An inhibitory role of the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression.

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but is also capable of inducing tissue factor, the prime initiator of coagulation, in endothelial cells. In this study we have analyzed the VEGF-elicited pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells. Using specific low molecular weight inhibitors we could demonstrate a crucial role of the p38 and Erk-1/2 mitogen-activated protein (MAP) kinases. In contrast, treatment with wortmannin or LY294002, inhibitors of phosphatidylinositol 3 (PI3)-kinase, resulted in a strong enhancement of the VEGF-induced tissue factor production, indicating a negative regulatory role of the PI3-kinase on tissue factor-inducing pathways. Accordingly, transduction with constitutively active Akt led to a reduction of VEGF-induced tissue factor production. Western blot analyses using antibodies specific for phosphorylated p38 showed an enhanced activation of this MAP kinase in human umbilical cord vein endothelial cells when stimulated with VEGF in the presence of wortmannin in comparison to either agent alone. Thus, the negative regulation of the PI3-kinase pathway on endothelial tissue factor activity can be explained at least in part by a suppression of this MAP kinase-signaling pathway. This is the first demonstration of a reciprocal relationship between procoagulant activity and the PI3-kinase-Akt signaling pathway, and it reveals a novel mechanism by which tissue factor expression can be controlled in endothelial cells.     

sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. We find that vascular endothelial growth factor (vegf) acts downstream of sonic hedgehog (shh) and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells.     

Hypoxia-mediated activation of Dll4-Notch-Hey2 signaling in endothelial progenitor cells and adoption of arterial cell fate

Adequate response to low oxygen levels (hypoxia) by hypoxia inducible factor (HIF) is essential for normal development and physiology, but this pathway may also contribute to pathological processes like tumor angiogenesis. Here we show that hypoxia is an inducer of Notch signaling. Hypoxic conditions lead to induction of the Notch ligand Dll4 and the Notch target genes Hey1 and Hey2 in various cell lines. Promoter analysis revealed that Hey1, Hey2 and Dll4 are induced by HIF-1alpha and Notch activation. Hypoxia-induced Notch signaling may also determine endothelial identity. Endothelial progenitor cells (EPCs) contain high amounts of COUP-TFII, a regulator of vein identity, while levels of the arterial regulators Dll4 and Hey2 are low. Hypoxia-mediated upregulation of Dll4 and Hey2 leads to repression of COUP-TFII in eEPCs. Finally, we show that Hey factors are capable of repressing HIF-1alpha-induced gene expression, suggesting a negative feedback loop to prevent excessive hypoxic gene induction. Thus, reduced oxygen levels lead to activation of the Dll4-Notch-Hey2 signaling cascade and subsequent repression of COUP-TFII in endothelial progenitor cells. We propose that this is an important step in the developmental regulation of arterial cell fate decision.     

Luteolin inhibits pyrogallol-induced apoptosis through the extracellular signal-regulated kinase signaling pathway

Luteolin is an antioxidative, antitumor and anti-inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol-induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0-400 μm) dose-dependently induced reactive oxygen species production, cytotoxicity, an annexin V-fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78-50 μm) in a concentration-dependent manner. Luteolin suppressed the poly (ADP-ribose) polymerase activation, caspase-8 cleavage and p38 mitogen-activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal-regulated kinase signaling pathway to counteract the pyrogallol-induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress-induced apoptosis via the extracellular signal-regulated kinase signaling pathway.     

