Vascular Stability Research Articles

Hemorrhagic progression of cavernous angiomas: a review

Cerebral cavernous malformations, also known as cerebral cavernous angiomas or cavernomas, are low-pressure berrylike vascular lesions with minimal to no intervening brain parenchyma. They occur with an incidence of 0.5% in the population, putting them at risk for epilepsy, neurological deficits, or hemorrhage. Genetic and inflammatory factors seem to be implicated in the development and rupture of cavernomas. Two forms of cavernomas have been described in the literature: the sporadic form, associated with single isolated lesions and the familial form that tends to result in multiple lesions. Both forms express loss of function mutations of endothelial genes that encode for proteins that play crucial roles in the proper formation of vascular lumen and maintenance of the vascular barrier, namely CCM1, CCM2 and CCM3. Therefore, cavernomas have been theorized to form due to vascular stabilization failure in angiogenesis which promotes the development of capillary dysplasia, weak intercellular junctions, and defective smooth muscle recruitment. These are also highly inflamed lesions laden with monocytes, macrophages, T cells, B cells, plasma cells and inflammatory cytokines. Further complicating this multi-factorial process is a new observation that chronic hypertension may be involved in the development or rupture of certain types of cerebral cavernous angioma lesions. We previously reported on a patient with new onset hypertension treated with medication that developed symptomatic sequential supra- and infratentorial cavernoma hemorrhages that caused hydrocephalus and cranial nerve deficits.One must theorize that this observation either represents an anomaly without causation in the process of cerebral cavernous malformation hemorrhage or possibly part of a multifactorial process in the physiology of certain cavernoma hemorrhage types. Studies of capillary physiology and successful treatment of cavernoma with beta blockers may suggest the latter in certain circumstances. Here we present a review of the literature in the development and treatment of hemorrhagic cavernous angiomas.

Autologous Bone Marrow Mesenchymal Stem Cells Improve the Quality and Stability of Vascularized Flap Surgery of Irradiated Skin in Pigs.

Cutaneous radiation syndrome has severe long‐term health consequences. Because it causes an unpredictable course of inflammatory waves, conventional surgical treatment is ineffective and often leads to a fibronecrotic process. Data about the long‐term stability of healed wounds, with neither inflammation nor resumption of fibrosis, are lacking. In this study, we investigated the effect of injections of local autologous bone marrow‐derived mesenchymal stromal cells (BM‐MSCs), combined with plastic surgery for skin necrosis, in a large‐animal model. Three months after irradiation overexposure to the rump, minipigs were divided into three groups: one group treated by simple excision of the necrotic tissue, the second by vascularized‐flap surgery, and the third by vascularized‐flap surgery and local autologous BM‐MSC injections. Three additional injections of the BM‐MSCs were performed weekly for 3 weeks. The quality of cutaneous wound healing was examined 1 year post‐treatment. The necrotic tissue excision induced a pathologic scar characterized by myofibroblasts, excessive collagen‐1 deposits, and inadequate vascular density. The vascularized‐flap surgery alone was accompanied by inadequate production of extracellular matrix (ECM) proteins (decorin, fibronectin); the low col1/col3 ratio, associated with persistent inflammatory nodules, and the loss of vascularization both attested to continued immaturity of the ECM. BM‐MSC therapy combined with vascularized‐flap surgery provided mature wound healing characterized by a col1/col3 ratio and decorin and fibronectin expression that were all similar to that of nonirradiated skin, with no inflammation, and vascular stability. In this preclinical model, vascularized flap surgery successfully and lastingly remodeled irradiated skin only when combined with BM‐MSC therapy. Stem Cells Translational Medicine 2018:569–582

Restoring blood vessel stability

Hypothesis What if a wide array of diseases that afflict blood vessels—from atherosclerosis to transplant vasculopathy and vascular malformations—had a common causal element? In a Perspective, Schwartz et al. propose that fundamental aspects of normal vascular remodeling promote disease if prolonged or exaggerated. They also suggest that unchecked vascular endothelial cell activation and extracellular matrix remodeling are common to multiple pathologies. If this is the case, interventions to restore vascular stability could improve health outcomes in multiple disease states. Science , this issue p. [270][1] [1]: /lookup/doi/10.1126/science.aat3470

Adenovirus-delivered angiopoietin-1 ameliorates phosgene-induced acute lung injury via inhibition of NLRP3 inflammasome activation

