Factors In Human Endothelial Cells Research Articles

Fibroblast activation in response to TGFβ1 is modulated by co-culture with endothelial cells in a vascular organ-on-chip platform.

Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFβ1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFβ1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFβ1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.

Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C

Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.

P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development.

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.

Nobiletin inhibits oxidized-LDL mediated expression of Tissue Factor in human endothelial cells through inhibition of NF-κB

IntroductionFlavonoids are nutrients usually included in human diet with several significant biological activities. Nobiletin is a flavonoid that, besides having anti-inflammatory and anti-tumoral activity, seems to exert protective effects on cardiovascular system. Several studies investigated nobiletin as a natural drug to antagonize the atherosclerotic disease. On the contrary, literature about its potential role in modulating the main acute complication of atherosclerosis, thrombosis, is still scanty.Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombotic events by triggering the formation of intracoronary thrombi. Oxidized-LDL have an important role in promoting athero-thrombotic events. This study investigates whether nobiletin might exert protective cardiovascular effects by preventing the oxidized-LDL mediated expression of TF in human endothelial cells in vitro. Moreover, we have studied whether the nobiletin effects might be modulated by the inhibition of the NF-κB pathway. Methods and resultsIn HUVEC, ox-LDL induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by Western-blot, FACS analysis and pro-coagulant activity assay. Nobiletin prevented these ox-LDL-mediated effects by exerting antioxidant effects, finally leading to inhibition of the transcription factor NF-κB. ConclusionsThese data suggest that nobiletin might be a potential antithrombotic agent of dietary origin. This flavonoid, through its antioxidant proprieties, might potentially exert an antithrombotic activity by inhibiting TF expression/activity in a cell population never investigated before in this context and that is normally represented in vessel wall such as endothelial cells.

Autoregulation of glypican-1 by intronic microRNA-149 fine-tunes the angiogenic response to fibroblast growth factor in human endothelial cells

MicroRNA-149 (miR-149) is located within the first intron of the glypican-1 (GPC1) gene. GPC1 is a low affinity receptor for fibroblast growth factor (FGF2) that enhances FGF2 binding to its receptor (FGFR1), subsequently promoting FGF2-FGFR1 activation and signaling. Using bioinformatic approaches, both GPC1 and FGFR1 were identified and subsequently validated as targets for miR-149 (both the mature strand, miR-149, and the passenger strand, miR-149*) in endothelial cells (ECs). As a consequence of their targeting activity towards GPC1 and FGFR1, both miR-149 and miR-149* regulated FGF2 signaling and FGF2-induced responses in ECs, namely proliferation, migration and cord formation. Moreover, lentiviral overexpression of miR-149 reduced in vivo tumor-induced neovascularization. Importantly, FGF2 transcriptionally stimulated the expression of miR-149 independently of its host gene, therefore assuring the steady state of FGF2-induced responses through the regulation of the GPC1-FGFR1 binary complex in ECs.

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells

AbstractA variety of endothelial agonist–induced responses are mediated by rises in intracellular Ca2+, suggesting that different Ca2+ signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca2+ release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca2+ from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca2+-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca2+ mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca2+-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.

Effects of PCB126 and 17β-oestradiol on endothelium-derived vasoactive factors in human endothelial cells

Epidemiological and experimental studies suggest an association between elevated serum levels of co-planar PCBs and hypertension, and one study indicate that this effect is dependent on the level of oestrogen. This study investigated the effects of 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and 17β-oestradiol (E 2) on vasoactive factors in human umbilical vein endothelial cells (HUVEC). The results reveal that PCB126 stimulated the vasoconstriction factors COX-2 and PGF 2α in HUVEC. An up-regulation of COX-2 expression was demonstrated using qRT-PCR, western blot and immunofluorescence and increased production of PGF 2α was demonstrated using LC/MS 2 and enzyme immunoassay. Also, PCB126 slightly increased ROS production and decreased NO production in HUVEC. The addition of E 2 enhanced PCB126-induced transcription of CYP1A1, CYP1B1 and COX-2 in HUVEC whereas an increased transcription of eNOS only occurred following combined treatment with E 2 and PCB126. Immunofluorescence demonstrated that HUVEC expressed AHR and ERβ but lacked ERα and the involvement of AHR and ERβ on the effects of PCB126 was examined by the addition of AHR and ER antagonists. The binding of PCB126 to AHR was critical for the effects of PCB126 whereas the role of ERβ was equivocal. In conclusion, these studies suggest that PCB126 induced changes in human endothelial cells that are characteristic for endothelial dysfunction in human hypertension and that PCB126-induced transcription of genes important for vascular function in human endothelial cells can be elevated by increased oestrogen levels. These findings may help understanding the mechanism for the association between PCB126 exposure and hypertension reported in human subjects and experimental animals.

Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells

Tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-alpha. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-alpha. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-alpha-stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions.

Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and α-Sarcin

Treatment of human endothelial cells with Shiga toxin 1 and 2 leads to the upregulation of genes encoding proinflammatory molecules involved in the pathogenesis of hemolytic-uremic syndrome. The paradoxical effect of inhibitors of mRNA translation, such as Shiga toxins, that at the same time induce protein expression was investigated by studying the relationship between their enzymatic activity (abstraction of adenine from nucleic acids) and the induction of interleukin-8 and granulocyte-macrophage colony-stimulating factor in human endothelial cells. As a positive control, the fungal toxin alpha-sarcin, acting on the same rRNA sequence targeted by Shiga toxins with a different mechanism (RNase activity), was used. The three toxins caused ribosomal lesions that, in turn, induced the activation of p38 stress kinase with kinetics that paralleled the inhibition of translation. Alpha-sarcin was devoid of activity on DNA. Shiga toxin 2 targeted nuclear DNA with more rapid kinetics than did Shiga toxin 1. Since the fungal ribotoxin was fully effective in the induction of proinflammatory proteins, we conclude that damage to ribosomes is indispensable and sufficient to activate protein expression via induction of the stress-kinase cascade. However, gene upregulation events induced by Shiga toxin 2 were much more efficient than those triggered by Shiga toxin 1, although the two toxins impaired translation to the same extent and had overlapping time courses of stress kinase activation. Regulations independent of the ribotoxic stress were assumed to operate in intoxicated cells. We hypothesized that the two bacterial toxins recognize different DNA sequences inducing different regulating effects on gene expression.

PO-09 Expression of alternatively spliced tissue factor in human endothelial cells: induction by inflammatory stimuli and modulation by heparins

PO-09 Expression of alternatively spliced tissue factor in human endothelial cells: induction by inflammatory stimuli and modulation by heparins

Vascular Endothelial Growth Factor Signaling to Endothelial Nitric Oxide Synthase

See related article, pages 715–722 There is substantial evidence supporting the idea that the process of angiogenesis requires the synthesis of endothelium-derived nitric oxide (NO). NO, characterized as a major endothelial-derived relaxing factor, exerts paracrine and autocrine roles in maintaining cardiovascular homeostasis, vascular tone, and microvascular permeability. During the early steps of angiogenesis in which new blood vessels sprout from existing vascular beds, there is a persistence of vasodilation and increase in vascular permeability, suggesting that these hemodynamic changes in the existing vasculature are indispensable during an angiogenic process. A number of angiogenic factors can triggers the release of NO, synthesized by the endothelial isoform of NO synthase (eNOS). One such factor is the vascular endothelial growth factor (VEGF), which as its name implies, is a critical mitogenic, chemoattractant, and survival factor for endothelial cells1 in addition to being characterized as a potent vascular permeability factor.2 Early studies have demonstrated that VEGF can readily stimulate NO production in cultured cells and isolated blood vessels and that NO is essential in mediating the VEGF-induced endothelial cell proliferation and organization into tubes in 3D cultures.3,4 Subsequent in vivo studies have also demonstrated that eNOS knockout mice had significantly attenuated VEGF and ischemia induced angiogenesis and vascular permeability.5,6 These studies place eNOS derived NO as a key mediator of VEGF induced angiogenesis in postnatal mice. Since the discovery of VEGF, or VEGF-A, and subsequent members of the VEGF family (VEGF-B, -C, -D, -E, and placenta growth factor, PlGF), intense research has been performed to elucidate their modes of action. These growth factor ligands bind to 3 receptor tyrosine kinases (RTKs), namely VEGF receptor-1, -2, and -3 as well as to coreceptors such as heparan sulfate proteoglycans or neuropilins. Like many other RTKs, these receptors are able to form …

