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ePubs

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

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ePubs

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

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

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

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

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

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

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

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

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

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

SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling

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

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

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

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

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

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

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

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

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

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

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

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

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

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

RASA1 regulates the function of lymphatic vessel valves in mice

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Engineering Vascularized Flaps Using Adipose-Derived Microvascular EC and MSC

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

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

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

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

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

Authors:
Weddell JC, Imoukhuede PI

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

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

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

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

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

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

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

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

Development of the larval lymphatic system in the zebrafish

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

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

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

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

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

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

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

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

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

Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling

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

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

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

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

Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

Authors:
Leppänen VM, Saharinen P, Alitalo K

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

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

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

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

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

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

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

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

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

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

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

Citation:
Circulation Research. 2017;CIRCRESAHA.116.310535

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

Lymphatic deletion of calcitonin receptor–like receptor exacerbates intestinal inflammation

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

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

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

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

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

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

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

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

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

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Citation:
J Clin Invest. doi:10.1172/JCI90086

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

5,6-δ-DHTL, a stable metabolite of arachidonic acid, is a potential EDHF that mediates microvascular dilation

Authors:
Reut Levi-Rosenzvig, Andreas M. Beyer, Joseph Hockenberry, Rotem Ben-Shushan, Dimitry Chuyun, Shahar Atiya, Snait Tamir, David D. Gutterman, Andrea Szuchman-Sapir

Abstract:
Objective: Prominent among the endothelium-derived hyperpolarizing factors (EDHFs) are the Cytochrome P450 (CYP) epoxygenase-derived arachidonic acid metabolites—the epoxyeicosatrienoic acids (EETs), that are known as vasodilators in the microcirculation. Among the EET isomers, 5,6-EET undergoes rapid lactonization in aqueous solution to the more stable 5,6-δ DHTL (5,6-dihydroxytrienoic lactone) isomer. It is unclear whether this metabolic transformation maintains its vasodilator potential and what is the mechanism of action. Thus, the aim of this study was to investigate the capacity of the lactone isomer, 5,6- δ DHTL, to induce dilation of arterioles and explore the endothelial Ca2+ response mechanism. Approach and Results: In isolated human microvessels 5,6- δ DHTL induced a dose dependent vasodilation, that was inhibited by mechanical denudation of the endothelial layer. This 5,6- δ DHTL -dependent dilation was partially reduced in the presence of L-NAME (NOS inhibitor) or the NO-scavenger, cPTIO (by 19.7%, which was not statistically significantly). In human endothelial cells, 5,6- δ DHTL induced an increase in intracellular Ca2+ ([Ca2+]i) in a dose dependent manner. This increase in [Ca2+]i was similar to that induced by the 5,6-EET isomer, and significantly higher than observed by administering the hydrolytic dihydroxy isomer, 5,6-DHET. Further experiments aimed to investigate the mechanism of action revealed, that the 5,6-δ DHTL-mediated ([Ca2+]i elevation was reduced by IP3 and ryanodine antagonists, but not by antagonists to the TRPV4 membrane channel. Similar to their effect on the dilation response in the arteries, NO inhibitors reduced the 5,6-δ DHTL-mediated ([Ca2+]i elevation by 20%. Subsequent 5,6-δ DHTL -dependent K+ ion efflux from endothelial cells, was abolished by the inhibition of small and intermediate conductance KCa. Conclusions: The present study shows that 5,6-δ DHTL is a potential EDHF, that dilates microvessels through a mechanism that involves endothelial dependent Ca2+ entry, requiring endothelial hyperpolarization. These results suggest the existence of additional lactone-containing metabolites that can be derived from the PUFA metabolism and which may function as novel EDHFs.

Citation:
Free Radical Biology and Medicine. Available online 19 December 2016

http://www.sciencedirect.com/science/article/pii/S0891584916311224

TSP1-CD47 Signaling is Upregulated in Clinical Pulmonary Hypertension and Contributes to Pulmonary Arterial Vasculopathy and Dysfunction

Authors:
Rogers NM, Sharifi-Sanjani M, Yao M, Ghimire K, Bienes-Martinez R, Mutchler SM, Knupp HE, Baust J, Novelli EM, Ross M, St Croix C, Kutten JC, Czajka CA, Sembrat JC, Rojas M, Labrousse-Arias D, Bachman TN, Vanderpool RR, Zuckerbraun BS, Champion HC, Mora AL, Straub AC, Bilonick RA, Calzada MJ, Isenberg JS

Abstract:
Aims: Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and Results: We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n=23) and without (n=16) PH. Compared to controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47 and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions: In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Citation:
Cardiovasc Res. 2016 Oct 13. pii: cvw218. [Epub ahead of print]

http://cardiovascres.oxfordjournals.org/content/early/2016/10/12/cvr.cvw218

Nitric oxide: What's new to NO?

Authors:
Ghimire K, Altmann HM, Straub A, Isenberg JS

Abstract:
Nitric oxide (NO) is one of the critical components of the vasculature, regulating key signaling pathways in health. In macro-vessels NO functions to suppress cell inflammation as well as adhesion. In this way it inhibits thrombosis and promotes blood flow. It also functions to limit vessel constriction and vessel wall remodeling. In micro-vessels and particularly capillaries NO, along with certain growth factors, is important in promoting new vessels formation, a process termed angiogenesis. With age and cardiovascular disease animal and human studies confirm that NO is dysregulated at multiple levels including decreased production, decreased tissue half-life and decreased potency. NO has also been implicated in diseases that are related to neurotransmission and cancer although it is likely that these processes involve NO at higher concentrations and from non-vascular cell sources. Conversely, NO and drugs that directly or indirectly increase NO signaling have found clinical applications in both age-related diseases and in younger individuals. This focused review considers recently reported advances being made in the field of NO signaling regulation at several levels including enzymatic production, receptor function, interacting partners, localization of signaling, matrix-cellular and cell-to-cell cross-talk, as well as the possible impact these newly described mechanisms have upon health and disease.

Citation:
Am J Physiol Cell Physiol. 2016 Dec 14:ajpcell.00315.2016. doi:10.1152/ajpcell.00315.2016. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/27974299

CD47 and Nox1 Mediate Dynamic Fluid-Phase Macropinocytosis of Native LDL

Authors:
Csanyi G, Feck DM, Ghoshal P, Singla B, Lin HP, Nagarajan S, Meijles DN, Al Ghouleh I, Cantu-Medellin NC, Kelley EE, Mateuszuk LM, Isenberg JS, Watkins SC, Pagano PJ

Abstract:
AIMS: Macropinocytosis has been implicated in cardiovascular and other disorders yet the physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved. RESULTS:TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and Nox1 signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser 3, actin remodeling, and macropinocytotic uptake of unmodified native LDL, leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE-/- mice in vivo. INNOVATION: Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct, receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to the early propagation of vascular inflammatory disease. CONCLUSIONS: The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated.

Citation:
Antioxid Redox Signal. 2016 Dec 13. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/?term=CD47+and+Nox1+Mediate+Dynamic+Fluid-Phase+Macropinocytosis+of+Native+LDL.

Endothelium in the pharyngeal arches 3, 4 and 6 is derived from the second heart field

Authors:
Xia Wang, Dongying Chen, Kelley Chen, Ali Jubran, AnnJosette Ramirez, Sophie Astrof

Abstract:
Oxygenated blood from the heart is directed into the systemic circulation through the aortic arch arteries (AAAs). The AAAs arise by remodeling of three symmetrical pairs of pharyngeal arch arteries (PAAs), which connect the heart with the paired dorsal aortae at mid-gestation. Aberrant PAA formation results in defects frequently observed in patients with lethal congenital heart disease. How the PAAs form in mammals is not understood. The work presented in this manuscript shows that the second heart field (SHF) is the major source of progenitors giving rise to the endothelium of the pharyngeal arches 3 – 6, while the endothelium in the pharyngeal arches 1 and 2 is derived from a different source. During the formation of the PAAs 3 – 6, endothelial progenitors in the SHF extend cellular processes toward the pharyngeal endoderm, migrate from the SHF and assemble into a uniform vascular plexus. This plexus then undergoes remodeling, whereby plexus endothelial cells coalesce into a large PAA in each pharyngeal arch. Taken together, our studies establish a platform for investigating cellular and molecular mechanisms regulating PAA formation and alterations that lead to disease.

Citation:
Dev Biol. 2016 Dec 9. pii: S0012-1606(16)30359-1. doi: 10.1016/j.ydbio.2016.12.010. [Epub ahead of print]

http://www.sciencedirect.com/science/article/pii/S0012160616303591

Functional Definition of Progenitors Versus Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process

Authors:
Patel J, Seppanen EJ, Rodero MP, Wong HY, Donovan P, Neufeld Z, Fisk NM, François M, Khosrotehrani K

Abstract:
BACKGROUND: During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neo-vessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, as well as wound healing, tumors and placenta. METHODS: Based on protein expression levels of common endothelial markers using flow cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. RESULTS: Lineage tracing using Cdh5creERt2/Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony forming capacity in limiting dilution and by transplantation in MatrigelTM plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr and Pdfgrα Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf and Notch The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. SOX18's role in the differentiation process was validated using lineage-tracing experiments based on Sox18CreERt2/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. CONCLUSIONS: Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.

Citation:
Circulation. 2016 Nov 29. pii: CIRCULATIONAHA.116.024754. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2016/11/29/CIRCULATIONAHA.116.024754

PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia

Authors:
Roxana Ola, Alexandre Dubrac, Jinah Han, Jennifer S. Fang, Bruno Larrivee, Monica Lee, Ana A. Urarte, Jan R. Kraehling, Gael Genet, Karen K. Hirschi, William C. Sessa, Francesc V. Canals, Mariona Graupera, Minhong Yan, Lawrence H. Young, Paul S. Oh, Anne Eichmann

Abstract:
Activin receptor-like kinase 1 (ALK1) is an endothelial serine–threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

Citation:
Nature Communications 7, Article number: 13650 (2016)doi:10.1038/ncomms13650

http://www.nature.com/articles/ncomms13650#results

Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice

Authors:
Kritikou E, van Puijvelde GH, van der Heijden T, van Santbrink PJ, Swart M, Schaftenaar FH, Kröner MJ, Kuiper J, Bot I

Abstract:
Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

Citation:
Sci Rep. 2016 Nov 24;6:37585. doi: 10.1038/srep37585

https://www.ncbi.nlm.nih.gov/pubmed/27883026

Biofluids, cell mechanics and epigenetics: Flow-induced epigenetic mechanisms of endothelial gene expression

Authors:
Davies PF, Manduchi E, Jiménez JM, Jiang, Y-Z

Abstract:
Epigenetics is the regulation of gene expression (transcription) in response to changes in the cell environment through genomic modifications that largely involve the non-coding fraction of the human genome and that cannot be attributed to modification of the primary DNA sequence. Epigenetics is dominant in establishing cell fate and positioning during programmed embryonic development. However the same pathways are used by mature postnatal and adult mammalian cells during normal physiology and are implicated in disease mechanisms. Recent research demonstrates that blood flow and pressure are cell environments that can influence transcription via epigenetic pathways. The principal epigenetic pathways are chemical modification of cytosine residues of DNA (DNA methylation) and of the amino tails of histone proteins associated with DNA in nucleosomes. They also encompass the post-transcriptional degradation of mRNA transcripts by non-coding RNAs (ncRNA). In vascular endothelium, epigenetic pathways respond to temporal and spatial variations of flow and pressure, particularly hemodynamic disturbed blood flow, with important consequences for gene expression. The biofluid environment is linked by mechanotransduction and solute transport to cardiovascular cell phenotypes via signaling pathways and epigenetic regulation for which there is an adequate interdisciplinary infrastructure with robust tools and methods available. Epigenetic mechanisms may be less familiar than acute genomic signaling to Investigators at the interface of biofluids, biomechanics and cardiovascular biology. Here we introduce a biofluids / cellular biomechanics readership to the principal epigenetic pathways and provide a contextual overview of endothelial epigenetic plasticity in the regulation of flow-responsive transcription.