Objective: Angiopoietin-1 (Ang1) is reported to have the ability to attenuate endothelial permeability and inflammation during the stress condition and is considered to play a critical role in vascular stabilization. The aim of this study was to investigate the mechanisms involved in the protective effects of adenovirus-delivered Ang1 in phosgene-induced acute lung injury (ALI).Methods: ALI was induced in rats by phosgene exposure at 8.33 g/m3 for 5 min, followed by an intravenous injection of adenovirus-Ang1 (Ad/Ang1). The histologic changes of the lung were evaluated with H&E staining. The levels of cytokines in the serum and bronchoalveolar lavage fluid (BALF) were determined by ELISA. NLRP3 inflammasome activation was assessed with immunohistochemistry, RT-PCR, Western blotting and TUNEL staining.Results: Histologic analyses suggested that reduced severity in phosgene-induced ALI with Ad/Ang1 treatment. Reduced levels of IL-1β, IL-18 and IL-33 were found in both serum and BALF samples from Ad/Ang1-treated ALI rats induced by phosgene. Moreover, immunohistochemistry analysis revealed that Ad/Ang1 treatment inhibited the NLRP3 inflammasome activation. Decreased mRNA and protein levels of NLRP3 and caspase-1 were found in phosgene-exposed rats treated with Ad/Ang1. In addition, TUNEL staining indicated a decrease in pyroptosis in phosgene-exposed rats treated with Ad/Ang1.Conclusions: Ang1 exerts beneficial effects on phosgene-induced lung injury via inhibition of NLRP3 inflammasome activation. Disruption of NLRP3 inflammasome activation might be served as therapeutic modality for the treatment of phosgene-induced ALI.

NOTCH regulation of the endothelial cell phenotype.

Purpose of reviewThe formation of a hierarchical vascular network is a complex process that requires precise temporal and spatial integration of several signaling pathways. Amongst those, Notch has emerged as a key regulator of multiple steps that expand from endothelial sprouting to arterial specification and remains relevant in the adult. This review aims to summarize major concepts and rising hypotheses on the role of Notch signaling in the endothelium.Recent findingsA wealth of new information has helped to clarify how Notch signaling cooperates with other pathways to orchestrate vascular morphogenesis, branching, and function. Endothelial vascular endothelial growth factor, C-X-C chemokine receptor type 4, and nicotinamide adenine dinucleotide phosphate oxidase 2 have been highlighted as key regulators of the pathway. Furthermore, blood flow forces during vascular development induce Notch1 signaling to suppress endothelial cell proliferation, enhance barrier function, and promote arterial specification. Importantly, Notch1 has been recently recognized as an endothelial mechanosensor that is highly responsive to the level of shear stress to enable differential Notch activation in distinct regions of the vessel wall and suppress inflammation.SummaryAlthough it is well accepted that the Notch signaling pathway is essential for vascular morphogenesis, its contributions to the homeostasis of adult endothelium were uncovered only recently. Furthermore, its exquisite regulation by flow and impressive interface with multiple signaling pathways indicates that Notch is at the center of a highly interactive web that integrates both physical and chemical signals to ensure vascular stability.

Protein compositions changes of circulating microparticles in patients with valvular heart disease and cardiac surgery