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Novel angiogenic inhibitor DN-9693 that inhibits post-transcriptional induction of connective tissue growth factor (CTGF/CCN2) by vascular endothelial growth factor in human endothelial cells

Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)-mediated induction of the ctgf/ccn2 gene was a post-transcriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.

Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells.

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes. Notably, the genes significantly up- and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up- and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL-8, IL-6, IL-11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr-1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb-EGF, FGF16, heuregulin beta 1, c-kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin-1 and flt-1 were also upregulated. At 24 h, a clear 'cell cycle' signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c-met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

Fatty acid mobilized by the vascular endothelial growth factor in human endothelial cells.

Release of FFA from membrane phospholipids was observed after incubation of umbilical cord vein-derived endothelial cells (HUVEC) with vascular endothelial growth factor (VEGF). In particular, we found an increase of arachidonate, stearate, and palmitate in a time-dependent manner with a peak at 30 min. The maximum increase was reached by arachidonate (4.4-fold), followed by stearate (2.2-fold) and palmitate (1.3-fold). The arachidonate increase can be ascribed to the activation of phospholipase A2 (PLA2). In fact, cells preincubated with arachidonyl trifluoromethyl ketone, a PLA2 inhibitor, showed a marked reduction in arachidonate mobilization. The role of Ca2+ in PLA2 activation was also investigated. Cells incubated with VEGF in the presence of EGTA showed a marked decrease in arachidonate mobilization, whereas incubation with the calcium ionophore A23187 alone produced an increase in arachidonate, although to a lesser extent compared with the VEGF stimulation. Incubation with A23187 in association with PMA produced the same increase in arachidonate as the VEGF treatment. Mitogen-activated protein kinase (MAPK) activity was also found to increase as a consequence of VEGF stimulation. Taken together, these results suggest that the VEGF-mediated activation of PLA2 in HUVEC is dependent on both MAPK-mediated phosphorylation and Ca2+ increase. Furthermore, the increase in stearate and palmitate likely is brought about by the activation of a pathway involving phospholipase D, phosphatidate phosphohydrolase (PAP), and DAG lipase. In fact, the increase in those FFA was prevented when HUVEC were stimulated with VEGF in the presence of ethanol (which inhibits the formation of phosphatidate), propranolol (a specific inhibitor of PAP), or RHC-80267 (a specific inhibitor of DAG lipase).

Ca 2+-regulated secretion of tissue-type plasminogen activator and von Willebrand factor in human endothelial cells

von Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells which are secreted into the bloodstream upon a stimulus-induced rise in intracellular Ca 2+. Although the release of both factors appears to be regulated similarly, they exhibit opposing physiological effects in the vasculature with vWF inducing coagulation and platelet aggregation and tPA triggering fibrinolysis and thrombolysis. To analyze possible differences in the regulated secretion of vWF and tPA in more detail, we recorded the Ca 2+-triggered exocytosis of both factors in cultured human endothelial cells. We demonstrate that vWF and tPA which are stored in different granules within endothelial cells are released with different kinetics following endothelial stimulation with histamine or the Ca 2+ ionophore A23187. While the stimulus-induced release of vWF increases with time over a course of 30 min, maximal acute secretion of tPA is observed 5 min following stimulation and subsequently drops to background levels. In the case of vWF, secretion can also be monitored indirectly through an antibody-reinternalization assay which indicates an incomplete release of vWF during single exocytotic fusion events. Our data thus point to differences in the Ca 2+-triggered secretion of vWF and tPA which could allow a fine-tuning of their release thereby ensuring a balanced physiological action.

Soluble egg antigens from Schistosoma mansoni induce angiogenesis-related processes by up-regulating vascular endothelial growth factor in human endothelial cells.