Citation:
J. Biomechanics. 2016. ePub Nov 15; Doi: 10.1016/ j.jbiomech.2016.11.017

http://www.jbiomech.com/article/S0021-9290(16)31185-X/abstract

Fine-tuning vascular fate during endothelial-mesenchymal transition

Authors:
Xiao L, Dudley AC

Abstract:
In the heart and other organs, endothelial-mesenchymal transition (EndMT) has emerged as an important developmental process that involves coordinated migration, differentiation, and proliferation of the endothelium. In multiple disease states including cancer angiogenesis and cardiovascular disease, the processes that regulate EndMT are recapitulated, albeit in an uncoordinated and dysregulated manner. Members of the transforming growth factor beta (TGFβ) super-family are well known to impart cellular plasticity during EndMT by the timely activation (or repression) of transcription factors and miRNAs in addition to epigenetic regulation of gene expression. On the other hand, fibroblast growth factors (FGFs) are reported to augment or oppose TGFβ-driven EndMT in specific contexts. Here, we have synthesized the currently understood roles of TGFβ and FGF signalling during EndMT and have provided a new, comprehensive paradigm that delineates how an autocrine and paracrine TGFβ/FGF axis coordinates endothelial cell specification and plasticity. We also provide new guidelines and nomenclature that considers factors such as endothelial cell heterogeneity to better define EndMT across different vascular beds. This perspective should therefore help to clarify why TGFβ and FGF can both cooperate with or oppose one another during the complex process of EndMT in both health and disease.

Citation:
J Pathol. 2016 Oct 4. doi: 10.1002/path.4814. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/27701751

Regulation of embryonic neurogenesis by germinal zone vasculature

Authors:
Mathew Tata, Ivan Walla, Andy Joyce, Joaquim M. Vieira, Nicoletta Kessaris, and Christiana Ruhrberg

Abstract:
In the adult rodent brain, new neurons are born in two germinal regions that are associated with blood vessels, and blood vessels and vessel-derived factors are thought to regulate the activity of adult neural stem cells. Recently, it has been proposed that a vascular niche also regulates prenatal neurogenesis. Here we identify the mouse embryo hindbrain as a powerful model to study embryonic neurogenesis and define the relationship between neural progenitor cell (NPC) behavior and vessel growth. Using this model, we show that a subventricular vascular plexus (SVP) extends through a hindbrain germinal zone populated by NPCs whose peak mitotic activity follows a surge in SVP growth. Hindbrains genetically defective in SVP formation owing to constitutive NRP1 loss showed a premature decline in both NPC activity and hindbrain growth downstream of precocious cell cycle exit, premature neuronal differentiation, and abnormal mitosis patterns. Defective regulation of NPC activity was not observed in mice lacking NRP1 expression by NPCs, but instead in mice lacking NRP1 selectively in endothelial cells, yet was independent of vascular roles in hindbrain oxygenation. Therefore, germinal zone vascularization sustains NPC proliferation in the prenatal brain.

Citation:
Published online before print November 7, 2016, doi: 10.1073/pnas.1613113113, PNAS November 7, 2016

http://www.pnas.org/content/early/2016/11/04/1613113113.abstract

Alterations in plasma biochemical composition in NO deficiency induced by L-NAME in mice analysed by Fourier Transform Infrared Spectroscopy

Authors:
Staniszewska-Slezak E, Mateuszuk L, Chlopicki S, Baranska M, Malek K

Abstract:
Mouse model of nitric oxide deficiency, induced by prolonged treatment with NG -nitro-L-arginine methyl ester (L-NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L-NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO-deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L-NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS-DA method provided >95% classification accuracy. Spectral features characteristic for L-NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 - 0.005) were observed between spectra collected for each time-point of the L-NAME treatment versus control subjects. We demonstrated for the first time that NO-deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.

Citation:
J Biophotonics. 2016 Jul 21. doi: 10.1002/jbio.201600141. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27440215

Heparan sulfate proteoglycans mediate renal carcinoma metastasis

Authors:
Qazi H, Shi ZD, Song JW, Cancel LM, Huang P, Zeng Y, Roberge S, Munn LL, Tarbell JM

Abstract:
The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive tumor cells with high metastatic potential have enhanced invasion rates in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) in the glycocalyx plays an important role in this flow mediated mechanostransduction and upregulation of invasive and metastatic potential. In this study, highly metastatic renal cell carcinoma cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chamber and microfluidic assays, we show that flow-enhanced invasion is suppressed in HS deficient cells. To assess the ability of these cells to metastasize in vivo, parental or knockdown cells expressing fluorescence reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from the NDST1 knockdown cells compared to control cells with intact HS. The ability of these cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx in tumor progression.

Citation:
Int J Cancer. 2016 Aug 20. doi: 10.1002/ijc.30397. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed?linkname=pubmed_pubmed_reviews&from_uid=27543953

Autocrine VEGF Isoforms Differentially Regulate Endothelial Cell Behavior

Authors:
Hideki Yamamoto, Helene Rundqvist, Cristina Branco and Randall S. Johnson

Abstract:
Vascular endothelial growth factor A (VEGF) is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration, and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2). We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO) synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell homeostasis in normoxia and hypoxia.

Citation:
Front. Cell Dev. Biol., 21 September 2016 | http://dx.doi.org/10.3389/fcell.2016.00099

http://journal.frontiersin.org/article/10.3389/fcell.2016.00099/full

Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation

Authors:
Zhou HJ, Qin L, Zhang H, Tang, W, Ji, W, He Y, Liang, X, Wang Z, Yuan Q, Vortmeyer A, Toomre D, Fuh G, Yan M, Kluger MS, Wu D, Min W

Abstract:
Cerebral cavernous malformations (CCMs) are vascular malformations that affect the central nervous system and result in cerebral hemorrhage, seizure and stroke. CCMs arise from loss-of-function mutations in one of three genes: KRIT1 (also known as CCM1), CCM2 or PDCD10 (also known as CCM3). PDCD10 mutations in humans often result in a more severe form of the disease relative to mutations in the other two CCM genes, and PDCD10-knockout mice show severe defects, the mechanistic basis for which is unclear. We have recently reported that CCM3 regulates exocytosis mediated by the UNC13 family of exocytic regulatory proteins. Here, in investigating the role of endothelial cell exocytosis in CCM disease progression, we found that CCM3 suppresses UNC13B- and vesicle-associated membrane protein 3 (VAMP3)-dependent exocytosis of angiopoietin 2 (ANGPT2) in brain endothelial cells. CCM3 deficiency in endothelial cells augments the exocytosis and secretion of ANGPT2, which is associated with destabilized endothelial cell junctions, enlarged lumen formation and endothelial cell-pericyte dissociation. UNC13B deficiency, which blunts ANGPT2 secretion from endothelial cells, or treatment with an ANGPT2-neutralizing antibody normalizes the defects in the brain and retina caused by endothelial-cell-specific CCM3 deficiency, including the disruption of endothelial cell junctions, vessel dilation and pericyte dissociation. Thus, enhanced secretion of ANGPT2 in endothelial cells contributes to the progression of CCM disease, providing a new therapeutic approach for treating this devastating pathology.

Citation:
Nat Med. 2016 Aug 22. doi: 10.1038/nm.4169. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27548575

Point-of-Care Technologies for Precision Cardiovascular Care and Clinical Research : National Heart, Lung, and Blood Institute Working Group

Authors:
Kevin R. King, Luanda P. Grazette, Dina N. Paltoo, John T. McDevitt, Samuel K. Sia, Paddy M. Barrett, Fred S. Apple, Paul A. Gurbel, Ralph Weissleder, Hilary Leeds, Erin J. Iturriaga, Anupama K. Rao, Bishow Adhikari, Patrice Desvigne-Nickens, Zorina S. Galis, Peter Libby

Abstract:
Point-of-care technologies (POC or POCT) are enabling innovative cardiovascular diagnostics that promise to improve patient care across diverse clinical settings. The National Heart, Lung, and Blood Institute convened a working group to discuss POCT in cardiovascular medicine. The multidisciplinary working group, which included clinicians, scientists, engineers, device manufacturers, regulatory officials, and program staff, reviewed the state of the POCT field; discussed opportunities for POCT to improve cardiovascular care, realize the promise of precision medicine, and advance the clinical research enterprise; and identified barriers facing translation and integration of POCT with existing clinical systems. A POCT development roadmap emerged to guide multidisciplinary teams of biomarker scientists, technologists, health care providers, and clinical trialists as they: 1) formulate needs assessments; 2) define device design specifications; 3) develop component technologies and integrated systems; 4) perform iterative pilot testing; and 5) conduct rigorous prospective clinical testing to ensure that POCT solutions have substantial effects on cardiovascular care.

Citation:
JACC: Basic to Translational Science, Volume 1, Issues 1–2, January–February 2016, Pages 73–86

http://www.sciencedirect.com/science/article/pii/S2452302X16000139

Combined non-invasive assessment of endothelial shear stress and molecular imaging of inflammation for the prediction of inflamed plaque in hyperlipidaemic rabbit aortas

Authors:
Gitsioudis G, Chatzizisis YS, Wolf P, Missiou A, Antoniadis AP, Mitsouras D, Bartling S, Arica Z, Stuber M, Rybicki FJ, Nunninger M, Erbel C, Karvounis H, Libby P, Giannoglou GD, Katus HA, Korosoglou G

Abstract:
AIMS: To evaluate the incremental value of low endothelial shear stress (ESS) combined with high-resolution magnetic resonance imaging (MRI)- and computed tomography angiography (CTA)-based imaging for the prediction of inflamed plaque. METHODS AND RESULTS: Twelve hereditary hyperlipidaemic rabbits underwent quantitative analysis of plaque in the thoracic aorta with 256-slice CTA and USPIO-enhanced (ultra-small superparamagnetic nanoparticles, P904) 1.5-T MRI at baseline and at 6-month follow-up. Computational fluid dynamics using CTA-based 3D reconstruction of thoracic aortas identified the ESS patterns in the convex and concave curvature subsegments of interest. Subsegments with low baseline ESS exhibited significant increase in wall thickness and plaque inflammation by MRI, in non-calcified plaque burden by CTA, and developed increased plaque size, lipid and inflammatory cell accumulation (high-risk plaque features) at follow-up by histopathology. Multiple regression analysis identified baseline ESS and inflammation by MRI to be independent predictors of plaque progression, while receiver operating curve analysis revealed baseline ESS alone or in combination with inflammation by MRI as the strongest predictor for augmented plaque burden and inflammation (low ESS at baseline: AUC = 0.84, P < 0.001; low ESS and inflammation by molecular MRI at baseline: AUC = 0.89, P < 0.001). CONCLUSION: Low ESS predicts progression of plaque burden and inflammation as assessed by non-invasive USPIO-enhanced MRI. Combined non-invasive assessment of ESS and imaging of inflammation may serve to predict plaque with high-risk features.

Citation:
Eur Heart J Cardiovasc Imaging. 2016 Mar 24. pii: jew048. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/?term=Combined+Non-Invasive+Assessment+of+Endothelial+Shear+Stress+and+Molecular+Imaging+of+Inflammation+for+the+Prediction+of+Inflamed+Plaque+in+Hyperlipidemic+Rabbit+Aortas.

Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH)

Authors:
Atangana E, Schneider UC, Blecharz K, Magrini S, Wagner J, Nieminen-Kelhä M, Kremenetskaia I, Heppner FL, Engelhardt B, Vajkoczy P

Abstract:
Activation of innate immunity contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH). Microglia accumulation and activation within the brain has recently been shown to induce neuronal cell death after eSAH. In isolated mouse brain capillaries after eSAH, we show a significantly increased gene expression for intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Hence, we hypothesized that extracerebral intravascular inflammatory processes might initiate the previously reported microglia accumulation within the brain tissue. We therefore induced eSAH in knockout mice for ICAM-1 (ICAM-1-/-) and P-selectin glycoprotein ligand-1 (PSGL-1-/-) to find a significant decrease in neutrophil-endothelial interaction within the first 7 days after the bleeding in a chronic cranial window model. This inhibition of neutrophil recruitment to the endothelium results in significantly ameliorated microglia accumulation and neuronal cell death in knockout animals in comparison to controls. Our results suggest an outside-in activation of the CNS innate immune system at the vessel/brain interface following eSAH. Microglia cells, as part of the brain's innate immune system, are triggered by an inflammatory reaction in the microvasculature after eSAH, thus contributing to neuronal cell death. This finding offers a whole range of new research targets, as well as possible therapy options for patients suffering from eSAH.

