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Purpose We have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells. Methods PC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/μL) or VEGF-B (50 ng/μL) or “vehicle” (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells. Results TIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells. Conclusions Differences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Placentas from gestational diabetes mellitus (GDM) are often hypervascularized; however, participation of vascular endothelial growth factor (VEGF) and its receptors in this placental adaptation is unclear. We aimed to test whether changes in phosphorylation of tyrosine 951 or tyrosine 1175 (pY951 or pY1175) of the vascular endothelial growth factor receptor 2 (KDR) are associated with the proangiogenic state observed in placentas from GDM. We obtained placental samples from women with normal pregnancies (n = 24) or GDM (n = 18). We measured the relative expression of markers for endothelial cell number (CD31, CD34), VEGF, vascular endothelial growth factor receptor 1 (Flt-1), KDR, pY951 and pY1175 of KDR in placental homogenate. Immunohistochemistry of placental blood vessels were performed using CD34. Proliferation and migration of human umbilical vein endothelial cells (HUVEC) obtained from normal pregnancy and GDM were determined in absence or presence of conditioned medium (CM) harvested from GDM or normoglycemic HUVEC cultures. GDM was associated with more CD31 and CD34 protein compared to normal pregnancy. High number, but reduced area of placental blood vessels was found in GDM. Reduced Flt-1 levels (mRNA and protein) are associated with reduced KDR mRNA, but higher KDR protein levels in placentas from GDM. No significant changes in Y951-or Y1175-phosphorylation of KDR in placentas from GDM were found. GDM did not alter proliferation of HUVECs, but enhanced migration. Conditioned medium harvested from GDM HUVEC cultures enhanced KDR protein amount, tube formation capacity and cell migration in HUVEC isolated from normoglycemic pregnancies. The data indicate that GDM is associated with reduced expression of Flt-1 but high pro-migratory activation of KDR reflecting a proangiogenic state in GDM.

The sialomucins CD34 and podocalyxin (PODXL) are anti-adhesive molecules expressed at the luminal membrane of endothelial cells of small blood vessels and facilitate vascular lumen formation in the developing mouse aorta. CD34 transcript and protein levels are increased during human angiogenesis, its expression is particularly enriched on endothelial tip cell filopodia and CD34 is a marker for tip cells in vitro. Here, we investigated whether CD34 merely marks endothelial tip cells or has a functional role in tip cells and angiogenesis. We assessed that silencing CD34 in human microvascular endothelial cells has little effect on endothelial cell migration or invasion, but has a significant effect on vascular-endothelial growth factor-induced angiogenic sprouting activity in vitro. In vivo, the absence of CD34 reduced the density of filopodia on retinal endothelial tip cells in neonatal mice, but did not influence the overall architecture of the retinal vascular network. In oxygen-induced retinopathy, Cd34-/- mice showed normal intra-retinal regenerative angiogenesis but the number of pathological epi-retinal neovascular tufts were reduced. We conclude that CD34 is not essential for developmental vascularization in the retina, but its expression promotes the formation of pathological, invasive vessels during neovascularization.

Background Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. Methods VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Results Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Conclusions Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal dysfunction or disease.

CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand) upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF) are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

Background: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. Methodology/Principal Findings: We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI- 1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VEcadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. Conclusions/Significance: Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and “back table” preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2’(3’)-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury.

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microscopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity.

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3′ UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.

The vasculature of the central nervous system (CNS) is composed of vascular endothelial and mural cells which interact closely with glial cells and neurons. The development of the CNS vascularisation is a unique process which requires the contribution of specific regulators in addition to the classical angiogenic factors. The egfl7 gene is mainly detected in endothelial cells during physiological and pathological angiogenesis. Egfl7 codes for a secreted protein which predominantly accumulates into the extracellular space where it controls vascular elastin deposition or the Notch pathway. Egfl7 is the host gene of the microRNA miR126 which is also expressed in endothelial cells and which plays major functions during blood vessel development. While the expression of egfl7 and that of miR126 were well described in endothelial cells during development, their pattern of expression during the establishment of the CNS vasculature is still unknown. By analysing the expression of egfl7 and miR126 during mouse retina vascularisation, we observed that while expression of miR126 is detected in all endothelia, egfl7 is initially expressed in all endothelial cells and then is progressively restricted to veins and to their neighbouring capillaries. The recruitment of mural cells around retina arteries coincides with the down-regulation of egfl7 in the arterial endothelial cells, suggesting that this recruitment could be involved in the loss of egfl7 expression in arteries. However, the expression pattern of egfl7 is similar when mural cell recruitment is prevented by the injection of a PDGFRβ blocking antibody, suggesting that vessel maturation is not responsible for egfl7 down-regulation in retinal arteries.