We previously demonstrated that circulating microparticles from patients with valvular heart disease (VHD) and cardiac surgery with cardiopulmonary bypass(CPB) impaired endothelial function and vasodilation. Protein composition of circulating microparticles is critical in influencing the endothelial function and vasodilation. However, whether the protein compositions of circulating microparticles were changed in patients with VHD and cardiac surgery remains unknown. Circulating microparticles were isolated from age‐matched control subjects (n=50) and patients (n=50) with VHD before and 72 h after cardiac surgery with CPB. Proteomic study was performed by liquid chromatography and mass spectrometry (LC/MS) combined with isobaric tags for relative and absolute quantification (iTRAQ) technique. The different proteins were identified by ProteinPilot™, some of which were considered affecting vascular function and hemodynamic stability were validated by Western blotting. Bio‐informatic analysis of different proteins was also carried out. We found that 849 proteins were identified significantly change in circulating microparticles (p<0.05). Among these, 453 (53.36%) proteins were found in all three groups. 165 (19.43%), 39 (4.59%) and 80 (9.42%) proteins were unique in control group, pre‐operation group and post‐operation group respectively. Compared to control group, there were 181 up‐regulated and 175 down‐regulated proteins in pre‐operation group. Compared to pre‐operation group, there were 89 up‐regulated and 76 down‐regulated proteins in post‐operation group. Bio‐informatic analysis found that these different proteins were different in localization, molecular function and biological process. Western blot confirmed that the pro‐inflammatory proteins in circulating microparticles were significantly increased in VHD patients, which increased even more postoperatively. Pro‐coagulation proteins in circulating microparticles were also dramatically increased in preoperation, but decreased in postoperation. Our data demonstrated that the protein compositions of circulating microparticles were significantly changed in patients with VHD and cardiac surgery. These protein composition changes of circulating microparticles may lead to systemic inflammatory response and disorder of coagulation which may cause difficulties in maintaining the stability of circulation postoperatively.Support or Funding InformationNational Natural Science Foundation of China (Grants 81370370, 81670392, 81600382 and Distinguished Young Scholar Grant 81325001), International cooperation project(2015DFA31070) and National Major Research Program (2016YFC0903000) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee (Grant 2015A030312009), the Changjiang Scholars Program from the Ministry of Education of China, the Guangdong Pearl River Scholars Program, the Sun Yat‐sen University Clinical Research 5010 Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New Therapies of Neovascular AMD beyond Anti-VEGF Injections.

Neovascular age-related macular degeneration is a leading cause of vision loss among the aging population. The current standard of care to treat neovascular age-related macular degeneration is inhibiting vascular endothelial growth factor (VEGF) through intravitreal injections. Recent studies have demonstrated that the tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2 (Tie2) pathway also plays a critical role in angiogenesis and vascular stability. Additionally, newly developed treatment delivery systems have been designed to greatly reduce the frequency of injections. In targeting the Tie2 pathway and utilizing a sustained release delivery system, patients may experience improved visual outcomes and a reduced burden of treatment.

Circulating bone morphogenetic protein-9 levels are associated with hypertension and insulin resistance in humans

It has been demonstrated that bone morphogenetic protein-9 (BMP-9) may have an important role in vascular development and stability. However, the association of circulating BMP-9 with essential hypertension (HTN) has not been established in humans. The objective of this study is to observe the changes of circulating BMP-9 levels in patients with HTN and to investigate the association of circulation BMP-9 and insulin resistance (IR) in a cross-sectional study. Two hundred twenty-five individuals, including 132 patients with hypertension, and 93 healthy controls, were included in the present study. Circulating BMP-9 concentrations were measured with an ELISA kit. The association of circulating BMP-9 with other parameters was analyzed. When compared with healthy subjects, circulating BMP-9 concentrations were markedly lower in HTN patients (46.20 [31.85–62.80] vs. 77.21 [39.33–189.15], P < .01) and correlated negatively with blood pressure and the homeostasis model assessment of insulin resistance (P < .05 or P < .01). Decreasing levels of BMP-9 were independently and markedly related to HTN. In a multiple linear regression analysis, only systolic blood pressure and free fatty acid concentrations were independently associated with circulating BMP-9. Our findings suggest that BMP-9 may be a serum biomarker for HTN and IR.

Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain

The murine embryonic blood-brain barrier (BBB) consists of endothelial cells (ECs), pericytes (PCs), and basement membrane. Although PCs are critical for inducing vascular stability, signaling pathways in PCs that regulate EC morphogenesis during BBB development remain unexplored. Herein, we find that murine embryos lacking the transforming growth factor β (TGF-β) receptor activin receptor-like kinase5 (Alk5) in brain PCs (mutants) develop gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH). The germinal matrix (GM) is a highly vascularized structure rich in neuronal and glial precursors. We show that GM microvessels of mutants display abnormal dilation, reduced PC coverage, EC hyperproliferation, reduced basement membrane collagen, and enhanced perivascular matrix metalloproteinase activity. Furthermore, ALK5-depleted PCs downregulate tissue inhibitor of matrix metalloproteinase 3 (TIMP3), and TIMP3 administration to mutants improves endothelial morphogenesis and attenuates GMH-IVH. Overall, our findings reveal a key role for PC ALK5 in regulating brain endothelial morphogenesis and a substantial therapeutic potential for TIMP3 during GMH-IVH.