Metabolite control of angiogenesis: angiogenic effect of citrate

Endothelial cells respond to hypoxic changes with resultant accumulation of several metabolites and switch over to angiogenic phenotype. Although certain intermediates of glycolytic and oxidative metabolic pathways have been known to affect angiogenesis, the effect of citrate, which accumulates in certain tumors, on angiogenesis is not known. Therefore, the effect of citrate on angiogenesis was studied using different model systems. Increased vascularization in chorioallantoic membrane assay, increased endothelial sprouting in rat aortic rings, and increased expression of CD31, E-selectin in endothelial cells suggested a possible proangiogenic effect of citrate. Upregulation of angiogenic factors such as vascular endothelial growth factor and fibroblast growth factor suggested that the effect of citrate involves modulation of expression of angiogenic growth factors. LY 294002, an inhibitor of PI3K–Akt pathway, and wortmannin, an inhibitor of Akt pathway, reversed the effect of citrate in human umbilical vein endothelial cells. Citrate induced significant upregulation and activation of Akt in endothelial cells. Rapamycin, an inhibitor of mTOR, also reversed the effect of citrate in human umbilical vein endothelial cells and sprouting of aortic rings suggesting that the angiogenic effect of citrate involves activation of PI3K–Akt–mTOR pathway.     

NADPH oxidase activity selectively modulates vascular endothelial growth factor signaling pathways

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular physiology and pathophysiology. We have demonstrated previously that NADPH oxidase-derived ROS are required for VEGF-mediated migration and proliferation of endothelial cells. The goal of this study was to determine the extent to which VEGF signaling is coupled to NADPH oxidase activity. Human umbilical vein endothelial cells and/or human coronary artery endothelial cells were transfected with short interfering RNA against the p47(phox) subunit of NADPH oxidase, treated in the absence or presence of VEGF, and assayed for signaling, gene expression, and function. We show that NADPH oxidase activity is required for VEGF activation of phosphoinositide 3-kinase-Akt-forkhead, and p38 MAPK, but not ERK1/2 or JNK. The permissive role of NADPH oxidase on phosphoinositide 3-kinase-Akt-forkhead signaling is mediated at post-VEGF receptor levels and involves the nonreceptor tyrosine kinase Src. DNA microarrays revealed the existence of two distinct classes of VEGF-responsive genes, one that is ROS-dependent and another that is independent of ROS levels. VEGF-induced, thrombomodulin-dependent activation of protein C was dependent on NADPH oxidase activity, whereas VEGF-induced decay-accelerating factor-mediated protection of endothelial cells against complement-mediated lysis was not. Taken together, these findings suggest that NADPH oxidase-derived ROS selectively modulate some but not all the effects of VEGF on endothelial cell phenotypes.     

Factor X/Xa Elicits Protective Signaling Responses in Endothelial Cells Directly via PAR-2 and Indirectly via Endothelial Protein C Receptor-dependent Recruitment of PAR-1

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.     

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Notch signaling is required for arterial-venous differentiation during embryonic vascular development

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line     

Differences and similarities between mesenchymal stem cell and endothelial progenitor cell immunoregulatory properties against T cells

Bone-marrow-derived mesenchymal stem cells and endothelial progenitor cells have some interesting biological properties that make them unique for cell therapy of degenerative and cardiovascular disorders. Although both cell populations have been already studied and used for their regenerative potentials, recently their special immunoregulatory features have brought much more attention. Mesenchymal stem cells and endothelial progenitor cells have both proangiogenic functions and have been shown to suppress the immune response, particularly T cell proliferation, activation, and cytokine production. This makes them suitable choices for allogeneic stem cell transplantation. Nevertheless, these two cells do not have equal immunoregulatory activities. Many elements including their extraction sources, age/passage, expression of different markers, secretion of bioactive mediators, and some others could change the efficiency of their immunosuppressive function. However, to our knowledge, no publication has yet compared mesenchymal stem cells and endothelial progenitor cells for their immunological interaction with T cells. This review aims to specifically compare the immunoregulatory effect of these two populations including their Tcell suppression, deactivation, cytokine production, and regulatory T cells induction capacities. Moreover, it evaluates the implications of the tumor necrosis factor alpha-tumor necrosis factor receptor 2 axis as an emerging immune checkpoint signaling pathway controlling most of their immunological properties.     

The role of Wingless signaling in establishing the anteroposterior and dorsoventral axes of the eye disc

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. We show here that mutations in axin prevent photoreceptor differentiation and lead to tissue overgrowth and that both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. We also show that signaling by Decapentaplegic and Hedgehog normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Decapentaplegic signaling can promote its downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors.     

Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.