Citation:
Transl Stroke Res. 2016 Aug 1. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27477569

Autocrine release of angiopoietin-2 mediates cerebrovascular disintegration in Moyamoya disease

Authors:
Blecharz KG, Frey D, Schenkel T, Prinz V, Bedini G, Krug SM, Czabanka M, Wagner J, Fromm M, Bersano A, Vajkoczy P

Abstract:
Moyamoya disease is a rare steno-occlusive cerebrovascular disorder often resulting in hemorrhagic and ischemic strokes. Although sharing the same ischemic stimulus with atherosclerotic cerebrovascular disease, Moyamoya disease is characterized by a highly instable cerebrovascular system which is prone to rupture due to pathological neovascularization. To understand the molecular mechanisms underlying this instability, angiopoietin-2 gene expression was analyzed in middle cerebral artery lesions obtained from Moyamoya disease and atherosclerotic cerebrovascular disease patients. Angiopoietin-2 was significantly up-regulated in Moyamoya vessels, while serum concentrations of soluble angiopoietins were not changed. For further evaluations, cerebral endothelial cells incubated with serum from these patients in vitro were applied. In contrast to atherosclerotic cerebrovascular disease serum, Moyamoya disease serum induced an angiopoietin-2 overexpression and secretion, accompanied by loss of endothelial integrity. These effects were absent or inverse in endothelial cells of non-brain origin suggesting brain endothelium specificity. The destabilizing effects on brain endothelial cells to Moyamoya disease serum were partially suppressed by the inhibition of angiopoietin-2. Our findings define brain endothelial cells as the potential source of vessel-destabilizing factors inducing the high plasticity state and disintegration in Moyamoya disease in an autocrine manner. We also provide new insights into Moyamoya disease pathophysiology that may be helpful for preventive treatment strategies in future.

Citation:
J Cereb Blood Flow Metab. 2016 Jul 5. pii: 0271678X16658301. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27381827

Resolvin D2 Enhances Post-Ischemic Revascularization While Resolving Inflammation

Authors:
Michael J. Zhang, Brian E. Sansbury, Jason Hellmann, James F. Baker, Luping Guo, Caitlin M. Parmer, Joshua C. Prenner, Daniel J. Conklin, Aruni Bhatnagar, Mark A. Creager and Matthew Spite

Abstract:
Background—Resolvins are lipid mediators generated by leukocytes during the resolution phase of inflammation. They have been shown to regulate the transition from inflammation to tissue repair; however, it is unknown whether resolvins play a role in tissue revascularization following ischemia. Methods—We used a murine model of hind limb ischemia (HLI), coupled with laser Doppler perfusion imaging, micro computed tomography (microCT) and targeted mass spectrometry, to assess the role of resolvins in revascularization and inflammation-resolution. Results—In mice undergoing HLI, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass spectrometry (n=4-7 per group). We also identified RvD2 in skeletal muscle biopsies from humans with peripheral artery disease. Monocytes were recruited to skeletal muscle during HLI and isolated monocytes produced RvD2 in a lipoxygenase-dependent manner. Exogenous RvD2 enhanced perfusion recovery in HLI and microCT of limb vasculature revealed greater volume, with evidence of tortuous arterioles indicative of arteriogenesis (n=6-8 per group). Unlike other treatment strategies for therapeutic revascularization that exacerbate inflammation, RvD2 did not increase vascular permeability, but reduced neutrophil accumulation and the plasma levels of TNF-α and GM-CSF. In mice treated with RvD2, histopathological analysis of skeletal muscle of ischemic limbs showed more regenerating myocytes with centrally located nuclei. RvD2 enhanced endothelial cell migration in a Rac-dependent manner, via its receptor, GPR18, and Gpr18-deficient mice had an endogenous defect in perfusion recovery following HLI. Importantly, RvD2 rescued defective revascularization in diabetic mice. Conclusions—RvD2 stimulates arteriogenic revascularization during HLI suggesting that resolvins may be a novel class of mediators that both resolve inflammation and promote arteriogenesis.

Citation:
Circulation. 2016 Aug 9. pii: CIRCULATIONAHA.116.021894. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2016/08/09/CIRCULATIONAHA.116.021894

MYOSLID Is a Novel Serum Response Factor-Dependent Long Noncoding RNA That Amplifies the Vascular Smooth Muscle Differentiation Program

Authors:
Zhao J, Zhang W, Lin M, Wu W, Jiang P, Tou E, Xue M, Richards A, Jourd'heuil D, Asif A, Zheng D, Singer HA, Miano JM, Long X

Abstract:
OBJECTIVE: Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is virtually unknown. APPROACH AND RESULTS: To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced smooth muscle lncRNA, inducer of differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-β/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-β1-induced SMAD2 phosphorylation. CONCLUSIONS: We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-β/SMAD pathways.

Citation:
Arterioscler Thromb Vasc Biol. 2016 Jul 21. pii: ATVBAHA.116.307879. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27444199

VEGF-A acts via PDGFRα to promote viability of cells enduring hypoxia

Authors:
Pennock S, Kim LA, Kazlauskas A

Abstract:
Vascular endothelial cells growth factor A (VEGF) is a biologically and therapeutically important growth factor because it promotes angiogenesis in response to hypoxia, which underlies a wide variety of both physiological and pathological settings. Herein we report that both VEGF receptor 2 (VEGFR2)-positive, and -negative cells depended on VEGF to endure hypoxia. VEGF enhanced the viability of platelet-derived growth factor receptor α (PDGFRα)-positive/VEGFR2-negative cells by enabling indirect activation of PDGFRα and thereby reducing the level of p53. We conclude that the breadth of VEGF's influence extends beyond VEGFR-positive cells, and propose a plausible mechanistic explanation of this phenomenon.

Citation:
Mol Cell Biol. 2016 Jun 20. pii: MCB.01019-15. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27325673

Blue Rubber Bleb Nevus (BRBN) Syndrome is caused by Somatic TEK (TIE2) Mutations

Authors:
Soblet J, Kangas J, Nätynki M, Mendola A, Helaers R, Uebelhoer M, Kaakinen M, Cordisco M, Dompmartin A, Enjolras O, Holden S, Irvine AD, Kangesu L, Léauté-Labrèze C, Lanoel A, Lokmic Z, Maas S, McAleer MA, Penington A, Rieu P, Syed S, van der Vleuten C, Watson R, Fishman SJ, Mulliken JB, Eklund L, Limaye N, Boon LM, Vikkula M.

Abstract:
Blue Rubber Bleb Nevus syndrome (Bean syndrome, BRBN) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations (VMs); gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with BRBN. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal VMs. In contrast to common unifocal VM, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, i.e., two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in BRBN, while Y897C-R915C is recurrent in sporadically occurring multifocal VM: both cause ligand-independent activation of TIE2, and increase survival, invasion and colony-formation when expressed in human umbilical vein endothelial cells (HUVECs.

Citation:
J Invest Dermatol. 2016 Aug 9. pii: S0022-202X(16)32252-7. doi: 10.1016/j.jid.2016.07.034. [Epub ahead of print]

http://www.jidonline.org/article/S0022-202X(16)32252-7/abstract

Selective serotonin reuptake inhibitor (SSRI) exposure constricts the mouse ductus arteriosus in utero

Authors:
Hooper CW, Delaney C, Streeter T, Yarboro MT, Poole SD, Brown N, Slaughter JC, Cotton RB, Reese J, Shelton EL.

Abstract:
Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN), however a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

Citation:
Am J Physiol Heart Circ Physiol. 2016 Jul 1:ajpheart.00822.2015. doi: 10.1152/ajpheart.00822.2015. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27371685

NADPH Oxidase 2 Regulates LPS-induced Inflammation and Alveolar Remodeling in the Developing Lung

Authors:
Menden HL, Xia S, Mabry SM, Navarro A, Nyp MF, Sampath V

Abstract:
Sepsis is associated with alveolar simplification manifesting as Bronchopulmonary Dysplasia (BPD) in premature infants. The redox-dependent mechanisms underlying sepsis-induced inflammation and alveolar remodeling in the immature lung remain unclear. We developed a neonatal mouse model of sepsis-induced lung injury to investigate whether NADPH Oxidase 2 (NOX2) regulates Toll-Like Receptor (TLR)-mediated inflammation and alveolar remodeling. 6d-old NOX2+/+ and NOX2-/- mice were injected with intraperitoneal lipopolysaccharide (LPS) to induce sepsis. Lung inflammation and canonical TLR signaling were assessed 24hr after LPS. Alveolar development was examined in 15d-old mice after LPS on day 6. The in vivo efficacy of a NOX2-inhibitor (NOX2-I) on NOX2 complex assembly and sepsis-induced lung inflammation were examined. Lung cytokine expression and neutrophil influx induced with sepsis in NOX2+/+ mice were decreased >50% in NOX2-/- mice. LPS-induced TLR4 signaling evident by IKK-β and MAPK phosphorylation, and nuclear NF-κB/AP-1 translocation were attenuated in NOX2-/- mice. LPS increased MMP-9, while decreasing elastin and KGF levels in NOX2+/+ mice. LPS-induced increase in MMP-9, and decrease in FGF-7 and elastin were not evident in NOX2-/- mice. LPS-induced reduction in radial alveolar counts and increased mean linear intercepts were attenuated in NOX2-/- mice. LPS-induced NOX2 assembly evident by p67phox/gp91phox co-immunoprecipitation was disrupted with NOX2-I. NOX2-I also mitigated LPS-induced cytokine expression, TLR pathway signaling, and alveolar simplification. In a mouse model of neonatal sepsis, NOX2 regulates pro-inflammatory TLR signaling and alveolar remodeling induced by a single dose of LPS. Our results provide mechanistic insight into the regulation of sepsis-induced alveolar remodeling in the developing lung.

Citation:
Am J Respir Cell Mol Biol. 2016 Jul 20. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27438994

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10

Authors:
Lin J, He S, Sun X, Franck G, Deng Y, Yang D, Haemmig S, Wara AK, Icli B, Li D, Feinberg MW

Abstract:
Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA-dependent targets in response to ligand-specific signaling in ECs.-Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Citation:
FASEB J. 2016 Jun 13. pii: fj.201500163R. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27297585

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo

Authors:
Pan J, Dinh TT, Rajaraman A, Lee M, Scholz A, Czupalla CJ, Kiefel H, Zhu L, Xia L, Morser J, Jiang H, Santambrogio L, Butcher EC

Abstract:
Circulating factor VIII (FVIII) is derived from liver and from extra-hepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 mRNA. Transcriptomic profiling of subsets of stromal cells including endothelial cells (ECs) from mouse LNs and other tissues showed that F8 mRNA is expressed by lymphatic EC (LEC) but not by capillary ECs (CapECs), fibroblastic reticular cells, or hematopoietic cells. Among blood EC profiled, F8 expression was seen only in fenestrated EC (liver sinusoidal and renal glomerular EC), and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in CapECs, but not in LEC; it was co-expressed with F8 mRNA in postcapillary high endothelial venules. Purified LEC and liver sinusoidal EC but not CapECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII:C activity. Our results reveal localized vascular expression of FVIII and vWF, and identify LECs as a major cellular source of FVIII in extra-hepatic tissues.

Citation:
Blood. 2016 May 12. pii: blood-2015-12-684688. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27207787

Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation

Authors:
Lopez-Ramirez MA, Reijerkerk A, de Vries HE, Romero IA

Abstract:
Brain endothelial cells constitute the major cellular element of the highly specialized blood-brain barrier (BBB) and thereby contribute to CNS homeostasis by restricting entry of circulating leukocytes and blood-borne molecules into the CNS. Therefore, compromised function of brain endothelial cells has serious consequences for BBB integrity. This has been associated with early events in the pathogenesis of several disorders that affect the CNS, such as multiple sclerosis, HIV-associated neurologic disorder, and stroke. Recent studies demonstrate that brain endothelial microRNAs play critical roles in the regulation of BBB function under normal and neuroinflammatory conditions. This review will focus on emerging evidence that indicates that brain endothelial microRNAs regulate barrier function and orchestrate various phases of the neuroinflammatory response, including endothelial activation in response to cytokines as well as restoration of inflamed endothelium into a quiescent state. In particular, we discuss novel microRNA regulatory mechanisms and their contribution to cellular interactions at the neurovascular unit that influence the overall function of the BBB in health and during neuroinflammation.-Lopez-Ramirez, M. A., Reijerkerk, A., de Vries, H. E., Romero, I. A. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

Citation:
FASEB J. 2016 Apr 26. pii: fj.201600435RR. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27118674

Current Understanding of the Pathways Involved in Adult Stem and Progenitor Cell Migration for Tissue Homeostasis and Repair

Authors:
Goichberg P.