Survivin regulated by autophagy mediates hyperglycemia-induced vascular endothelial cell dysfunction

Diabetic vascular complications are often defined by vascular endothelial lesions. However, as a plastic cell type, whether endothelial cells could transit from quiescence to hyper-active status and hamper vascular stability upon hyperglycemia stimulation and whether this process is involved in diabetic vascular complications remain obscure. Survivin has been identified as an anti-apoptotic protein in tumor or epithelial cells by either promoting proliferation or inhibiting apoptosis. Therefore, this study aims at investigating the effects of hyperglycemia on endothelial cell status and the potential involvement of survivin. We found that high glucose (25 mM) did not cause endothelial injuries, instead, it evidently promotes endothelial proliferation and tube formation capacity indicating endothelial cell dysfunction upon hyperglycemia characterized by its preference to hyper-active status. Concomitantly, an upregulation of survivin was detected accompanied by the key component elevations of autophagy pathway including LC3, Beclin1, and p62. YM155, a specific inhibitor of survivin, could abrogate hyperglycemia-induced endothelial hyper-activation. Application of the autophagy inhibitor (3MA) and agonist (rapamycin) supported that survivin could be as a downstream effect or of autophagy. Thus, our results suggested that survivin/autophagy axis a potential therapeutic target in treatment of diabetic vascular complications.

Dual roles of endothelial FGF-2\u2013FGFR1\u2013PDGF-BB and perivascular FGF-2\u2013FGFR2\u2013PDGFR\u03b2 signaling pathways in tumor vascular remodeling

Perivascular cells are important cellular components in the tumor microenvironment (TME) and they modulate vascular integrity, remodeling, stability, and functions. Here we show using mice models that FGF-2 is a potent pericyte-stimulating factor in tumors. Mechanistically, FGF-2 binds to FGFR2 to stimulate pericyte proliferation and orchestrates the PDGFRβ signaling for vascular recruitment. FGF-2 sensitizes the PDGFRβ signaling through increasing PDGFRβ levels in pericytes. To ensure activation of PDGFRβ, the FGF-2–FGFR1-siganling induces PDGF-BB and PDGF-DD, two ligands for PDGFRβ, in angiogenic endothelial cells. Thus, FGF-2 directly and indirectly stimulates pericyte proliferation and recruitment by modulating the PDGF–PDGFRβ signaling. Our study identifies a novel mechanism by which the FGF-2 and PDGF-BB collaboratively modulate perivascular cell coverage in tumor vessels, thus providing mechanistic insights of pericyte–endothelial cell interactions in TME and conceptual implications for treatment of cancers and other diseases by targeting the FGF-2–FGFR-pericyte axis.

Reductions in brain pericytes are associated with arteriovenous malformation vascular instability.

OBJECTIVEBrain arteriovenous malformations (bAVMs) are rupture-prone tangles of blood vessels with direct shunting of blood flow between arterial and venous circulations. The molecular and/or cellular mechanisms contributing to bAVM pathogenesis and/or destabilization in sporadic lesions have remained elusive. Initial insights into AVM formation have been gained through models of genetic AVM syndromes. And while many studies have focused on endothelial cells, the contributions of other vascular cell types have yet to be systematically studied. Pericytes are multifunctional mural cells that regulate brain angiogenesis, blood-brain barrier integrity, and vascular stability. Here, the authors analyze the abundance of brain pericytes and their association with vascular changes in sporadic human AVMs.METHODSTissues from bAVMs and from temporal lobe specimens from patients with medically intractable epilepsy (nonvascular lesion controls [NVLCs]) were resected. Immunofluorescent staining with confocal microscopy was performed to quantify pericytes (platelet-derived growth factor receptor-beta [PDGFRβ] and aminopeptidase N [CD13]) and extravascular hemoglobin. Iron-positive hemosiderin deposits were quantified with Prussian blue staining. Syngo iFlow post-image processing was used to measure nidal blood flow on preintervention angiograms.RESULTSQuantitative immunofluorescent analysis demonstrated a 68% reduction in the vascular pericyte number in bAVMs compared with the number in NVLCs (p < 0.01). Additional analysis demonstrated 52% and 50% reductions in the vascular surface area covered by CD13- and PDGFRβ-positive pericyte cell processes, respectively, in bAVMs (p < 0.01). Reductions in pericyte coverage were statistically significantly greater in bAVMs with prior rupture (p < 0.05). Unruptured bAVMs had increased microhemorrhage, as evidenced by a 15.5-fold increase in extravascular hemoglobin compared with levels in NVLCs (p < 0.01). Within unruptured bAVM specimens, extravascular hemoglobin correlated negatively with pericyte coverage (CD13: r = -0.93, p < 0.01; PDGFRβ: r = -0.87, p < 0.01). A similar negative correlation was observed with pericyte coverage and Prussian blue-positive hemosiderin deposits (CD13: r = -0.90, p < 0.01; PDGFRβ: r = -0.86, p < 0.01). Pericyte coverage positively correlated with the mean transit time of blood flow or the time that circulating blood spends within the bAVM nidus (CD13: r = 0.60, p < 0.05; PDGFRβ: r = 0.63, p < 0.05). A greater reduction in pericyte coverage is therefore associated with a reduced mean transit time or faster rate of blood flow through the bAVM nidus. No correlations were observed with time to peak flow within feeding arteries or draining veins.CONCLUSIONSBrain pericyte number and coverage are reduced in sporadic bAVMs and are lowest in cases with prior rupture. In unruptured bAVMs, pericyte reductions correlate with the severity of microhemorrhage. A loss of pericytes also correlates with a faster rate of blood flow through the bAVM nidus. This suggests that pericytes are associated with and may contribute to vascular fragility and hemodynamic changes in bAVMs. Future studies in animal models are needed to better characterize the role of pericytes in AVM pathogenesis.

