that, in mice, treatment with lithium reduces solid-stress-induced neuronal death and improves motor coordination. Our findings indicate that brain-tumour-generated solid stress impairs neurological function in patients, and that lithium as a therapeutic intervention could counter these effects.
Citation:
Nature Biomedical Engineering(2019)
CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells - role in fatty acid delivery
Authors:
Raheel H, Ghaffari S, Khosraviani N, Mintsopoulos V, Auyeung D, Wang C, Kim YH, Mullen B, Sung HK, Ho M, Fairn G, Neculai D, Febbraio M, Heit B, Lee WL
Abstract:
In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.
Citation:
Am J Physiol Lung Cell Mol Physiol. 2019 Jan 31. doi: 10.1152/ajplung.00127.2018. [Epub ahead of print]
Mature vessel networks in engineered tissue promote graft–host anastomosis and prevent graft thrombosis
Authors:
Shahar Ben-Shaul, Shira Landau, Uri Merdler, and Shulamit Levenberg
Abstract:
Graft vascularization remains one of the most critical challenges facing tissue-engineering experts in their attempt to create thick transplantable tissues and organs. In vitro prevascularization of engineered tissues has been suggested to promote rapid anastomosis between the graft and host vasculatures; however, thrombotic events have been reported upon graft implantation. Here, we aimed to determine whether in vitro vessel maturation in transplantable grafts can accelerate vascular integration and graft perfusion and prevent thrombotic events in the grafts. To this end, endothelial cells and fibroblasts were cocultured on 3D scaffolds for 1, 7, or 14 d to form vasculature with different maturation degrees. Monitoring graft–host interactions postimplantation demonstrated that the 14-d in vitro-cultured grafts, bearing more mature and complex vessel networks as indicated by elongated and branched vessel structures, had increased graft–host vessel anastomosis; host vessel penetration into the graft increased approximately eightfold, and graft perfusion increased sixfold. The presence of developed vessel networks prevented clot accumulation in the grafts. Conversely, short-term cultured constructs demonstrated poor vascularization and increased thrombus formation. Elevated expression levels of coagulation factors, von Willebrand factor (vWF), and tissue factor (TF), were demonstrated in constructs bearing less mature vasculature. To conclude, these findings demonstrate the importance of establishing mature and complex vessel networks in engineered tissues before implantation to promote anastomosis with the host and accelerate graft perfusion.
Citation:
PNAS published ahead of print February 4, 2019 https://doi.org/10.1073/pnas.1814238116
AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate
Authors:
Gu Q, Yang X, Lv J, Zhang J, Xia B, Kim JD, Wang R, Xiong F, Meng S, Clements TP, Tandon B, Wagner DS, Diaz MF, Wenzel PL, Miller YI, Traver D, Cooke JP, Li W, Zon LI, Chen K, Bai Y, Fang L
Abstract:
Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here we report that a somite-derived pro-hematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch upregulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide ChIP-seq, RNA-seq, and ATAC-seq indicate that Srebp2 trans-regulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.