Abstract:
With the advancements in the field of adult stem and progenitor cells grows the recognition that the motility of primitive cells is a pivotal aspect of their functionality. There is accumulating evidence that the recruitment of tissue-resident and circulating cells is critical for organ homeostasis and effective injury responses, whereas the pathobiology of degenerative diseases, neoplasm and aging, might be rooted in the altered ability of immature cells to migrate. Furthermore, understanding the biological machinery determining the translocation patterns of tissue progenitors is of great relevance for the emerging methodologies for cell-based therapies and regenerative medicine. The present article provides an overview of studies addressing the physiological significance and diverse modes of stem and progenitor cell trafficking in adult mammalian organs, discusses the major microenvironmental cues regulating cell migration, and describes the implementation of live imaging approaches for the exploration of stem cell movement in tissues and the factors dictating the motility of endogenous and transplanted cells with regenerative potential.

Citation:
Stem Cell Rev. 2016 May 21. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27209167

Aging impairs ischemia-induced neovascularization by attenuating the mobilization of bone marrow-derived angiogenic cells

Authors:
Yuen Ting Lam, Laura Lecce, Zoe Clayton, Philippa J.L. Simpson, Richard H. Karas, Martin K.C. Ng

Abstract:
Background: Aging is associated with impaired ischemia-induced neovascularization. However, the effects of aging on bone marrow-derived angiogenic cell (BMDAC)-mediated vasculogenesis and on angiogenesis at the ischemic sites remain incompletely understood. Methods and results: Two- and 24-month old male C57Bl/6J mice were subjected to hindlimb ischemia. The levels of Sca1 +/CXCR4 + BMDACs were determined post-ischemia by flow cytometry. In young mice, ischemia increased Sca1 +/CXCR4 + BMDAC levels in the bone marrow and spleen at day 3 (p < 0.001) and in the circulating blood at day 7 (p < 0.01) post-ischemia. However, ischemia-induced elevation of progenitor cells was attenuated in the bone marrow, spleen and blood of old mice despite a preserved HIF-1α-mediated angiogenic response in the ischemic tissues. Irradiated young recipient mice engrafted with old bone marrow displayed reduced levels of Sca1 +/CXCR4 + BMDACs in the bone marrow and circulating blood post-ischemia compared to recipients with young bone marrow. Ex vivo cultured BMDACs from old mice exhibited reduced SDF-1-stimulated migration (p < 0.01) and a decrease in JAK-2 and AKT activation. However, the intrinsic angiogenic function of BMDACs, including VEGF secretion and promotion of endothelial cell tubule formation, was preserved with aging. Furthermore, facilitated mobilization of old bone marrow-derived mononuclear cells to the ischemic hindlimb by intramuscular injection enhanced ischemia-induced neovascularization in old mice in vivo (p < 0.001). Conclusions: The age-related impairment in ischemia-induced neovascularization is largely attributable to a marked attenuation of BMDAC mobilization with a preservation of intrinsic angiogenic function with age.

Citation:
IJC Metabolic & Endocrine, Volume 12, September 2016, Pages 19–29, doi:10.1016/j.ijcme.2016.05.005

http://www.sciencedirect.com/science/article/pii/S221476241630010X

Endothelial Lipid Phosphate Phosphatase-3 Deficiency that Disrupts the Endothelial Barrier Function is a Modifier of Cardiovascular Development

Authors:
Chatterjee I, Baruah J, Lurie EE, Wary KK

Abstract:
AIMS: Lipid phosphate phosphatase-3 (LPP3) is expressed at high levels in endothelial cells (ECs). Although LPP3 is known to hydrolyse the phosphate group from lysolipids such as spingosine-1-phosphate and its structural homologues, the function of Lpp3 in ECs is not completely understood. In this study, we investigated how tyrosine-protein kinase receptor (TEK or Tie2) promoter-dependent deletion of Lpp3 alters EC activities. METHODS AND RESULTS: Lpp3fl/fl mice were crossed with the tg.Tie2Cre transgenic line. Vasculogenesis occurred normally in embryos with Tie2Cre-mediated deletion of Lpp3 (called Lpp3ECKO), but embryonic lethality occurred in two waves, the first wave between E8.5 and E10.5, while the second between E11.5 and E13.5. Lethality in Lpp3ECKO embryos after E11.5 was accompanied by vascular leakage and haemorrhage, which likely resulted in insufficient cardiovascular development. Analyses of haematoxylin- and eosin-stained heart sections from E11.5 Lpp3ECKO embryos showed insufficient heart growth associated with decreased trabeculation, reduced growth of the compact wall, and absence of cardiac cushions. Staining followed by microscopic analyses of Lpp3ECKO embryos revealed the presence of apoptotic ECs. Furthermore, Lpp3-deficient ECs showed decreased gene expression and protein levels of Cyclin-D1, VE-cadherin, Fibronectin, Klf2, and Klf4. To determine the underlying mechanisms of vascular leakage and barrier disruption, we performed knockdown and rescue experiments in cultured ECs. LPP3 knockdown decreased transendothelial electrical resistance and increased permeability. Re-expression of β-catenin cDNA in LPP3-knockdown ECs partially restored the effect of the LPP3 loss, whereas re-expression of p120ctn cDNA did not. CONCLUSION: These findings demonstrate the essential roles of LPP3 in the maturation of EC barrier integrity and normal cardiovascular development.

Citation:
Cardiovasc Res. 2016 Apr 28. pii: cvw090. [Epub ahead of print]

http://cardiovascres.oxfordjournals.org/content/early/2016/04/28/cvr.cvw090

Intracellular RIG-I Signaling Regulates TLR4-independent Endothelial Inflammatory Responses to Endotoxin

Authors:
Jill Moser, Peter Heeringa, Rianne M. Jongman, Peter J. Zwiers, Anita E. Niemarkt, Rui Yan, Inge A. de Graaf, Ranran Li, Erzso R. Regan, Philipp Kümpers, William C. Aird, Geerten P. van Nieuw Amerongen, Jan G. Zijlstra, Grietje Molema, Matijs van Meurs

Abstract:
Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid–inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB–mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis.

Citation:
J Immunol 2016 196:4681-4691; published ahead of print April 29, 2016, doi:10.4049/jimmunol.1501819

http://www.jimmunol.org/content/early/2016/04/29/jimmunol.1501819.abstract

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease

Authors:
Agapova OA, Fang Y, Sugatani T, Seifert ME, Hruska KA

Abstract:
The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

Citation:
Kidney Int. 2016 Jun;89(6):1231-43. doi: 10.1016/j.kint.2016.02.002. Epub 2016 Mar 11.

http://www.sciencedirect.com/science/article/pii/S0085253816002908

Elevated Leukocyte Azurophilic Enzymes in Human Diabetic Ketoacidosis Plasma Degrade Cerebrovascular Endothelial Junctional Proteins

Authors:
Woo MM, Patterson EK, Clarson C, Cepinskas G, Bani-Yaghoub M, Stanimirovic DB, Fraser DD

Abstract:
OBJECTIVE: Diabetic ketoacidosis in children is associated with vasogenic cerebral edema, possibly due to the release of destructive polymorphonuclear neutrophil azurophilic enzymes. Our objectives were to measure plasma azurophilic enzyme levels in children with diabetic ketoacidosis, to correlate plasma azurophilic enzyme levels with diabetic ketoacidosis severity, and to determine whether azurophilic enzymes disrupt the blood-brain barrier in vitro. DESIGN: Prospective clinical and laboratory study. SETTING: The Children's Hospital, London Health Sciences Centre. SUBJECTS: Pediatric type 1 diabetes patients; acute diabetic ketoacidosis or age-/sex-matched insulin-controlled. MEASUREMENTS AND MAIN RESULTS: Acute diabetic ketoacidosis in children was associated with elevated polymorphonuclear neutrophils. Plasma azurophilic enzymes were elevated in diabetic ketoacidosis patients, including human leukocyte elastase (p < 0.001), proteinase-3 (p < 0.01), and myeloperoxidase (p < 0.001). A leukocyte origin of human leukocyte elastase and proteinase-3 in diabetic ketoacidosis was confirmed with buffy coat quantitative real-time polymerase chain reaction (p < 0.01). Of the three azurophilic enzymes elevated, only proteinase-3 levels correlated with diabetic ketoacidosis severity (p = 0.002). Recombinant proteinase-3 applied to human brain microvascular endothelial cells degraded both the tight junction protein occludin (p < 0.05) and the adherens junction protein VE-cadherin (p < 0.05). Permeability of human brain microvascular endothelial cell monolayers was increased by recombinant proteinase-3 application (p = 0.010). CONCLUSIONS: Our results indicate that diabetic ketoacidosis is associated with systemic polymorphonuclear neutrophil activation and degranulation. Of all the polymorphonuclear neutrophil azurophilic enzymes examined, only proteinase-3 correlated with diabetic ketoacidosis severity and potently degraded the blood-brain barrier in vitro. Proteinase-3 might mediate vasogenic edema during diabetic ketoacidosis, and selective proteinase-3 antagonists may offer future vascular- and neuroprotection.

Citation:
Crit Care Med. 2016 Apr 11. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27071071

PHD2 Deficiency in Endothelial Cells and Hematopoietic Cells Induces Obliterative Vascular Remodeling and Severe Pulmonary Arterial Hypertension in Mice and Humans through HIF-2α

Authors:
Zhiyu Dai, Ming Li, John Wharton, Maggie M. Zhu, You-Yang Zhao

Abstract:
Background—Vascular occlusion and complex plexiform lesions are hallmarks of the pathology of severe pulmonary arterial hypertension (PAH) in patients. However, mechanisms of obliterative vascular remodeling remain elusive and hence current therapies have not targeted the fundamental disease modifying mechanisms and result in only modest improvement in morbidity and mortality. Methods and Results—Mice with Tie2Cre-mediated disruption of Egln1 (encoding prolyl-4 hydroxylase 2, PHD2) (Egln1Tie2) in endothelial cells (ECs) and hematopoietic cells exhibited spontaneous severe PAH with extensive pulmonary vascular remodeling including vascular occlusion and plexiform-like lesions resembling the hallmarks of the pathology of clinical PAH. As seen in idiopathic PAH patients, Egln1Tie2 mice exhibited unprecedented right ventricular hypertrophy and failure and progressive mortality. Consistently, PHD2 expression was diminished in lung ECs of obliterated pulmonary vessels in idiopathic PAH patients. Genetic deletions of both Egln1 and Hif1a or Egln1 and Hif2a identified hypoxia-inducible factor-2α (HIF-2α) as the critical mediator of severe PAH seen in Egln1Tie2 mice. We also observed altered expression of many PH-causing genes in Egln1Tie2 lungs which was also normalized in Egln1Tie2/Hif2aTie2 lungs. PHD2-deficient ECs promoted smooth muscle cell proliferation in part through HIF-2α-activated CXCL12 expression. Genetic deletion of Cxcl12 attenuated PAH in Egln1Tie2 mice. Conclusions—These studies defined an unexpected role of PHD2 deficiency in the mechanisms of severe PAH and identified the first genetically modified mouse model with obliterative vascular remodeling and pathophysiology recapitulating clinical PAH. Thus, targeting PHD2/HIF-2α signaling is a promising strategy to reverse vascular remodeling for treatment of severe PAH.

Citation:
Circulation. 2016 Apr 25. pii: CIRCULATIONAHA.116.021494. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2016/04/22/CIRCULATIONAHA.116.021494

A novel chemo-mechano-biological model of arterial tissue growth and remodelling

Authors:
Aparício, Pedro, Mark S. Thompson, and Paul N. Watton

Abstract:
Arterial growth and remodelling (G&R) is mediated by vascular cells in response to their chemical and mechanical environment. To date, mechanical and biochemical stimuli tend to be modelled separately, however this ignores their complex interplay. Here, we present a novel mathematical model of arterial chemo-mechano-biology. We illustrate its application to the development of an inflammatory aneurysm in the descending human aorta. The arterial wall is modelled as a bilayer cylindrical non-linear elastic membrane, which is internally pressurised and axially stretched. The medial degradation that accompanies aneurysm development is driven by an inflammatory response. Collagen remodelling is simulated by adaption of the natural reference configuration of constituents; growth is simulated by changes in normalised mass-densities. We account for the distribution of attachment stretches that collagen fibres are configured to the matrix and, innovatively, allow this distribution to remodel. This enables the changing functional role of the adventitia to be simulated. Fibroblast-mediated collagen growth is represented using a biochemical pathway model: a system of coupled non-linear ODEs governs the evolution of fibroblast properties and levels of key biomolecules under the regulation of Transforming Growth Factor (TGF)-β, a key promoter of matrix deposition. Given physiologically realistic targets, different modes of aneurysm development can be captured, while the predicted evolution of biochemical variables is qualitatively consistent with trends observed experimentally. Interestingly, we observe that increasing the levels of collagen-promoting TGF-β results in arrest of aneurysm growth, which seems to be consistent with experimental evidence. We conclude that this novel Chemo-Mechano-Biological (CMB) mathematical model has the potential to provide new mechanobiological insight into vascular disease progression and therapy.