Related Transcriptional Enhancer Factor-1 (RTEF-1) has been suggested to induce angiogenesis through regulating target genes. Whether RTEF-1 has a direct role in angiogenesis and what specific genes are involved in RTEF-1 driven angiogenisis have not been elucidated. We found that over-expressing RTEF-1 in Human dermal microvascular endothelial cells-1 (HMEC-1) significantly increased endothelial cell aggregation, growth and migration while the processes were inhibited by siRNA of RTEF-1. In addition, we observed that Endothelial differentiation gene-1 (Edg-1) expression was up-regulated by RTEF-1 at the transcriptional level. RTEF-1 could bind to Edg-1 promoter and subsequently induce its activity. Edg-1 siRNA significantly blocked RTEF-1-driven increases in endothelial cell aggregation in a Matrigel assay and retarded RTEF-1-induced endothelial cell growth and migration. Pertussis Toxin (PTX), a Gi/Go protein sensitive inhibitor, was found to inhibit RTEF-1 driven endothelial cell aggregation and migration. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1 and is involved in RTEF-1-induced angiogenesis in endothelial cells. Gi/Go protein coupled receptor pathway plays a role in RTEF-1 driven angiogenesis in endothelial cells.

International audience; OBJECTIVE: The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of 'specialized endothelial cells'. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions. APPROACH AND RESULTS: First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers. CONCLUSIONS: We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays.

Forkhead box (Fox) transcription factors are important regulators of cardiovascular development and several Fox-proteins have recently been shown to modulate embryonic and post-natal angiogenesis. However, the role of the FoxP subfamily, which is highly expressed in cardiovascular tissue, has not been investigated so far. Here, we show that the transcription factor FoxP1 is the highest expressed FoxP-protein in endothelial cells and that it is upregulated at the site of neovascularization during hindlimb ischemia in mice. Silencing of FoxP1 results in a strong inhibition of proliferation, tube formation and migration of cultured endothelial cells. Accordingly, knockdown of FoxP1 in zebrafish was followed by a disruption of intersomitic vascular formation. Using gene expression profiling, we show that FoxP1 induces a specific change of the endothelial transcriptome and functions as a suppressor of semaphorin 5B, which has previously been described as a neuronal inhibitory factor. Our findings now demonstrate that semaphorin 5B also acts as a FoxP1- dependent suppressor of endothelial cell proliferation, migration and sprouting, mediating the effects of FoxP1. In summary, our data indicate that the transcription factor FoxP1 is essential for the angiogenic function of endothelial cells and functions as a suppressor of the inhibitory guidance cue semaphorin 5B, suggesting an important function of FoxP1 in the regulation of neovascularization.

OBJECTIVE: This study was to investigate the molecular mechanisms underlying the 27nt-miRNA-mediated regulation of expression of the endothelial nitric oxide synthase (eNOS) gene. METHODS: Cell lines overexpressing 27nt-miRNA or its mutant were established by transfecting the miRNA expression vector into the endothelial cells. eNOS mRNA and protein expression were examined by RT-PCR and Western Blotting, respectively. Luciferase activity reporter system was used to study the target of 27nt-miRNA. RESULTS: The results showed that overexpression of 27nt-miRNA significantly inhibited eNOS mRNA level and protein expression, and reduced the eNOS transcriptional efficiency. Such inhibitory effects of 27nt-miRNA were attenuated by the sequence mutations in 27nt-miRNA. Interestingly, the transcription factor SP-1 expression was reduced by 27nt-miRNA. Meanwhile, overxpression of SP-1 protein partially restored eNOS expression, and rescued the 27nt-miRNA-mediated reduction of endothelial cell proliferation. Moreover, certain sites in the SP-1 mRNA were found to be the direct target of 27nt-miRNA by a luciferase reporter system. CONCLUSIONS: These results demonstrate that the 27nt-miRNA suppresses eNOS gene expression and SP-1 expression in vascular endothelial cells. The 27nt-miRNA directly target to SP-1 mRNA, thereby contributing to proliferation of endothelial cells.

TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function.

Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage.