Citation:
Science. 2019 Jan 31. pii: eaav1749. doi: 10.1126/science.aav1749. [Epub ahead of print]
http://science.sciencemag.org/content/early/2019/01/30/science.aav1749
Visualization and Quantification of Mitochondrial Structure in the Endothelium of Intact Arteries
Authors:
Durand MJ, Ait-Aissa K, Levchenko V, Staruschenko A, Gutterman DD, Beyer AM
Abstract:
Aims: To quantify the mitochondrial structure of ECs in intact arteries vs. cultured cells. Methods and Results: Cre-stop mito-Dendra2 mice, expressing the fluorescent protein Dendra2 in the mitochondrial matrix only, were used to label EC mitochondria using Cre-recombinase under the control of the VE-cadherin promoter. Conduit arteries, resistance arterioles and veins were fixed, mounted on glass slides and fluorescent images were obtained using a laser scanning confocal microscope (ex 488 nm; em 550 nm). ImageJ was used to calculate form factor (FF) and aspect ratio (AR) of the mitochondrial segments. Mitochondrial fragmentation count (MFC) was calculated by counting non-contiguous mitochondrial particles and dividing by the number of pixels which comprise the mitochondrial network. Primary aortic EC cultures (48 h on culture plates) were generated to compare the mitochondrial structure of cultured ECs vs. intact arteries. Aortic segments were also exposed to high glucose overnight (33 mM) ex vivo, and separate groups of mice were either infused with a high glucose saline solution (300 mM) via tail vein catheter for one hour or injected with Streptozotocin (STZ; 50 mg/kg) to cause hyperglycemia. Compared to cultured ECs, the mitochondria of ECs from the intact aorta were more fragmented (MFC: 6.4±2.5 vs. 18.6±9.4, respectively; p < 0.05). The mitochondrial segments of ECs within the aorta were more circular in shape (FF: 3.5±0.75 vs. 1.8±0.30, respectively; p < 0.05) and had less branching (AR: 2.9±0.60 vs. 2.0±0.25, respectively; p < 0.05) compared to cultured ECs. Ex vivo exposure of the intact aorta to high glucose overnight caused mitochondrial fission compared to normal glucose conditions (5 mM; MFC: 25.5±11.1 high glucose vs. 11.0±3.6 normal glucose; p < 0.05). Both one-hour infusion of high glucose saline (MFC: 22.4±4.3) and STZ treatment (MFC: 40.3±14.2) caused mitochondrial fission compared to freshly fixed aortas from control mice (MFC: 18.6±9.4; p < 0.05 vs. high glucose infusion and STZ treatment). Conclusions: Using a novel mouse model, we were able to, for the first time, obtain high resolution images of EC mitochondrial structure in intact arteries. We reveal the endothelial mitochondrial network is more fragmented in the intact aorta compared to cultured ECs, indicating that mitochondria assume a more elongated and branched phenotype in cell culture.
Citation:
Cardiovasc Res. 2018 Nov 22. doi: 10.1093/cvr/cvy294. [Epub ahead of print]
https://academic.oup.com/cardiovascres/advance-article-abstract/doi/10.1093/cvr/cvy294/5198893
A dynamic and integrated epigenetic program at distal regions orchestrates transcriptional responses to VEGFA
Authors:
Wang S, Chen J, Garcia SP, Liang X, Zhang F, Yan P, Yu H, Wei W, Li Z, Wang J, Le H, Han Z, Luo X, Day DS, Stevens SM, Zhang Y, Park PJ, Liu ZJ, Sun K, Yuan GC, Pu WT, Zhang B
Abstract:
Cell behaviors are dictated by epigenetic and transcriptional programs. Little is known about how extracellular stimuli modulate these programs to reshape gene expression and control cell behavioral responses. Here, we interrogated the epigenetic and transcriptional response of endothelial cells to VEGFA treatment and found rapid chromatin changes that mediate broad transcriptomic alterations. VEGFA-responsive genes were associated with active promoters, but changes in promoter histone marks were not tightly linked to gene expression changes. VEGFA altered transcription factor occupancy and the distal epigenetic landscape, which profoundly contributed to VEGFA-dependent changes in gene expression. Integration of gene expression, dynamic enhancer, and transcription factor occupancy changes induced by VEGFA yielded a VEGFA-regulated transcriptional regulatory network, which revealed that the small MAF transcription factors are master regulators of the VEGFA transcriptional program and angiogenesis. Collectively these results revealed that extracellular stimuli rapidly reconfigure the chromatin landscape to coordinately regulate biological responses.