Pericytes Derived from Human Pluripotent Stem Cells.

Pericytes wrap blood microvessels and are believed to play important roles in vascular morphogenesis, maturation, and stability. In addition, pericytes have emerged as candidates for targeting cancer growth and for wound healing. In order to model these processes and test new therapies, it is desirable to have a reliable, scalable source of pericytes. Human pluripotent stem cells (hPSCs), which possess the ability to differentiate into any cell type in the body, have been used to generate pericytes in vitro quickly, consistently, and with high yields. In this chapter, we consider the differentiation of pericytes from hPSCs. We compare the approaches taken by multiple groups and discuss characterization of hPSC-pericytes. Studying pericyte differentiation in vitro provides the opportunity to identify factors influencing pericyte development and to establish the ontogenic relationships between pericytes and similar cells. The development of highly specific, defined pericyte populations from hPSCs will enable downstream applications requiring large quantities of cells, including tissue engineered models and cell therapies.

Pericyte Biology: Development, Homeostasis, and Disease.

In the nineteenth century, a French researcher, Charles-Marie Benjamin Rouget, revealed a population of contractile cells associated with small blood vessels, which were initially named after him as the Rouget cells. In the twentieth century, a German scientist, Karl Wilhelm Zimmermann, called these cells "pericytes" due to their anatomical position located in a perivascular position. The word pericyte was derived from "peri" meaning "around" and "cyte" from the word "kytos" (cell), illustrating a cell encircling a blood vessel. Until now, pericytes are still identified partially based on their specific anatomical location and morphology. Pericytes are present in all vascularized tissues, surrounding blood vessel walls. They encircle endothelial cells and communicate with them along the length of the blood vessels by paracrine signaling and physical contacts. Previously, the accurate distinction of pericytes from other perivascular cells was difficult, as electron and light microscopy were the sole available techniques capable to image these cells, limiting the information acquired from those works. This resulted in the misleading assumption that pericytes are merely inert supporting cells, limited exclusively to the physiological function of vascular stability. In the last 10years, the combination of fluorescent and confocal microscopy with genetic state-of-art techniques, such as fate lineage tracing, enabled remarkable progress in the discovery of multiple novel essential functions for pericytes in health and disease, before unexpected. Recently, the rapidly expanding understanding of the pathophysiological roles of pericytes drew the attention of several research groups. Now, we know, for instance, that pericytes may play immune functions: attract innate leukocytes to exit via sprouting blood vessels, regulate lymphocyte activation, and contribute to the clearance of toxic by-products, having direct phagocytic activity. Pericytes also may behave as stem cells, forming other cell populations, as well as regulate the behavior of other stem cells in their niches. Very little is known about the exact identity of pericyte ancestors within developing tissues, and there is evidence for multiple distinct developmental sources. Pericytes differ in their embryonic origins between tissues and also within the same organ. Importantly, pericytes from distinct tissues may differ in their distribution, morphology, expression of molecular markers, plasticity, and functions; and, even within the same organ, there are various pericyte subpopulations. This book describes the major contributions of pericytes to different organ biology in physiological and pathological conditions. Further insights into the biology of pericytes will have important implications for our understanding of organ development, homeostasis, and disease. This book's initial title was "Pericyte Biology: Development, Homeostasis, and Disease." However, due to the current great interest in this topic, we were able to assemble more chapters than would fit in one book, covering pericyte biology under distinct circumstances. Therefore, the book was subdivided into three volumes entitled: "Pericyte Biology: Novel Concepts," "Pericyte Biology in Different Organs," and "Pericyte Biology in Disease." Here, we present a selected collection of detailed chapters on what we know so far about pericytes. More than 30 chapters written by experts in the field summarize our present knowledge on pericyte biology.Here, we present a selected collection of detailed chapters on what we know so far about pericytes. More than 30 chapters written by experts in the field summarize our present knowledge on pericyte biology.

Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling.

The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization. We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak. Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling

Angiogenesis is central to both normal and pathologic processes. Endothelial cells (ECs) express O-glycoproteins that are believed to play important roles in vascular development and stability. Endomucin-1 (EMCN) is a type I O-glycosylated, sialic-rich glycoprotein, specifically expressed by venous and capillary endothelium. Evidence has pointed to a potential role for EMCN in angiogenesis but it had not been directly investigated. In this study, we examined the role of EMCN in angiogenesis by modulating EMCN levels both in vivo and in vitro. Reduction of EMCN in vivo led to the impairment of angiogenesis during normal retinal development in vivo. To determine the cellular basis of this inhibition, gain- and loss-of-function studies were performed in human retinal EC (HREC) in vitro by EMCN over-expression using adenovirus or EMCN gene knockdown by siRNA. We show that EMCN knockdown reduced migration, inhibited cell growth without compromising cell survival, and suppressed tube morphogenesis of ECs, whereas over-expression of EMCN led to increased migration, proliferation and tube formation. Furthermore, knockdown of EMCN suppressed VEGF-induced signaling as measured by decreased phospho-VEGFR2, phospho-ERK1/2 and phospho-p38-MAPK levels. These results suggest a novel role for EMCN as a potent regulator of angiogenesis and point to its potential as a new therapeutic target for angiogenesis-related diseases.

Standardized fraction of Xylocarpus moluccensis fruits improve vascular relaxation and plaque stability in dyslipidemic models of atherosclerosis

Ethnopharmacological relevanceXylocarpus moluccensis (Lamk.) M. Roem of family Meliaceae has triterpenoids rich fruits. Triterpenoids have been known to possess cardioprotection and anti-atherosclerotic activities (Han and Bakovic, 2015; Wu et al., 2009). Standardized fraction of these fruits exhibited anti-dyslipidemic (Srivastava et al., 2015), anti-inflammatory (Ravangpai et al., 2011) and CNS depressant activity (Sarker et al., 2007). However, there is no report in the literature on its cardiovascular effects. Aim of the studyThe present study was undertaken to assess vasoprotective, anti-atherosclerotic and further examine the anti-dyslipidemic effect of the standardized fraction of Xylocarpus moluccensis (F018) fruits in the mechanical injury and high fat diet (HFD) induced dyslipidemic/ atherosclerosis models. Materials and methodsGuinea pigs were fed 0.08% cholesterol + 15% fat diet for 3 weeks, while ApoE KO mice were fed high fat diet for 18 weeks to induce dyslipidemia and atherosclerosis. A combination of balloon injury and high fat diet (1% cholesterol, 6% peanut oil) for 5 weeks was used to accelerate atherosclerosis in NZW rabbits. F018 was administered once daily by oral route in guinea pigs (10, 25 or 50mg/kg/day for 3 weeks), ApoE KO mice (50mg/kg/day for 6 weeks) and in NZW rabbit (25mg/kg/day for 5 weeks) to monitor its effect on dyslipidemia, vasoreactivity and plaque composition by using standard methodologies. ResultsF018 treatment in guinea pigs (25 and 50mg/kg/day), ApoE mice (50mg/kg/day) and rabbits (25mg/kg/day) significantly reduced plasma lipids and improved ACh induced vasorelaxation. Anti-dyslipidemic effect of F018 seems to be due to the modulation of enterohepatic genes involved in the cholesterol absorption and excretion. Moreover, significant improvement in the acetylcholine (ACh) induced vasorelaxation was accompanied with reduced inflammatory burden and enhanced activation of eNOS in ApoE mice aortic tissue. Similarly inflammatory cytokines, immunolabeling of macrophage marker (CD68) and MMP-9 were reduced along with augmentation in vascular smooth muscle cells and collagen type I and III in the mechanically injured iliac artery segment in the rabbits. ConclusionsAltogether, F018 preserved vasoreactivity, reduced atherosclerotic plaque progression and enhanced plaque stability by reducing lipids, inflammatory cytokines, improving endothelial function and collagen content.