Citation:
Journal of Biomechanics, DOI: http://dx.doi.org/10.1016/j.jbiomech.2016.04.037

http://www.jbiomech.com/article/S0021-9290(16)30542-5/abstract

In Vivo Functional Evaluation of Tissue Engineered Vascular Grafts Fabricated Using Human Adipose-Derived Stem Cells from High-Cardiovascular Risk Populations

Authors:
Krawiec JT, Weinbaum JS, Liao HT, Ramaswamy A, Pezzone DJ, Josowitz AD, D'Amore A, Rubin JP, Wagner WR, Vorp DA

Abstract:
Many preclinical evaluations of autologous small diameter tissue-engineered vascular grafts (TEVGs) utilize cells from healthy humans or animals. However, these models hold minimal relevance for clinical translation, as the main targeted demographic is patients at high cardiovascular risk such as individuals with diabetes mellitus or the elderly. Stem cells such as adipose-derived mesenchymal stem cells (AD-MSCs) represent a clinically ideal cell type for TEVGs, as these can be easily and plentifully harvested and offer regenerative potential. To understand whether AD-MSCs sourced from diabetic and elderly donors are as effective as those from young, non-diabetics (i.e. "healthy") in the context of TEVG therapy, we implanted TEVGs constructed with human AD-MSCs from each donor type as an aortic interposition graft in a rat model. The key failure mechanism observed was thrombosis, and this was most prevalent in grafts using cells from diabetic patients. The remainder of the TEVGs were able to generate robust vascular-like tissue consisting of smooth muscle cells, endothelial cells, collagen, and elastin. We further investigated a potential mechanism for the thrombotic failure of AD-MSCs from diabetic donors; we found that these cells have a diminished potential to promote fibrinolysis compared to those from healthy donors. Together, this study served as proof-of-concept for the development of a TEVG based on human AD-MSCs, illustrated the importance of testing cells from realistic patient populations, and highlighted one possible mechanistic explanation as to the observed thrombotic failure of our diabetic AD-MSC based-TEVGs.

Citation:
Tissue Eng Part A. 2016 Apr 14. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/27079751

Endogenous Transmembrane TNF-Alpha Protects Against Premature Senescence in Endothelial Colony Forming Cells

Authors:
Linden A Green, Victor Njoku, Julie A Mund, Jamie Case, Merv C Yoder, Michael P Murphy, and Matthias Clauss

Abstract:
Rationale: Transmembrane TNF-α (tmTNF-α) is the prime ligand for TNFR2, which has been shown to mediate angiogenic and blood vessel repair activities in mice. We have previously reported that the angiogenic potential of highly proliferative endothelial colony forming cells (ECFCs) can be explained by the absence of senescent cells, which in mature endothelial cells occupy more than 30% of the population, and that exposure to a chronic inflammatory environment induced premature, telomere-independent senescence in ECFCs. Objective: The goal of this study was to determine the role of transmembrane TNF-α in the proliferation of ECFCs. Methods and Results: Here we show that tmTNF-α expression on ECFCs selects for higher proliferative potential and when removed from the cell surface promotes ECFC senescence. Moreover, the induction of premature senescence by chronic inflammatory conditions is blocked by inhibition of tmTNF-α cleavage. Indeed, the mechanism of chronic inflammation-induced premature senescence involves an abrogation of tmTNF/TNFR2 signaling. This process is mediated by activation of the tmTNF cleavage metalloprotease TACE via p38 MAP kinase activation and its concurrent export to the cell surface by means of increased iRhom2 expression. Conclusions: Thus we conclude that tmTNF-α on the surface of highly proliferative ECFCs plays an important role in the regulation of their proliferative capacity.

Citation:
Circulation Research. CIRCRESAHA.116.308332. Published online before print April 13, 2016

http://circres.ahajournals.org/content/early/2016/04/13/CIRCRESAHA.116.308332.abstract

Smooth Muscle Enriched Long Non-Coding RNA (SMILR) Regulates Cell Proliferation

Authors:
Ballantyne MD, Pinel K, Dakin R, Vesey AT, Diver L, Mackenzie R, Garcia R, Welsh P, Sattar N, Hamilton G, Joshi N, Dweck MR, Miano JM, McBride MW, Newby DE, McDonald RA, Baker AH

Abstract:
BACKGROUND: Phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic state is implicated in diverse vascular pathologies including atherogenesis, plaque stabilisation, and neointimal hyperplasia. However, very little is known as to the role of long non coding RNA (lncRNA) during this process. Here we investigated a role for long non-coding (lnc)RNAs in VSMC biology and pathology. METHODS AND RESULTS: Using RNA-sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein (HSV) VSMCs following stimulation with IL1α and PDGF. We focused on a novel lncRNA (Ensembl: RP11-94A24.1) which we termed smooth muscle induced lncRNA enhances replication (SMILR). Following stimulation,SMILRexpression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown ofSMILRmarkedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal toSMILRwere also altered by IL1α/PDGF treatment, and HAS2 expression was reduced bySMILRknockdown. In human samples, we observed increased expression ofSMILRin unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. CONCLUSIONS: These results identifySMILRas a driver of VSMC proliferation and suggest that modulation ofSMILRmay be a novel therapeutic strategy to reduce vascular pathologies.

Citation:
Circulation. 2016 Apr 6. pii: CIRCULATIONAHA.115.021019. [Epub ahead of print]

http://circ.ahajournals.org/content/early/2016/04/06/CIRCULATIONAHA.115.021019.abstract

A role for the long non-coding RNA SENCR in commitment and function of endothelial cells

Authors:
Boulberdaa M, Scott E, Ballantyne M, Garcia R, Descamps B, Angelini GD, Brittan M, Hunter A, McBride M, McClure J, Miano JM, Emanueli C, Mills NL, Mountford JC, Baker AH

Abstract:
Despite the increasing importance of long non-coding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long non-coding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and haemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In HUVEC, SENCR induced proliferation, migration and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.

Citation:
Mol Ther. 2016 Feb 22. doi: 10.1038/mt.2016.41. [Epub ahead of print]

http://www.nature.com/mt/journal/vaop/naam/abs/mt201641a.html

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice

Authors:
Howatt DA, Balakrishnan A, Moorleghen JJ, Muniappan L, Rateri DL, Uchida HA, Takano J, Saido TC, Chishti AH, Baud L, Subramanian V

Abstract:
Objective—Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)–derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. Approach and Results—To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor−/− mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. Conclusions—The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Citation:
Arterioscler Thromb Vasc Biol. 2016 Mar 10. pii: ATVBAHA.116.307285. [Epub ahead of print]

http://atvb.ahajournals.org/content/early/2016/03/10/ATVBAHA.116.307285.abstract

Purified and recombinant hemopexin: protease activity and effect on neutrophil chemotaxis

Authors:
Lin T, Liu J, Huang F, van Engelen T, Thundivalappil SR, Riley FE, Super M, Watters AL, Smith A, Brinkman N, Ingber DE and Warren HS

Abstract:
OBJECTIVE: Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. METHODS: Amidolytic assay was performed to measure the protease activity of several plasma derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of LPS, and also injected into mice intravenously 30 minutes before inducing neutrophil migration via intraperitoneal injection of thioglycolate. Control groups received the same amount of albumin. RESULTS: Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on TNF production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. CONCLUSIONS: Hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

Citation:
Mol Med. 2016 Jan 8. doi: 10.2119/molmed.2016.00006. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26772775

Morphogenesis of 3D vascular networks is regulated by tensile forces

Authors:
Rosenfeld D, Landau S, Shandalov Y, Raindel N, Freiman A, Shor E, Blinder Y, Vandenburgh HH, Mooney DJ, Levenberg S

Abstract:
Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair.

Citation:
Proc Natl Acad Sci U S A. 2016 Mar 7. pii: 201522273. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26951667

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance

Authors:
Sachiko Nishimoto, Daiju Fukuda, Yasutomi Higashikuni, Kimie Tanaka, Yoichiro Hirata, Chie Murata, Joo-ri Kim-Kaneyama, Fukiko Sato, Masahiro Bando, Shusuke Yagi, Takeshi Soeki, Tetsuya Hayashi, Issei Imoto, Hiroshi Sakaue, Michio Shimabukuro and Masataka Sata

Abstract:
Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9−/−) macrophages. Fat-fed Tlr9−/− mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9−/− mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography–determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

Citation:
Science Advances 25 Mar 2016:Vol. 2, no. 3, e1501332, DOI: 10.1126/sciadv.1501332

http://advances.sciencemag.org/content/2/3/e1501332

Decreased blood vessel leakage can improve cancer therapy and reduce tumour spread

Authors:
Xiujuan Li, Narendra Padhan, Elisabet O. Sjostrom, Francis P. Roche, Chiara Testini, Naoki Honkura, Miguel Sainz-Jaspeado, Emma Gordon, Katie Bentley, Andrew Philippides, Vladimir Tolmachev, Elisabetta Dejana, Radu V. Stan, Dietmar Vestweber, Kurt Ballmer-Hofer, Christer Betsholtz, Kristian Pietras, Leif Jansson & Lena Claesson-Welsh

Abstract:
Cancer therapy is often hampered by the accumulation of fluids in and around the tumor, which is caused by leakage from the blood vessels in the tumor. Researchers now show how leakage from blood vessels is regulated. They have identified a novel mechanism whereby leakage can be suppressed to improve the result of chemotherapy and reduce the spread of tumors in mice.

Citation:
Nature Communications

http://www.mynewsdesk.com/uk/uu/pressreleases/decreased-blood-vessel-leakage-can-improve-cancer-therapy-and-reduce-tumour-spread-1328482?utm_campaign=send_list&utm_medium=email&utm_source=sendgrid

Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling

Authors:
Franco, C.A., M.L. Jones, M.O. Bernabeu, A.C. Vion, P. Barbacena, J. Fan, T. Mathivet, C.G. Fonseca, A. Ragab, T.P. Yamaguchi, P.V. Coveney, R.A. Lang, and H. Gerhardt

Abstract:
Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.

Citation:
Elife. 2016 Feb 4;5. pii: e07727. doi: 10.7554/eLife.07727

http://elifesciences.org/content/5/e07727v2

Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo

Authors:
Gebala, V., R. Collins, I. Geudens, L.K. Phng, and H. Gerhardt

Abstract:
How vascular tubes build, maintain and adapt continuously perfused lumens to meet local metabolic needs remains poorly understood. Recent studies showed that blood flow itself plays a critical role in the remodelling of vascular networks1, 2, and suggested it is also required for the lumenization of new vascular connections3, 4. However, it is still unknown how haemodynamic forces contribute to the formation of new vascular lumens during blood vessel morphogenesis. Here we report that blood flow drives lumen expansion during sprouting angiogenesis in vivo by inducing spherical deformations of the apical membrane of endothelial cells, in a process that we have termed inverse blebbing. We show that endothelial cells react to these membrane intrusions by local and transient recruitment and contraction of actomyosin, and that this mechanism is required for single, unidirectional lumen expansion in angiogenic sprouts. Our work identifies inverse membrane blebbing as a cellular response to high external pressure. We show that in the case of blood vessels such membrane dynamics can drive local cell shape changes required for global tissue morphogenesis, shedding light on a pressure-driven mechanism of lumen formation in vertebrates.

Citation:
Nat Cell Biol. 2016 Feb 29. doi: 10.1038/ncb3320. [Epub ahead of print]

http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3320.html

A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance

Authors:
Jang C, Oh SF, Wada S, Rowe GC, Liu L, Chan MC, Rhee J, Hoshino A, Kim B, Ibrahim A, Baca LG, Kim E, Ghosh CC, Parikh SM, Jiang A, Chu Q, Forman DE, Lecker SH, Krishnaiah S, Rabinowitz JD, Weljie AM, Baur JA, Kasper DL, Arany Z

Abstract:
Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1α; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1α to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.