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, > 30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

Growth differentiation factor 15 (GDF15), a direct target gene of p53, is a multifunctional member of the TGF-β/BMP superfamily. GDF15 can be induced and is implicated as a key secretory cytokine in response to multiple cellular stimuli. Accumulating evidence indicates that GDF15 is associated with the development and prognosis of diabetes mellitus, while whether GDF15 can be induced by high glucose is unknown. In the present study, we revealed that high glucose could induce GDF15 expression and secretion in cultured human umbilical vein endothelial cells in a ROS- and p53-dependent manner. Inhibition of high glucose-induced GDF15 expression by siRNA demonstrated that adaptively induced GDF15 played a protective role against high glucose-induced human umbilical vein endothelial cell apoptosis via maintaining the active state of PI3K/Akt/eNOS pathway and attenuating NF-κB/JNK pathway activation. The protective effects of GDF15 were probably achieved by inhibiting ROS overproduction in high glucose-treated human umbilical vein endothelial cells in a negative feedback manner. Our results suggest that high glucose can promote GDF15 expression and secretion in human umbilical vein endothelial cells, which in turn attenuates high glucose-induced endothelial cell apoptosis.

Introduction The endothelial function plays key roles in both promoting and protecting against atherosclerotic disease. Several methods, including peripheral arterial tonometry (PAT), have been reported to be useful for investigating endothelial dysfunction. Furthermore, the level of thrombomodulin (TM) is assumed to reflect the endothelial dysfunction. In the present study, we investigated endothelial dysfunction in patients with peripheral arterial disease (PAD) by measuring their TM levels, and evaluated the correlation between TM and various parameters. Materials and Methods We measured the TM levels and performed PAT in 17 patients with PAD. We also recorded the patients’ demographic information, including comorbidities, and investigated their hemodynamic status by measuring the ankle brachial pressure index (ABI). The PAT results were used to calculate the reactive hyperemia index (RHI), which reflected the patients’ level of endothelial dysfunction. Results The RHI and ABI values and the serum level of creatinine were found to be significantly correlated with the TM level (P = 0.040, 0.041 and 0.005, respectively). After setting an RHI value of 1.67 as the cut-off, the patients with RHI values of

Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy.

Background The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury. Methodology We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated. Results VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2−/− and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect. Conclusion Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis.

Vascular endothelial growth factor A (VEGF-A) binds to the VEGFR2 receptor tyrosine kinase, regulating endothelial function, vascular physiology and angiogenesis. However, the mechanism underlying VEGFR2 turnover and degradation in this response is unclear. Here, we tested a role for heat-shock proteins in regulating the presentation of VEGFR2 to a degradative pathway. Pharmacological inhibition of HSP90 stimulated VEGFR2 degradation in primary endothelial cells and blocked VEGF-A-stimulated intracellular signaling via VEGFR2. HSP90 inhibition stimulated the formation of a VEGFR2-HSP70 complex. Clathrin-mediated VEGFR2 endocytosis is required for this HSP-linked degradative pathway for targeting VEGFR2 to the endosome-lysosome system. HSP90 perturbation selectively inhibited VEGF-A-stimulated human endothelial cell migration in vitro. A mouse femoral artery model showed that HSP90 inhibition also blocked blood vessel repair in vivo consistent with decreased endothelial regeneration. Depletion of either HSP70 or HSP90 caused defects in blood vessel formation in a transgenic zebrafish model. We conclude that perturbation of the HSP70-HSP90 heat-shock protein axis stimulates degradation of endothelial VEGFR2 and modulates VEGF-A-stimulated intracellular signaling, endothelial cell migration, blood vessel development and repair.

TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFβ receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFβ signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.

Endothelial injury related to oxidative stress is a key event in cardiovascular diseases, such as hypertension and atherosclerosis. The activation of the redox-sensitive Kv1.5 potassium channel mediates mitochondrial reactive oxygen species (ROS)-induced apoptosis in vascular smooth muscle cells and some cancer cells. Kv1.5 channel is therefore taken as a new potential therapeutic target for pulmonary hypertension and cancers. Although Kv1.5 is abundantly expressed in vascular endothelium, there is little knowledge of its role in endothelial injury related to oxidative stress. We found that DPO-1, a specific inhibitor of Kv1.5, attenuated H(2)O(2)-evoked endothelial cell apoptosis in an in vivo rat carotid arterial model. In human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells (HPAECs), angiotensin II and oxLDL time- or concentration-dependently enhanced Kv1.5 protein expression in parallel with the production of intracellular ROS and endothelial cell injury. Moreover, siRNA-mediated knockdown of Kv1.5 attenuated, whereas adenovirus-mediated Kv1.5 cDNA overexpression enhanced oxLDL-induced cellular damage, NADPH oxidase and mitochondria-derived ROS production and restored the decrease in protein expression of mitochondria uncoupling protein 2 (UCP2). Collectively, these data suggest that Kv1.5 may play an important role in oxidative vascular endothelial injury.