Sequential delivery of VEGF, FGF-2 and PDGF from the polymeric system enhance HUVECs angiogenesis in vitro and CAM angiogenesis

Angiogenesis is an organized series of events, beginning with vessel destabilization, followed by endothelial cell re-organization, and ending with vessel maturation. The formation of a mature vascular network requires precise spatial and temporal regulation of a large number of angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor (PDGF). VEGF aids in vascular permeability and endothelial cell recruitment, FGF-2 activates endothelial cell proliferation and migration while PDGF stimulates vascular stability. Accordingly, VEGF may inhibit vessel stabilization while PDGF may inhibit endothelial cell recruitment. Therefore, a new polymeric system was prepared by the supercritical carbon dioxide foaming technology, which realized sequential delivery of two or more growth factors with the controlled dose and rate. Increased release of VEGF (71.10%) and FGF-2 (69.76%) compared to PDGF (43.17%) was observed for the first 7 days. Thereafter, up till 21 days, an increased rate of release of BMP-2 compared to VEGF 165 was observed. The effects of PDGF-PLAms/VEGF-FGF-2-PLGA scaffolds on angiogenesis were investigated by human umbilical vein endothelial cells (HUVECs) angiogenic differentiation in vitro and chorioallantoic membrane (CAM) angiogenesis in vivo. Sequential delivery of VEGF, FGF-2 and PDGF from structural polymer scaffolds with distinct kinetics resulted in significant angiogenic differentiation of HUVECs and rapid formation of mature vascular networks in chorioallantoic membrane. This study reported a composite scaffold with distinct release kinetics, and these results clearly indicated the importance of sequential delivery of multiple growth factors in tissue regeneration and engineering.

TIMP3 is Regulated by Pericytes upon Shear Stress Detection Leading to a Modified Endothelial Cell Response

Atherosclerosis is a hallmark of cardiovascular disease. Shear stress on endothelial cells has been linked to atherogenesis and to fibrous cap thinning and rupture. Pericytes reside in the sub-endothelial space of vessels and have vasoprotective effects. They are subjected to shear stress when endothelial cell integrity is disrupted. The aim was to investigate the susceptibility and response of pericytes to shear stress. Endothelial cells and pericytes were seeded in two dimensional monocultures and co-cultures, and in a novel three dimensional co-culture system and were subjected to no, low and high shear stress (0, 10, 30 dyne/cm2) for 48 h. The morphological response to flow was assessed by histology and the expression of extracellular matrix proteins was analysed using quantitative polymerase chain reaction, immunoblotting, and ELISA. While endothelial cells aligned into flow direction, pericytes aligned perpendicularly (p < .001), indicating that they must be capable of sensing flow. When pericytes were embedded into a 3D matrix they showed similar alignment and pericytes built long processes towards the lumen. Under shear stress endothelial cells upregulated “a disintegrin and metalloproteinase with thrombospondin motif 1” (ADAMTS-1) (p < .01) and pericytes upregulated “tissue inhibitor of matrix metalloproteinase” (TIMP) 3 (p < .05), an inhibitor of ADAMTS-1, meanwhile differential expression of extracellular matrix (ECM) proteins could be detected in co-cultures of both cells. For TIMP3 expression direct cell–cell contact between endothelial cells and pericytes was required. The experiments highlight that pericytes are able to sense direct flow thereby regulating ECM proteins known to be involved in vascular remodelling. Furthermore, pericytes counter-regulate endothelial ADAMTS-1 by protective TIMP3 expression to prevent matrix degradation and maintain vascular stability. For this protective effect direct cell contact was necessary. This observation might represent an adaptive, protective mechanism of pericytes to counteract endothelial damage in the onset of atherosclerosis.