Citation:
Nat Med. 2016 Mar 7. doi: 10.1038/nm.4057. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/26950361

Relaxin-2 and Soluble Flt1 Levels in Peripartum Cardiomyopathy: Results of the Multicenter IPAC Study

Authors:
Damp J, Givertz MM, Semigran M, Alharethi R, Ewald G, Felker GM, Bozkurt B, Boehmer J, Haythe J, Skopicki H, Hanley-Yanez K, Pisarcik J, Halder I, Gorcsan J 3rd, Rana S, Arany Z, Fett JD, McNamara DM; IPAC Investigators

Abstract:
OBJECTIVES: This study explored the association of vascular hormones with myocardial recovery and clinical outcomes in peripartum cardiomyopathy (PPCM). BACKGROUND: PPCM is an uncommon disorder with unknown etiology. Angiogenic imbalance may contribute to its pathophysiology. METHODS: In 98 women with newly diagnosed PPCM enrolled in the Investigation in Pregnancy Associated Cardiomyopathy study, serum was obtained at baseline for analysis of relaxin-2, prolactin, soluble fms-like tyrosine kinase 1 (sFlt1), and vascular endothelial growth factor (VEGF). Left ventricular ejection fraction (LVEF) was assessed by echocardiography at baseline and 2, 6, and 12 months. RESULTS: Mean age was 30 ± 6 years, with a baseline of LVEF 0.35 ± 0.09. Relaxin-2, prolactin, and sFlt1 were elevated in women presenting early post-partum, but decreased rapidly and were correlated inversely with time from delivery to presentation. In tertile analysis, higher relaxin-2 was associated with smaller left ventricular systolic diameter (p = 0.006) and higher LVEF at 2 months (p = 0.01). This was particularly evident in women presenting soon after delivery (p = 0.02). No relationship was evident for myocardial recovery and prolactin, sFlt1 or VEGF levels. sFlt1 levels were higher in women with higher New York Heart Association functional class (p = 0.01) and adverse clinical events (p = 0.004). CONCLUSIONS: In women with newly diagnosed PPCM, higher relaxin-2 levels soon after delivery were associated with myocardial recovery at 2 months. In contrast, higher sFlt1 levels correlated with more severe symptoms and major adverse clinical events. Vascular mediators may contribute to the development of PPCM and influence subsequent myocardial recovery.

Citation:
JACC Heart Fail. 2016 Feb 27. pii: S2213-1779(16)00018-4. doi: 10.1016/j.jchf.2016.01.004. [Epub ahead of print]

https://www.ncbi.nlm.nih.gov/pubmed/26970832

Tau pathology-dependent remodelling of cerebral arteries precedes Alzheimer’s disease-related microvascular cerebral amyloid angiopathy

Authors:
Mario Merlini, Debora Wanner and Roger M. Nitsch

Abstract:
Alzheimer’s disease (AD) is characterised by pathologic cerebrovascular remodelling. Whether this occurs already before disease onset, as may be indicated by early Braak tau-related cerebral hypoperfusion and blood–brain barrier (BBB) impairment found in previous studies, remains unknown. Therefore, we systematically quantified Braak tau stage- and cerebral amyloid angiopathy (CAA)-dependent alterations in the alpha-smooth muscle actin (α-SMA), collagen, and elastin content of leptomeningeal arterioles, small arteries, and medium-sized arteries surrounding the gyrus frontalis medialis (GFM) and hippocampus (HIPP), including the sulci, of 17 clinically and pathologically diagnosed AD subjects (Braak stage IV–VI) and 28 non-demented control subjects (Braak stage I–IV). GFM and HIPP paraffin sections were stained for general collagen and elastin with the Verhoeff–van Gieson stain; α-SMA and CAA/amyloid β (Aβ) were detected using immunohistochemistry. Significant arterial elastin degradation was observed from Braak stage III onward and correlated with Braak tau pathology (ρ = 0.909, 95 % CI 0.370 to 0.990, p < 0.05). This was accompanied by an increase in neutrophil elastase expression by α-SMA-positive cells in the vessel wall. Small and medium-sized arteries exhibited significant CAA-independent α-SMA loss starting between Braak stage I and II–III, along with accumulation of phosphorylated paired helical filament (PHF) tau in the perivascular space of intraparenchymal vessels. α-SMA remained at the decreased level throughout the later Braak stages. In contrast, arterioles exhibited significant α-SMA loss only at Braak stage V and VI/in AD subjects, which was CAA-dependent/correlated with CAA burden (ρ = −0.422, 95 % CI −0.557 to −0.265, p < 0.0001). Collagen content was only significantly changed in small arteries. Our data indicate that vessel wall remodelling of leptomeningeal arteries is an early-onset, Braak tau pathology-dependent process unrelated to CAA and AD, which potentially may contribute to downstream CAA-dependent microvascular pathology in AD.

Citation:
Acta Neuropathol. 2016 Mar 17. [Epub ahead of print]

http://link.springer.com/article/10.1007%2Fs00401-016-1560-2

Temporal modulation of collective cell behavior controls vascular network topology

Authors:
Kur E, Kim J, Tata A, Comin CH, Harrington KI, Costa LD, Bentley K, Gu C

Abstract:
Vascular network density determines the amount of oxygen and nutrients delivered to host tissues, but how the vast diversity of densities is generated is unknown. Reiterations of endothelial-tip-cell selection, sprout extension and anastomosis are the basis for vascular network generation, a process governed by the VEGF/Notch feedback loop. Here, we find that temporal regulation of this feedback loop, a previously unexplored dimension, is the key mechanism to determine vascular density. Iterating between computational modeling and in vivo live imaging, we demonstrate that the rate of tip-cell selection determines the length of linear sprout extension at the expense of branching, dictating network density. We provide the first example of a host tissue-derived signal (Semaphorin3E-Plexin-D1) that accelerates tip cell selection rate, yielding a dense network. We propose that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators may exist to sculpt vascular topology.

Citation:
Elife. 2016 Feb 24;5. pii: e13212. doi: 10.7554/eLife.13212.

https://elifesciences.org/content/5/e13212v2/article-info

Hypertension: The Missing WNKs

Authors:
Hashem A Dbouk, Chou-Long Huang, Melanie H Cobb

Abstract:
The With no Lysine [K] (WNK) family of enzymes are central in regulation of blood pressure. WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight not only into the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. Defining the roles of WNKs in in the cardiovascular system will provide clues into additional mechanisms by which WNKs can regulate blood pressure. This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.

Citation:
American Journal of Physiology - Renal Physiology Published 23 March 2016 Vol. no. , DOI: 10.1152/ajprenal.00358.2015

http://ajprenal.physiology.org/content/early/2016/03/17/ajprenal.00358.2015

Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis

Authors:
Rahman HN, Wu H, Dong Y, Pasula S, Wen A, Sun Y, Brophy ML, Tessneer KL, Cai X, McManus J, Chang B, Kwak S, Rahman NS, Xu W, Fernandes C, Mcdaniel JM, Xia L, Smith L, Srinivasan S, Chen H

Abstract:
RATIONALE: We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE: The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS: Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Citation:
Circ Res. 2016 Mar 18;118(6):957-69. doi: 10.1161/CIRCRESAHA.115.307679. Epub 2016 Feb 15.

http://circres.ahajournals.org/content/early/2016/02/12/CIRCRESAHA.115.307679.abstract.html?ijkey=1fRITlUNqwwuYnI&keytype=ref

MicroRNA-181b Improves Glucose Homeostasis and Insulin Sensitivity by Regulating Endothelial Function in White Adipose Tissue

Authors:
Sun X, Lin J, Zhang Y, Kang S, Belkin N, Wara AK, Icli B, Hamburg NM, Li D, Feinberg MW

Abstract:
RATIONALE: The pathogenesis of insulin resistance involves dysregulated gene expression and function in multiple cell types, including endothelial cells (ECs). Post-transcriptional mechanisms such as microRNA-mediated regulation of gene expression could affect insulin action by modulating EC function. OBJECTIVE: To determine whether microRNA-181b (miR-181b) affects the pathogenesis of insulin resistance by regulating EC function in white adipose tissue during obesity. METHODS AND RESULTS: MiR-181b expression was reduced in adipose tissue ECs of obese mice, and rescue of miR-181b expression improved glucose homeostasis and insulin sensitivity. Systemic intravenous delivery of miR-181b robustly accumulated in adipose tissue ECs, enhanced insulin-mediated Akt phosphorylation at Ser473, and reduced endothelial dysfunction, an effect that shifted macrophage polarization toward an M2 anti-inflammatory phenotype in epididymal white adipose tissue. These effects were associated with increased endothelial nitric oxide synthase and FoxO1 phosphorylation as well as nitric oxide activity in epididymal white adipose tissue. In contrast, miR-181b did not affect insulin-stimulated Akt phosphorylation in liver and skeletal muscle. Bioinformatics and gene profiling approaches revealed that Pleckstrin homology domain leucine-rich repeat protein phosphatase, a phosphatase that dephosphorylates Akt at Ser473, is a novel target of miR-181b. Knockdown of Pleckstrin homology domain leucine-rich repeat protein phosphatase increased Akt phosphorylation at Ser473 in ECs, and phenocopied miR-181b's effects on glucose homeostasis, insulin sensitivity, and inflammation of epididymal white adipose tissue in vivo. Finally, ECs from diabetic subjects exhibited increased Pleckstrin homology domain leucine-rich repeat protein phosphatase expression. CONCLUSIONS: Our data underscore the importance of adipose tissue EC function in controlling the development of insulin resistance. Delivery of miR-181b or Pleckstrin homology domain leucine-rich repeat protein phosphatase inhibitors may represent a new therapeutic approach to ameliorate insulin resistance by improving adipose tissue endothelial Akt-endothelial nitric oxide synthase-nitric oxide signaling.

Citation:
Circ Res. 2016 Mar 4;118(5):810-21. doi: 10.1161/CIRCRESAHA.115.308166. Epub 2016 Jan 7.

http://circres.ahajournals.org/content/118/5/810

NRP1 function and targeting in neurovascular development and eye disease

Authors:
Raimondi C, Brash JT, Fantin A, Ruhrberg C

Abstract:
Neuropilin 1 (NRP1) is expressed by neurons, blood vessels, immune cells and many other cell types in the mammalian body and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function. In recent years, several types of mouse knockout models have been developed that have provided useful tools for experimental investigation of NRP1 function, and a multitude of therapeutics targeting NRP1 have been designed, mostly with the view to explore them for cancer treatment. This review provides a general overview of current knowledge of the signalling pathways that are modulated by NRP1, with particular focus on neuronal and vascular roles in the brain and retina. This review will also discuss the potential of NRP1 inhibitors for the treatment for neovascular eye diseases.

Citation:
Prog Retin Eye Res. 2016 Feb 27. pii: S1350-9462(16)30006-4. doi: 10.1016/j.preteyeres.2016.02.003. [Epub ahead of print]

http://www.sciencedirect.com/science/article/pii/S1350946216300064

Endothelial cell survival during angiogenesis requires the pro-survival protein MCL1

Authors:
Watson EC, Whitehead L, Adams RH, Dewson G, Coultas L

Abstract:
Angiogenesis is essential to match the size of blood vessel networks to the metabolic demands of growing tissues. While many genes and pathways necessary for regulating angiogenesis have been identified, those responsible for endothelial cell (EC) survival during angiogenesis remain largely unknown. We have investigated the in vivo role of myeloid cell leukemia 1 (MCL1), a pro-survival member of the BCL2 family, in EC survival during angiogenesis. EC-specific deletion of Mcl1 resulted in a dose-dependent increase in EC apoptosis in the angiogenic vasculature and a corresponding decline in vessel density. Our results suggest this apoptosis was independent of the BH3-only protein BIM. Despite the known link between apoptosis and blood vessel regression, this was not the cause of reduced vessel density observed in the absence of endothelial MCL1. Rather, the reduction in vessel density was linked to ectopic apoptosis in regions of the angiogenic vasculature where EC proliferation and new vessel growth occurs. We have therefore identified MCL1 as an essential survival factor for ECs that is required for blood vessel production during angiogenesis.