BACKGROUND: Sirtuin 1 (SIRT1) acts as a key regulator of vascular endothelial homeostasis, angiogenesis, and endothelial dysfunction. However, the underlying mechanism for SIRT1-mediated lung carcinoma angiogenesis remains unknown. Herein, we report that the nicotinamide adenine dinucleotide 1 (NAD1)-dependent deacetylase SIRT1 can function as an intrinsic negative modulator of Delta-like ligand 4 (DLL4)/Notch signaling in Lewis lung carcinoma (LLC) xenograft-derived vascular endothelial cells (lung cancer-derived ECs). PRINCIPAL FINDINGS: SIRT1 negatively regulates Notch1 intracellular domain (N1IC) and Notch1 target genes HEY1 and HEY2 in response to Delta-like ligand 4 (DLL4) stimulation. Furthermore, SIRT1 deacetylated and repressed N1IC expression. Quantitative chromatin immunoprecipitation (qChIP) analysis and gene reporter assay demonstrated that SIRT1 bound to one highly conserved region, which was located at approximately -500 bp upstream of the transcriptional start site of Notch1,and repressed Notch1 transcription. Inhibition of endothelial cell growth and sprouting angiogenesis by DLL4/Notch signaling was enhanced in SIRT1-silenced lung cancer-derived EC and rescued by Notch inhibitor DAPT. In vivo, an increase in proangiogenic activity was observed in Matrigel plugs from endothelial-specific SIRT1 knock-in mice. SIRT1 also enhanced tumor neovascularization and tumor growth of LLC xenografts. CONCLUSIONS: Our results show that SIRT1 facilitates endothelial cell branching and proliferation to increase vessel density and promote lung tumor growth through down-regulation of DLL4/Notch signaling and deacetylation of N1IC. Thus, targeting SIRT1 activity or/and gene expression may represent a novel mechanism in the treatment of lung cancer.

Background Angiogenesis is essential for the progression of osteoarthritis (OA). Hepatocyte growth factor (HGF) is an angiogenic mediator, and it shows elevated levels in regions of OA. However, the relationship between HGF and vascular endothelial growth factor (VEGF-A) in OA synovial fibroblasts (OASFs) is mostly unknown. Methodology/Principal Findings Here we found that stimulation of OASFs with HGF induced concentration- and time-dependent increases in VEGF-A expression. Pretreatment with PI3K inhibitor (Ly294002), Akt inhibitor, or mTORC1 inhibitor (rapamycin) blocked the HGF-induced VEGF-A production. Treatment of cells with HGF also increased PI3K, Akt, and mTORC1 phosphorylation. Furthermore, HGF increased the stability and activity of HIF-1 protein. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that c-Met, PI3K, Akt, and mTORC1 signaling pathways were potentially required for HGF-induced HIF-1α activation. Conclusions/Significance Taken together, our results provide evidence that HGF enhances VEGF-A expression in OASFs by an HIF-1α-dependent mechanism involving the activation of c-Met/PI3K/Akt and mTORC1 pathways.

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.

BackgroundThe family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb.Methodology/principal findingsEctopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors.Conclusions/significanceTaken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.

Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

Many aspects of development, tumor growth and metastasis depend upon the provision of an adequate vasculature. This can be a result of regulated angiogenesis, recruitment of circulating endothelial progenitors and/or vascular trans-differentiation. The present study demonstrates that treatment of SKBR-3 breast cancer cells with retinoic acid (RA), an important regulator of embryogenesis, cancer and other diseases, stimulates the formation of networks in Matrigel. RA-treatment of SKBR-3 cells co-cultured with human umbilical vein endothelial cells resulted in the formation of mixed structures. RA induces expression of many endothelial genes including vascular endothelial (VE) cadherin. VE-cadherin was also induced by RA in a number of other breast cancer cells. We show that RA-induced VE-cadherin is responsible for the RA-induced morphological changes. RA rapidly induced the expression of Sox-9 and ER81, which in turn form a complex on the VE-cadherin promoter and are required to mediate the transcriptional regulation of VE-cadherin by RA. These data indicate that RA may promote the expression of endothelial genes resulting in endothelial-like differentiation, or provide a mechanism whereby circulating endothelial progenitor cells could be incorporated into a growing organ or tumor.