Schistosomiasis mansoni is characterized by hepatic granuloma formation. Endothelial cell activation within these granulomas may contribute to their development and to increased vascularization in the granuloma periphery. The earliest event in granuloma formation is the lodging of schistosome eggs within presinusoidal capillaries. The eggs secrete factors that may activate endothelial cells. This study investigated the effects of Schistosoma mansoni soluble egg antigen (SEA) on angiogenic processes: proliferation, tube formation, and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs require serum and growth factors to proliferate in vitro. Proliferation occurred when SEA or live eggs were substituted for growth factors, but not for serum. SEA increased HUVEC tube formation and decreased HUVEC apoptosis after serum and growth factor deprivation. Messenger RNA for vascular endothelial growth factor (VEGF) increased 2-fold in SEA-treated HUVECs. These findings suggest that products secreted by schistosome eggs may promote angiogenesis within hepatic granulomas by up-regulating endothelial cell VEGF.

Identification of Membrane Calcium Channels Essential for Cytoplasmic and Nuclear Calcium Elevations Induced by Vascular Endothelial Growth Factor in Human Endothelial Cells

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the fmt stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

Effects of Binding of Ligand (FVIIa) to Induced Tissue Factor in Human Endothelial Cells

The tissue factor protein is structurally related to the cytokine receptors and ligand binding (factor VIIa) has been reported to give an intracellular calcium signal, thus indicating that tissue factor is a true receptor. In view of the attempts to use recombinant factor VIIa as a therapeutic agent in hemophilia, its binding effects may be of clinical interest. We have studied the effect of ligand binding to human endothelial cells that were stimulated with interleukin-1 to express tissue factor. Human umbilical cord vein endothelial cells produce and release a wide variety of proteins that participate in coagulation and fibrinolysis, and we have investigated whether binding of recombinant factor VIIa to tissue factor altered the release of some of these compounds. Three main findings are reported. (1) After an initial increase, the measurable tissue factor activity in endothelial cells decreased more rapidly in the presence of factor VIIa (half-life 3.7±0.7 hours) than in its absence (half-life 7.4±1.5 hours). This difference was not seen when tissue factor antigen was measured, indicating that ligand binding did not increase the degradation of the protein. (2) Tissue factor pathway inhibitor was detected on the cell surface, in cell homogenates, and in cell medium. When recombinant factor VIIa was added to the cells there was a significant decrease in the release of tissue factor pathway inhibitor to the medium. Four hours after recombinant factor VIIa was added, the levels were 7.5-fold higher in the medium of untreated cells compared to the medium of cells treated with recombinant factor VIIa. (3) We observed increased release of von Willebrand factor (vWF). After 1 and 6 hours with recombinant FVIIa the release was significantly greater than in controls without FVIIa. We did not detect significant differences in the release of tissue plasminogen activator or tissue factor pathway inhibitor.

Akt Takes Center Stage in Angiogenesis Signaling

The serine/threonine kinase Akt, also named protein kinase B, plays a central role in promoting the survival of a wide range of cell types.1 In this issue of Circulation Research , Kim et al2 report that angiopoietin-1 (Ang1) activates this survival kinase and thereby inhibits endothelial cell apoptosis. Ang1 was recently identified as the specific ligand for the Tie receptor family, which during embryonic blood vessel formation is required for vascular remodeling and vessel integrity.3 Mechanistically, Ang1 acts via stimulation of the Tie2 receptor. Upon activation, tyrosine phosphorylation of the Tie2 receptor (most probably at Tyr 11014 ) induces the association and activation of the phosphatidylinositol 3′kinase with subsequent activation of Akt.2 These findings complement previous studies, which demonstrated that vascular endothelial growth factor (VEGF) via the KDR/Flt-1 receptor also stimulates Akt and thereby promotes endothelial cell survival.5 6 Moreover, potent inhibitors of endothelial cell apoptosis such as shear stress or insulin activate Akt.7 8 Thus, activation of Akt seems to be a general antiapoptotic mechanism induced by proangiogenic stimuli. Because apoptosis of endothelial cells counteracts angiogenesis, one may …