Citation:
Cell Death Differ. 2016 Mar 4. doi: 10.1038/cdd.2016.20. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26943318

Restoration of lymphatic function rescues obesity in Prox1-haploinsufficient mice

Authors:
Noelia Escobedo, Steven T. Proulx, Sinem Karaman, Miriam E. Dillard, Nicole Johnson, Michael Detmar, and Guillermo Oliver

Abstract:
Prox1 heterozygous mice have a defective lymphatic vasculature and develop late-onset obesity. Chyle abnormally leaks from those vessels, accumulates in the surrounding tissues, and causes an increase in adipose tissue. We characterized the lymphatics of Prox1+/– mice to determine whether the extent of obesity correlated with the severity of lymphatic defects. The lymphatic vasculature in Prox1+/– mice exhibited reduced tracer clearance from the ear skin, dysfunctional perfusion of the lower legs, and reduced tracer uptake into the deep lymphatic collectors during mechanostimulation prior to the onset of obesity. Ear lymphatic vessels and leg collectors in Prox1+/– mice were disorganized and irregular, further confirming that defective lymphatic vessels are associated with obesity in Prox1+/– mice. We now provide conclusive in vivo evidence that demonstrates that leaky lymphatics mediate obesity in Prox1+/– mice, as restoration of lymphatic vasculature function was sufficient to rescue the obesity features in Prox1+/– mice. Finally, depth-lipomic profiling of lymph contents showed that free fatty acids induce adipogenesis in vitro.

Citation:
Citation Information: JCI Insight. 2016;1(2):e85096. doi:10.1172/jci.insight.85096

http://insight.jci.org/articles/view/85096

Integrin β3 inhibition is a therapeutic strategy for supravalvular aortic stenosis

Authors:
Misra A, Sheikh AQ, Kumar A, Luo J, Zhang J, Hinton RB, Smoot L, Kaplan P, Urban Z, Qyang Y, Tellides G, Greif DM

Abstract:
The aorta is the largest artery in the body, yet processes underlying aortic pathology are poorly understood. The arterial media consists of circumferential layers of elastic lamellae and smooth muscle cells (SMCs), and many arterial diseases are characterized by defective lamellae and excess SMCs; however, a mechanism linking these pathological features is lacking. In this study, we use lineage and genetic analysis, pharmacological inhibition, explant cultures, and induced pluripotent stem cells (iPSCs) to investigate supravalvular aortic stenosis (SVAS) patients and/or elastin mutant mice that model SVAS. These experiments demonstrate that multiple preexisting SMCs give rise to excess aortic SMCs in elastin mutants, and these SMCs are hyperproliferative and dedifferentiated. In addition, SVAS iPSC-derived SMCs and the aortic media of elastin mutant mice and SVAS patients have enhanced integrin β3 levels, activation, and downstream signaling, resulting in SMC misalignment and hyperproliferation. Reduced β3 gene dosage in elastin-null mice mitigates pathological aortic muscularization, SMC misorientation, and lumen loss and extends survival, which is unprecedented. Finally, pharmacological β3 inhibition in elastin mutant mice and explants attenuates aortic hypermuscularization and stenosis. Thus, integrin β3-mediated signaling in SMCs links elastin deficiency and pathological stenosis, and inhibiting this pathway is an attractive therapeutic strategy for SVAS.

Citation:
J Exp Med. 2016 Feb 8. pii: jem.20150688. [Epub ahead of print]

http://jem.rupress.org/content/early/2016/02/03/jem.20150688.abstract

Gentiana lutea exerts anti-atherosclerotic effects by preventing endothelial inflammation and smooth muscle cell migration

Authors:
R. Kesavan, S. Chandel, S. Upadhyay, R. Bendre, R. Ganugula, U.R. Potunuru, H. Giri, G. Sahu, P. Uday Kumar, G. B. Reddy, G. Joksic, A.K. Bera and M. Dixit

Abstract:
Background and aims: Studies suggest that Gentiana lutea (GL), and its component isovitexin, may exhibit anti-atherosclerotic properties. In this study we sought to investigate the protective mechanism of GL aqueous root extract and isovitexin on endothelial inflammation, smooth muscle cell migation, and on the onset and progression of atherosclerosis in streptozotocin (STZ)-induced diabetic rats. Methods and results:Our results show that both GL extract and isovitexin, block leukocyte adhesion and generation of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells (RASMCs), following TNF-alpha and platelet derived growth factor-BB (PDGF-BB) challenges respectively. Both the extract and isovitexin blocked TNF-α induced expression of ICAM-1 and VCAM-1 in HUVECs. PDGF-BB induced migration of RASMCs and phospholipase C-γ activation, were also abrogated by GL extract and isovitexin. Fura-2 based ratiometric measurements demonstrated that, both the extact, and isovitexin, inhibit PDGF-BB mediated intracellular calcium rise in RASMCs. Supplementation of regular diet with 2% GL root powder for STZ rats, reduced total cholesterol in blood. Oil Red O staining demonstrated decreased lipid accumulation in aortic wall of diabetic animals upon treatment with GL. Medial thickness and deposition of collagen in the aortic segment of diabetic rats were also reduced upon supplementation. Immunohistochemistry demonstrated reduced expression of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and vascular endothelial cadherin (VE-cadherin) in aortic segments of diabetic rats following GL treatment. Conclusions:Thus, our results support that GL root extract/powder and isovitexin exhibit anti-atherosclerotic activities.

Citation:
Nutr Metab Cardiovasc Dis. 2016 Jan 6. pii: S0939-4753(15)00264-1. doi: 10.1016/j.numecd.2015.12.016. [Epub ahead of print]

http://www.sciencedirect.com/science/article/pii/S0939475315002641

Recent insights into endothelial control of leukocyte extravasation

Authors:
Hordijk PL

Abstract:
In the process of leukocyte migration from the circulation across the vascular wall, the crosstalk with endothelial cells that line the blood vessels is essential. It is now firmly established that in endothelial cells important signaling events are initiated upon leukocyte adhesion that impinge on the regulation of cell-cell contact and control the efficiency of transendothelial migration. In addition, several external factors such as shear force and vascular stiffness were recently identified as important regulators of endothelial signaling and, consequently, leukocyte transmigration. Here, I review recent insights into endothelial signaling events that are linked to leukocyte migration across the vessel wall. In this field, protein phosphorylation and Rho-mediated cytoskeletal dynamics are still widely studied using increasingly sophisticated mouse models. In addition, activation of tyrosine phosphatases, changes in endothelial cell stiffness as well as different vascular beds have all been established as important factors in endothelial signaling and leukocyte transmigration. Finally, I address less-well-studied but interesting components in the endothelium that also control transendothelial migration, such as the ephrins and their Eph receptors, that provide novel insights in the complexity associated with this process.

Citation:
Cell Mol Life Sci. 2016 Jan 21. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26794844

Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress

Authors:
Christopher G. Scheitlin, Justin A. Julian, Santhanam Shanmughapriya, Muniswamy Madesh, Nikolaos M Tsoukias, B. Rita Alevriadou

Abstract:
Shear stress is known to stimulate an intracellular free calcium concentration ([Ca2+]i) response in vascular endothelial cells (ECs). [Ca2+]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca2+]i response is, in part, due to Ca2+ release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca2+ store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca2+]i in sheared ECs. Cultured ECs, loaded with a Ca2+-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca2+]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs and oscillation frequencies depended on the shear level. [Ca2+]i transients/oscillations were present when experiments were conducted in Ca2+-free solution (plus lanthanum), but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca2+]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca2+ uniporter also inhibited the shear-induced [Ca2+]i transients/oscillations compared to controls. Hence, EC mitochondria, through Ca2+ uptake/release, regulate the temporal profile of shear-induced ER Ca2+ release. [Ca2+]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca2+]i response.

Citation:
Am J Physiol Cell Physiol. 2016 Jan 6:ajpcell.00171.2015. doi: 10.1152/ajpcell.00171.2015. [Epub ahead of print]

http://ajpcell.physiology.org/content/early/2016/01/04/ajpcell.00171.2015

Age-dependent impact of CaV 3.2 T-type calcium channel deletion on myogenic tone and flow-mediated vasodilatation in small arteries

Authors:
Mikkelsen MF, Björling K, Jensen LJ

Abstract:
The myogenic response and flow-mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. CaV 3.2 T-type calcium channels are expressed in both VSMCs and ECs of small arteries. The T-type channels are important drug targets but due to the lack of specific antagonists our understanding of the role of CaV 3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow-mediated vasodilatation, structural remodeling, and mRNA + protein expression in small mesenteric arteries from CaV 3.2 knock-out vs. wild-type mice at young vs. mature adult age. In young mice, only, deletion of CaV 3.2 led to enhanced myogenic response and ∼50 % reduction of flow-mediated vasodilatation. Ni2+ had both CaV 3.2-dependent and -independent effects. No changes in mRNA expression of several important K+ and Ca2+ channel genes were induced by CaV 3.2 knock-out. However, the expression of the other T-type channel isoform (CaV 3.1) was reduced at the mRNA and protein level in mature adult compared to young WT arteries. Our study shows important roles of the CaV 3.2 T-type calcium channels in myogenic tone and flow-mediated vasodilation that disappear with aging. Since increased arterial tone is a risk factor for cardiovascular disease we conclude that CaV 3.2 channels, by modulating pressure- and flow-mediated vasomotor responses to prevent excess arterial tone, protect against cardiovascular disease.

Citation:
J Physiol. 2016 Jan 11. doi: 10.1113/JP271470. [Epub ahead of print]

http://onlinelibrary.wiley.com/doi/10.1113/JP271470/full

LDL-Related Intolerance to Glucose, Diastolic Hypertension and Additive Effects of Smoking Were Found with Three Female Study Groups

Authors:
Ruth-Maria Korth

Abstract:
Initial prodiabetic risk profiles were invented here with three female study groups consisting of primarily healthy women (A1: 1990-1999, n = 160; A2: 2009, n = 88; A: n = 248, 36 ± 14 years; B: 2014: n = 65, aged 37± 11 years). Significantly higher blood pressure was found comparing intolerance versus tolerance to glucose (p < 0.05, IGTT, 22 of 68). High LDL-cholesterol (LDL-C) showed additive effects as LDL-related intolerance was further related with rise of blood pressure (p < 0.05), of triglycerides (p = 0.02), of fasting blood glucose (p = 0.07) and of urine pathology (p = 0.07). High LDL-C of women who reported smoking at baseline was correlated with diastolic hypertension whereby alcohol problems overlapped (p = 0.036, A). Unhealthy combinations were found consisting of LDL-related intolerance to glucose, LDL-related smoking, of alcohol-related hypertriglyceridemia or of combined drinking and smoking testing urine pathology over the course of time. Obese women were at direct risk for hypertension in the presence of high LDL-C and submaximal ratio of serum albumin to triglycerides (Alb/Trig). Obese women reacted highly sensitive to critical alcohol consumption showing then macroalbuminuria. Current participants who disowned daily alcohol consumption showed healthy morning urines and normal fasting blood glucose. Mild decrease of HDL-C was observed during heavy smoking of relatively young women who had normal biomarkers. Women with intolerance to glucose were at direct risk for hypertension whereby high LDL-C and/or smoking triggered prodiabetic risk profiles. Obese women had elevated LDL-C during hypertension and reacted highly sensitive to alcohol-related proteinuria and/or hematuria.

Citation:
Scientific Research, Vol.8 No.3, February 2016, DOI: 10.4236/health.2016.83026

http://www.scirp.org/Journal/PaperInformation.aspx?PaperID=63584

Angiocrine functions of organ-specific endothelial cells

Authors:
Shahin Rafii, Jason M. Butler & Bi-Sen Ding

Abstract:
Endothelial cells that line capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establishes specialized vascular niches that deploy sets of growth factors, known as angiocrine factors. These cues participate actively in the induction, specification, patterning and guidance of organ regeneration, as well as in the maintainance of homeostasis and metabolism. When upregulated following injury, they orchestrate self-renewal and differentiation of tissue-specific resident stem and progenitor cells into functional organs. Uncovering the mechanisms by which organotypic endothelium distributes physiological levels of angiocrine factors both spatially and temporally will lay the foundation for clinical trials that promote organ repair without scarring.