Purpose This study was aimed to identify the expression pattern of vascular endothelial growth factor (VEGF) in non-small cell lung cancer (NSCLC) and to explore its potential correlation with the progression of NSCLC. Methods Gene expression profile GSE39345 was downloaded from the Gene Expression Omnibus database. Twenty healthy controls and 32 NSCLC samples before chemotherapy were analyzed to identify the differentially expressed genes (DEGs). Then pathway enrichment analysis of the DEGs was performed and protein-protein interaction networks were constructed. Particularly, VEGF genes and the VEGF signaling pathway were analyzed. The sub-network was constructed followed by functional enrichment analysis. Results Total 1666 up-regulated and 1542 down-regulated DEGs were identified. The down-regulated DEGs were mainly enriched in the pathways associated with cancer. VEGFA and VEGFB were found to be the initiating factor of VEGF signaling pathway. In addition, in the epidermal growth factor receptor (EGFR), VEGFA and VEGFB associated sub-network, kinase insert domain receptor (KDR), fibronectin 1 (FN1), transforming growth factor beta induced (TGFBI) and proliferating cell nuclear antigen (PCNA) were found to interact with at least two of the three hub genes. The DEGs in this sub-network were mainly enriched in Gene Ontology terms related to cell proliferation. Conclusion EGFR, KDR, FN1, TGFBI and PCNA may interact with VEGFA to play important roles in NSCLC tumorigenesis. These genes and corresponding proteins may have the potential to be used as the targets for either diagnosis or treatment of patients with NSCLC.

Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.

The activation of endothelial cells is essential to repair damage caused by atherosclerosis via endothelial cell proliferation and migration. Overexpression of VEGF (vascular endothelial growth factor) and the downstream gene, B-cell lymphoma-2 (BCL-2) could result in apoptosis-resistant endothelial cells, which are responsible for aggravated hyperplasia and instable plaques generation. Previous studies have shown that miRNA126 could regulate the expression of VEGF. Here, we verified the existence of a miRNA126 binding site in VEGF’s 3’UTR. Additionally, VEGF regulated BCL-2 expression via AP1 (Activator Protein 1) binding site in BCL-2’s promoter. Next, we established an apoptosis-resistant endothelial cell line and constructed a lentiviral vector to express miRNA126 under the control of the BCL-2 promoter to investigate whether conditional expression of miRNA126 could modulate VEGF and BCL-2 expression in apoptosis-resistant endothelial cells. This lentiviral system specifically expressed miRNA126 in cells with high BCL-2 levels, downregulated VEGF expression, inhibited MAPK pathway activation and downregulated BCL-2 expression via suppression of AP1, and as a whole, reduced apoptosis-resistant endothelial cells, while the effects of miRNA126 on normal endothelial cells were relatively small. Our results demonstrate that conditional miRNA126 overexpression under the control of the downstream BCL-2 promoter provides a flexible regulatory strategy for reducing the apoptosis-resistant endothelial cells without having a significant impact on normal endothelial cells.

Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (ENG) or ACVRL1 (also known as ALK1) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. ENG and ACVRL1 encode receptors for the TGFβ superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling in vivo. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity.

The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg), as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR), Kit, colony stimulating factor 1 receptor (CSF1R), and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+) mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use of Abl kinase inhibitors may have potential for the treatment of disorders involving pathological vascular leakage.

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.

We identified that the angiotensin receptor antagonist, candesartan, has profound neurovascular protective properties when administered after ischemic stroke and was associated with a proangiogenic state at least partly explained by vascular endothelial growth factor A (VEGFA). However, the spatial distribution of vascular endothelial growth factor (VEGF) isoforms and their receptors remained unknown. Protein analysis identified a significant increase in vascular endothelial grow factor B (VEGFB) in the cerebrospinal fluid (CSF) and the ischemic hemispheres (with increased VEGF receptor 1 activation) of treated animals (p

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF) has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling) and loss-of-function (Egr-1) approaches demonstrated the role of mitogen-activated protein kinases (MAPKs), PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

BackgroundMigration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1) phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood.Methodology/principal findingsIn this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1) to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1) to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold) in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a) leading to G(1) arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays.Conclusion/significanceThe results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear actin might be critical in the regulation of these fundamental endothelial functions.

The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α β γ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldosterone-modulated endothelial stiffness. Here we explore the functional role of the endothelial αENaC subunit in vascular function in vivo. Compared to littermates, mice with conditional αENaC subunit gene inactivation in the endothelium only (endo-αENaC Knock Out mice) had no difference in their physiological parameters such as systolic blood pressure or heart rate. Acute and long-term renal Na+ handlings were not affected, indicating that endothelial αENaC subunit is not involved in renal sodium balance. Pharmacological inhibition of ENaC with benzamil blunted acetylcholine-induced nitric oxide production in mesenteric arteries from wild type mice but not in endo-αENaC KO mice, suggesting a critical role of endothelial ENaC in agonist-induced nitric oxide production. In endo-αENaC KO mice, compensatory mechanisms occurred and steady state vascular function was not altered except for flow-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo.