Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.     

Effects of thrombin/thrombosis in angiogenesis and tumour progression.

Laboratory, histopathological, pharmacological and clinical evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. On the other hand, epidemiological studies provide evidence of an increased risk of cancer diagnosis following primary thromboembolism. Moreover, the metastatic ability of human breast cancer cells is correlated with the number of thrombin receptors of these cells, and thrombin treatment of B16 melanoma cells dramatically increases the number of lung metastases in rats. We have proposed that these tumour-promoting effects of thrombin can be explained by the ability of thrombin to activate angiogenesis, an essential requirement for tumour progression. Many of the cellular events involved in the angiogenic cascade can be activated by thrombin. At the molecular level, brief exposure of endothelial cells to thrombin causes an upregulation of the receptors (KDR and Flt-1) of VEGF, the key angiogenic mediator. This results in a synergistic effect of thrombin and VEGF in the activation of angiogenesis. In addition, thrombin activates cancer cells to secrete VEGF, thus causing a mutual stimulation between EC and CA cells. Cancer cells exposed to thrombin secrete metalloproteinase 92 KD and overexpress the integrin a(v)b(3), all of which are involved in tumour metastasis.     

The non-proteolytically active thrombin peptide TP508 stimulates angiogenic sprouting

Thrombin is a serine protease that promotes platelet aggregation, blood coagulation, and tissue repair. A peptide derived from a non-proteolytically active region of thrombin, TP508, also promotes tissue repair and increased vascularity, yet does not activate platelet and inflammatory cascades. TP508 binds to cells with high affinity and stimulates cells independent of the proteolytically active thrombin receptors (PARs) and thus is considered to activate a non-proteolytically active receptor (non-PAR) pathway. Using a model of angiogenic sprouting, we further defined the angiogenic potential of TP508 and investigated the role of non-proteolytic, thrombin-mediated pathways in angiogenesis. The assay involves measuring angiogenic sprouting from cultured, intact microvessel fragments. In this assay, TP508 stimulated angiogenic sprouting to an extent similar to or greater than the potent angiogenic factor, VEGF. However, TP508 had no significant effect on the number of sprouts that formed per vessel. In contrast to TP508, proteolytically active receptor agonists had no effect or inhibited angiogenic sprouting. The increased sprouting activity stimulated by TP508 was VEGF dependent but did not involve an increase in VEGF mRNA expression above baseline levels. These results suggest that TP508 acts early in angiogenesis and directly on microvascular cells to accelerate sprouting, but not to induce more sprouting, in a manner different than the intact thrombin molecule.     

Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms

Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by indirectly regulating and organizing a network of angiogenic molecules. In addition, it has been proposed that thrombin can directly activate endothelial cell proliferation. However, in this report it was shown that thrombin is a poor growth factor for human endothelial cells, and its modest mitogenic activity is mediated indirectly by the release of heparin-binding epidermal growth factor, subsequent to proteinase-activated receptor 1 (PAR1) activation. On the other hand, it was demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, pointing to a novel role of thrombin in vascular protection. Analysis by annexin V-propidium iodide double staining revealed that thrombin, specifically, promoted survival of serum-starved endothelial cells in a concentration-dependent manner. In contrast to its mitogenic effect, the anti-apoptotic effect of thrombin was largely independent of its catalytic activity and was mediated through interaction with alphanubeta3 and alpha5beta1 integrins, whereas the involvement of PAR1 was limited. These results provide new insights in understanding the role of thrombin in endothelial cell signaling and vascular biology.     

Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.     

Thrombin enhanced migration and MMPs expression of human chondrosarcoma cells involves PAR receptor signaling pathway

Thrombin is a multifunctional protease that can activate hemostasis and coagulation through the cleavage of fibrinogen to form fibrin clots. Thrombin also plays a crucial role in migration and metastasis of human cancer cells. However, the effect of thrombin on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that thrombin increased the migration and expression of matrix metalloproteinase (MMP)‐2 and MMP‐13 in human chondrosarcoma cells (JJ012 and SW1353 cells). By using pharmacological inhibitors or activators or genetic inhibition by the protease‐activated receptor (PAR), we found that the PAR1 and PAR4 receptor but not PAR3 receptor are involved in thrombin‐mediated cell migration and MMPs expression. Thrombin‐mediated migration and MMPs up‐regulation was attenuated by phospholipase C (PLC), protein kinase C, and c‐Src inhibitor. Activations of PLCβ, PKCα, c‐Src, and NF‐κB pathways after thrombin treatment was demonstrated, and thrombin‐induced MMPs expression and migration activity was inhibited by the specific inhibitors and mutants of PLC, PKC, c‐Src, and NF‐κB cascades. Taken together, our results indicated that thrombin enhances the migration of chondrosarcoma cells by increasing MMP‐2 and MMP‐13 expression through the PAR/PLC/PKCα/c‐Src/NF‐κB signal transduction pathway. J. Cell. Physiol. 223:737–745, 2010. © 2010 Wiley‐Liss, Inc.     

Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: a novel antiangiogenic therapy

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.     

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.     

On the mechanism of thrombin-induced angiogenesis: involvement of alphavbeta3-integrin

Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of alphavbeta3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with alphavbeta3 as demonstrated by direct binding of alphavbeta3 protein to immobilized thrombin. This interaction of thrombin with alphavbeta3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to alphavbeta3 or a specific peptide antagonist to alphavbeta3 can abolish all these alphavbeta3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to alphavbeta3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi.     

Vascular endothelial growth factor up-regulates expression of receptor activator of NF-kappa B (RANK) in endothelial cells. Concomitant increase of angiogenic responses to RANK ligand

Vascular endothelial growth factor (VEGF) is known as a key regulator of angiogenesis during endochondral bone formation. Recently, we demonstrated that TNF-related activation-induced cytokine (TRANCE or RANKL), which is essential for bone remodeling, also had an angiogenic activity. Here we report that VEGF up-regulates expression of receptor activator of NF-kappa B (RANK) and increases angiogenic responses of endothelial cells to TRANCE. Treatment of human umbilical vein endothelial cells (HUVECs) with VEGF increased both RANK mRNA and surface protein expression. Although placenta growth factor specific to VEGF receptor-1 had no significant effect on RANK expression, inhibition of downstream signaling molecules of the VEGF receptor-2 (Flk-1/KDR) such as Src, phospholipase C, protein kinase C, and phosphatidylinositol 3'-kinase suppressed VEGF-stimulated RANK expression in HUVECs. Moreover, the MEK inhibitor PD98059 or expression of dominant negative MEK1 inhibited induction of RANK by VEGF but not the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). VEGF potentiated TRANCE-induced ERK activation and tube formation via RANK up-regulation in HUVECs. Together, these results show that VEGF enhances RANK expression in endothelial cells through Flk-1/KDR-protein kinase C-ERK signaling pathway, suggesting that VEGF plays an important role in modulating the angiogenic action of TRANCE under physiological or pathological conditions.     

Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, plays an important role during angiogenesis. It is constitutively expressed by human endothelial cells as a latent enzyme and requires activation. Thrombin is the only described physiological inducer of gelatinase A in human endothelial cells. In this study, we investigated the mechanisms of gelatinase A activation by another physiological inducer, collagen. Endothelial cells were cultured on various ECM components for 24 h and the conditioned media were assessed for gelatinase A activity using gelatin zymography. The results demonstrated that type I collagen matrix specifically activates gelatinase A after 24 h in human umbilical vein and 48 h in neonatal foreskin endothelial cells. In contrast, thrombin activated gelatinase A after only 2 h. Activation by collagen was sustained over long periods of time in culture (96 h). Unlike thrombin-induced activation, collagen required active membrane type 1-MMP (MT1-MMP) on the endothelial cell surface to activate gelatinase A. In addition, collagen-induced activation of gelatinase A was inhibited by antibodies to the integrin receptor, alpha(2)beta(1), but not alpha(3)beta(1). Our findings, that collagen can provide long-term activation of gelatinase A are likely to be relevant to endothelial cell invasion during angiogenesis.     

Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is considered a pro-inflammatory molecule. We have previously demonstrated that differentiated monocytes express the proteolytically activated receptor for thrombin (PAR-1) and that thrombin enhances the release of interleukin (IL)-6 in human monocytes. In the present study we show that thrombin upregulates the production of both IL-1alpha and IL-1beta in phytohemagglutin (PHA)-activated human peripheral blood mononuclear cells (PBMC). Treating PHA-activated PBMC with the PAR-1 activation peptide, SFLLRN, mimics the effects of thrombin on IL-1alpha and IL-1beta production. Thus, it appears that these pro-inflammatory effects induced by thrombin may be mediated through activation of PAR-1. ELISA and RNase protection assays indicate that thrombin and SFLLRN peptide upregulates IL-1 expression at both protein and mRNA levels. Thrombin directly affects monocyte IL-1 expression, since treatment of differentiated U937 cells with thrombin and SFLLRN enhances IL-1 production. These results may help explain how thrombin can enhance IL-1 expression in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to inflammatory responses observed in several pathophysiological conditions.     

凝血酶与血管内皮细胞

凝血酶不仅在凝血过程中起主导作用,还可以引发多种凝血酶受体介导的分子和细胞间相互作用,在动脉粥样硬化和再狭窄形成中起着重要的作用。现就凝血酶及其受体的特性,以及对血管内皮细胞的作用,包括通透性改变、内皮生长因子、基质金属蛋白酶、黏附分子表达作一综述。     

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.     

R-Ras Protein Inhibits Autophosphorylation of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells and Suppresses Receptor Activation in Tumor Vasculature

Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.