Citation:
Genome Res. 2019 Jan 22. doi: 10.1101/gr.239053.118. [Epub ahead of print]
Bola3 Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension
Authors:
Yu Q, Tai YY, Tang Y, Zhao J, Negi V, Culley MK, Pilli J, Sun W, Brugger K, Mayr J, Saggar R, Saggar R, Wallace WD, Ross DJ, Waxman AB, Wendell SG, Mullett SJ, Sembrat J, Rojas M, Khan OF, Dahlman J, Sugahara M, Kagiyama N, Satoh T, Zhang M, Feng N, Gorcsan J, Vargas SO, Haley KJ, Kumar R, Graham BB, Langer R, Anderson DG, Wang B, Shiva S, Bertero T, Chan SY
Abstract:
Background and Hypothesis: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. The BolA Family Member 3 (BOLA3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. However, the exact mechanistic role of BOLA3 in PH remains undefined. Methods and Results: In cultured hypoxic PAECs as well as lung from human and multiple rodent models of PH, endothelial BOLA3 expression was down-regulated, which involved HIF-2α-dependent transcriptional repression via HDAC-mediated histone deacetylation. In vitro gain and loss of function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In addition, BOLA3 deficiency down-regulated the glycine cleavage system protein H (GCSH), thus bolstering intracellular glycine content. As a result of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction, while decreasing angiogenic potential. In vivo, pharmacologic knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 demonstrated that BOLA3 deficiency promotes histologic and hemodynamic manifestations of PH. Conclusion: In aggregate, BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic re-programming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, iron-sulfur biogenesis, and glycine biology, for diagnostic and therapeutic development in this disease.
Citation:
Originally published5 Nov 2018Circulation. 2018;138:A11422
https://www.ahajournals.org/doi/abs/10.1161/circ.138.suppl_1.11422
Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation
Authors:
Li X, Cai Y, Goines J, Pastura P, Brichta L, Lane A, Le Cras TD, Boscolo E
Abstract:
Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VM grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model of VM based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis, and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ and ERK activity.
Citation:
Arterioscler Thromb Vasc Biol. 2019 Jan 10:ATVBAHA118312315. doi: 10.1161/ATVBAHA.118.312315. [Epub ahead of print]
Loss of estrogen-related receptor alpha facilitates angiogenesis in endothelial cells
Authors:
Likhite N, Yadav V, Milliman EJ, Sopariwala DH, Lorca S, Narayana NP, Sheth M, Reineke EL, Giguère V, Narkar V
Abstract:
Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multi-faceted role in vasculature. Of the three ERR subtypes ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation, but increased migration and tube formation. ERRα depletion increased basal, VEGFA and ANG1/2 stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to wild type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets such as metabolism, mitochondrial biogenesis and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, pro-angiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis, and potentially fine-tunes growth factor-mediated angiogenesis.IMPORTANCE Impaired angiogenesis, the process of blood vessel formation, is central to various diseases including peripheral vascular disease, retinopathy, nephropathy, cardiac ischemia and cancer. While few angiogenic regulators have been described, full breadth of the cellular and molecular pathways that regulate this phenomenon are far from clear. Fundamental discoveries related to angiogenesis regulation could lead to new drugs that normalize blood vessels in the aforementioned diseases. Using a cell and molecular biology approach, our work highlights a gene regulator called ERRα in endothelial cells (the building blocks of the vasculature), which plays a role in limiting angiogenic activation by controlling endothelial gene expression. It can be envisioned that future pre-clinical and pharmaco/physiological studies could lead to advancement of ERRα as a therapeutic regulator of angiogenesis in vascular-linked diseases.
Citation:
Mol Cell Biol. 2019 Jan 2. pii: MCB.00411-18. doi: 10.1128/MCB.00411-18. [Epub ahead of print]
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]
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]
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]
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]
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]
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
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]
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.
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 (<100m diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100m 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.
Citation:
Diabetes Metab Res Rev. 2018 Aug 11:e3058. doi: 10.1002/dmrr.3058. [Epub ahead of print]
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]
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.
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]