Citation:
Nature 529, 316–325 (21 January 2016) doi:10.1038/nature17040

http://www.nature.com/nature/journal/v529/n7586/full/nature17040.html

The glycocalyx and its significance in human medicine

Authors:
J. M. Tarbell and L. M. Cancel

Abstract:
Cells are covered by a surface layer of glycans that is referred to as the ‘glycocalyx’. In this review, we focus on the role of the glycocalyx in vascular diseases (atherosclerosis, stroke, hypertension, kidney disease and sepsis) and cancer. The glycocalyx and its principal glycosaminoglycans [heparan sulphate (HS) and hyaluronic acid (HA)] and core proteins (syndecans and glypicans) are degraded in vascular diseases, leading to a breakdown of the vascular permeability barrier, enhanced access of leucocytes to the arterial intima that propagate inflammation and alteration of endothelial mechanotransduction mechanisms that protect against disease. By contrast, the glycocalyx on cancer cells is generally robust, promoting integrin clustering and growth factor signalling, and mechanotransduction of interstitial flow shear stress that is elevated in tumours to upregulate matrix metalloproteinase release which enhances cell motility and metastasis. HS and HA are consistently elevated on cancer cells and are associated with tumour growth and metastasis. Later, we will review the agents that might be used to enhance or protect the glycocalyx to combat vascular disease, as well as a different set of compounds that can degrade the cancer cell glycocalyx to suppress cell growth and metastasis. It is clear that what is beneficial for either vascular disease or cancer will not be so for the other. The overarching conclusions are that (i) the importance of the glycocalyx in human medicine is only beginning to be recognized, and (ii) more detailed studies of glycocalyx involvement in vascular diseases and cancer will lead to novel treatment modalities.

Citation:
Journal of Internal Medicine, Article first published online: 8 JAN 2016, DOI: 10.1111/joim.12465

http://onlinelibrary.wiley.com/doi/10.1111/joim.12465/abstract

Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques

Authors:
Hutcheson JD, Goettsch C, Bertazzo S, Maldonado N, Ruiz JL, Goh W, Yabusaki K, Faits T, Bouten C, Franck G, Quillard T, Libby P, Aikawa M, Weinbaum S, Aikawa E

Abstract:
Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification areas. We also show that calcification morphology and the plaque's collagen content-two determinants of atherosclerotic plaque stability-are interlinked.

Citation:
Nat Mater. 2016 Jan 11. doi: 10.1038/nmat4519. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26752654

Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling

Authors:
Basu S, Proweller A

Abstract:
Smooth muscle myosin light chain kinase (SM-MLCK) is the key enzyme responsible for phosphorylation of regulatory myosin light chain (MLC20), resulting in actin-myosin cross-bridging and force generation in vascular smooth muscle required for physiological vasoreactivity and blood pressure control. In this study, we investigated the combinatorial role of myocardin/serum response factor (SRF) and Notch signaling in the transcriptional regulation of MLCK gene expression. Promoter reporter analyses in rat A10 smooth muscle cells revealed a bimodal pattern of MLCK promoter activity and gene expression upon stimulation with constitutively active Notch1 in presence of myocardin or by Jagged1 ligand stimulation. An initial Notch1-induced increase in MLCK transcription was followed by loss in promoter sensitivity, which could be restored with further Notch1 dose escalation. Real-time PCR analyses revealed that endogenous levels of Hairy Related Transcription (HRT) factor 2 (HRT2) peaked concurrently with inhibitory concentrations of Notch1. Forced expression of HRT2 demonstrated simultaneous repression of both myocardin- and Notch1-induced MLCK promoter activity. HRT2-mediated repression was further confirmed by HRT2 truncations and siHRT2 treatments that rescued MLCK promoter activity and gene expression. Chromatin immunoprecipitation studies revealed both Jagged1 ligand- and Notch1-enhanced myocardin/SRF complex formation at the promoter CArG element. In contrast, heightened levels of HRT2 concomitantly disrupted myocardin/SRF and Notch transcription complex formation at respective CArG and CSL binding elements. Taken together, SM-MLCK promoter activity appears highly sensitive to the relative levels of Notch1 signaling, HRT2, and myocardin. These findings identify a novel Notch-dependent HRT2 autoregulatory circuit coordinating transcriptional regulation of SM-MLCK.

Citation:
J Biol Chem. 2015 Dec 24. pii: jbc.M115.679803. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26703474

Intravascular fibrin molecular imaging improves the detection of unhealed stents assessed by optical coherence tomography in vivo

Authors:
Hara T, Ughi GJ, McCarthy JR, Erdem SS, Mauskapf A, Lyon SC, Fard AM, Edelman ER, Tearney GJ, Jaffer FA.

Abstract:
Aims Fibrin deposition and absent endothelium characterize unhealed stents that are at heightened risk of stent thrombosis. Optical coherence tomography (OCT) is increasingly used for assessing stent tissue coverage as a measure of healed stents, but cannot precisely identify whether overlying tissue represents physiological neointima. Here we assessed and compared fibrin deposition and persistence on bare metal stent (BMS) and drug-eluting stent (DES) using near-infrared fluorescence (NIRF) molecular imaging in vivo, in combination with simultaneous OCT stent coverage. Methods and results Rabbits underwent implantation of one BMS and one DES without overlap in the infrarenal aorta (N = 20 3.5 × 12 mm). At Days 7 and/or 28, intravascular NIRF-OCT was performed following the injection of fibrin-targeted NIRF molecular imaging agent FTP11-CyAm7. Intravascular NIRF-OCT enabled high-resolution imaging of fibrin overlying stent struts in vivo, as validated by histopathology. Compared with BMS, DES showed greater fibrin deposition and fibrin persistence at Days 7 and 28 (P < 0.01 vs. BMS). Notably, for edge stent struts identified as covered by OCT on Day 7, 92.8 ± 9.5% of DES and 55.8 ± 23.6% of BMS struts were NIRF fibrin positive (P < 0.001). At Day 28, 18.6 ± 10.6% (DES) and 5.1 ± 8.7% (BMS) of OCT-covered struts remained fibrin positive (P < 0.001). Conclusion Intravascular NIRF fibrin molecular imaging improves the detection of unhealed stents, using clinically translatable technology that complements OCT. A sizeable percentage of struts deemed covered by OCT are actually covered by fibrin, particularly in DES, and therefore such stents might remain prothrombotic. These findings have implications for the specificity of standalone clinical OCT assessments of stent healing.

Citation:
Eur Heart J, DOI: http://dx.doi.org/10.1093/eurheartj/ehv677 ehv677 First published online: 18 December 2015

http://eurheartj.oxfordjournals.org/content/early/2015/12/17/eurheartj.ehv677

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

Authors:
Andreas M. Beyer, Julie K. Freed, Matthew J. Durand, Michael Riedel, Karima Ait-Aissa, Paula Green, Joseph C. Hockenberry, R. Garret Morgan, Anthony J. Donato, Refael Peleg, Mario Gasparri, Chris K. Rokkas, Janine H. Santos, Esther Priel, David D. Gutterman

Abstract:
Rationale: Telomerase is a nuclear regulator of telomere elongation with recent reports suggesting a role in regulation of mitochondrial reactive oxygen species (mtROS). Since mtROS (H2O2) mediate flow-mediated dilation (FMD) in the human microcirculation. Objective: We examined the hypothesis that telomerase activity modulates microvascular FMD and loss of telomerase activity contributes to the change of mediator from nitric oxide (NO) to mtH2O2 in patients with coronary artery disease (CAD). Methods and Results: Human coronary and adipose arterioles were isolated for videomicroscopy. FMD was measured in vessels pre-treated with the telomerase inhibitor BIBR-1532 or vehicle. Statistical differences between groups were determined using a two-way ANOVA (n≥4; P<0.05). L-NAME (NO synthase inhibitor) abolished FMD in arterioles from subjects without CAD, while PEG-Catalase (H2O2 scavenger) had no effect. Following exposure to BIBR-1532, arterioles from non-CAD subjects maintained the magnitude of dilation but changed the mediator from NO to mtH2O2 (% max diameter at 100 cm H2O: Vehicle 74.6±4.1, L-NAME 37.0±2.0*, PEG-Catalase 82.1±2.8; BIBR-1532 69.9±4.0, L-NAME 84.7±2.2, PEG-Catalase 36.5±6.9*). Conversely, treatment of microvessels from CAD patients with the telomerase activator AGS-499 converted the PEG-catalase-inhibitable dilation to one mediated by NO (% max diameter at 100 cm H2O: Adipose, AGS 499 78.5±3.9; L-NAME 10.9±17.5*; Peg-Catalase 79.2±4.9). Endothelial-independent dilation was not altered with either treatment. Conclusions: We have identified a novel role for telomerase in re-establishing a physiological mechanism of vasodilation in arterioles from subjects with CAD. These findings suggest a new target for reducing the oxidative milieu in the microvasculature of patients with CAD.

Citation:
CIRCRESAHA.115.307918, Published online before print December 23, 2015, doi: 10.1161/CIRCRESAHA.115.307918

http://circres.ahajournals.org/content/early/2015/12/22/CIRCRESAHA.115.307918.abstract

Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies

Authors:
Ware JS, Li J, Mazaika E, Yasso CM, DeSouza T, Cappola TP, Tsai EJ, Hilfiker-Kleiner D, Kamiya CA, Mazzarotto F, Cook SA, Halder I, Prasad SK, Pisarcik J, Hanley-Yanez K, Alharethi R, Damp J, Hsich E, Elkayam U, Sheppard R, Kealey A, Alexis J, Ramani G, Safirstein J, Boehmer J, Pauly DF, Wittstein IS, Thohan V, Zucker MJ, Liu P, Gorcsan J 3rd, McNamara DM, Seidman CE, Seidman JG, Arany Z; IMAC-2 and IPAC Investigators.

Abstract:
Background Peripartum cardiomyopathy shares some clinical features with idiopathic dilated cardiomyopathy, a disorder caused by mutations in more than 40 genes, including TTN, which encodes the sarcomere protein titin. Methods In 172 women with peripartum cardiomyopathy, we sequenced 43 genes with variants that have been associated with dilated cardiomyopathy. We compared the prevalence of different variant types (nonsense, frameshift, and splicing) in these women with the prevalence of such variants in persons with dilated cardiomyopathy and with population controls. Results We identified 26 distinct, rare truncating variants in eight genes among women with peripartum cardiomyopathy. The prevalence of truncating variants (26 in 172 [15%]) was significantly higher than that in a reference population of 60,706 persons (4.7%, P=1.3×10-7) but was similar to that in a cohort of patients with dilated cardiomyopathy (55 of 332 patients [17%], P=0.81). Two thirds of identified truncating variants were in TTN, as seen in 10% of the patients and in 1.4% of the reference population (P=2.7×10-10); almost all TTN variants were located in the titin A-band. Seven of the TTN truncating variants were previously reported in patients with idiopathic dilated cardiomyopathy. In a clinically well-characterized cohort of 83 women with peripartum cardiomyopathy, the presence of TTN truncating variants was significantly correlated with a lower ejection fraction at 1-year follow-up (P=0.005). Conclusions The distribution of truncating variants in a large series of women with peripartum cardiomyopathy was remarkably similar to that found in patients with idiopathic dilated cardiomyopathy. TTN truncating variants were the most prevalent genetic predisposition in each disorder.

Citation:
N Engl J Med. 2016 Jan 6. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26735901

Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress

Authors:
Christopher G. Scheitlin, Justin A. Julian, Santhanam Shanmughapriya, Muniswamy Madesh, Nikolaos M Tsoukias, B. Rita Alevriadou

Abstract:
Shear stress is known to stimulate an intracellular free calcium concentration ([Ca2+]i) response in vascular endothelial cells (ECs). [Ca2+]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca2+]i response is, in part, due to Ca2+ release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca2+ store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca2+]i in sheared ECs. Cultured ECs, loaded with a Ca2+-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca2+]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs and oscillation frequencies depended on the shear level. [Ca2+]i transients/oscillations were present when experiments were conducted in Ca2+-free solution (plus lanthanum), but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca2+]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca2+ uniporter also inhibited the shear-induced [Ca2+]i transients/oscillations compared to controls. Hence, EC mitochondria, through Ca2+ uptake/release, regulate the temporal profile of shear-induced ER Ca2+ release. [Ca2+]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca2+]i response.

Citation:
Am J Physiol Cell Physiol. 2016 Jan 6:ajpcell.00171.2015. doi: 10.1152/ajpcell.00171.2015. [Epub ahead of print]

http://ajpcell.physiology.org/content/early/2016/01/04/ajpcell.00171.2015