Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin alpha(v)beta3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow.     

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.     

Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin alpha(v)beta3 to glycosaminoglycans

Tumor endothelial marker (TEM) 5 is a member of the adhesion family of G-protein-coupled receptors and up-regulated in endothelial cells during tumor and physiologic angiogenesis. Here, we report that TEM5 is expressed on the surface of endothelial cells. A soluble TEM5 (sTEM5) fragment is shed by endothelial cells during capillary-like network formation and upon growth factor stimulation. We found that sTEM5 binds to several glycosaminoglycans. Furthermore, sequence analysis and functional and biochemical studies revealed that sTEM5 contains a cryptic RGD-binding site for integrin alpha(v)beta3. Matrix metalloprotease 9-processed, but not full-length, sTEM5 mediated endothelial cell adhesion by direct interaction with integrin alpha(v)beta3. Adhesion to proteolytically processed sTEM5 (ppsTEM5) or glycosaminoglycan-bound ppsTEM5 promoted survival of growth factor deprived endothelial cells. ppsTEM5-mediated cell survival was inhibited by a function blocking integrin alpha(v)beta3 antibody. Based on our results we conclude that sTEM5 is shed by endothelial cells during angiogenesis and binds to glycosaminoglycans present on extracellular matrix and cell surface proteoglycans. Further proteolytic processing of sTEM5 leads to exposure of its RGD motif mediating endothelial cell survival by linking integrin alpha(v)beta3 to glycosaminoglycans.     

Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin.

The Tat protein of HIV-1, a transactivator of viral gene expression, is released by acutely infected T cells and, in this form, exerts angiogenic activities. These have linked the protein to the pathogenesis of Kaposi's sarcoma (KS), a vascular tumor frequent and aggressive in HIV-1-infected individuals (AIDS-KS). In this study, we show that a combination of the same inflammatory cytokines increased in KS lesions, namely IL-1 beta, TNF-alpha, and IFN-gamma, synergizes with Tat to promote in nude mice the development of angioproliferative KS-like lesions that are not observed with each factor alone. Inflammatory cytokines induce the tissue expression of both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two angiogenic molecules highly produced in primary KS lesions. However, bFGF, but not VEGF, synergizes with Tat in vivo and induces endothelial cells to migrate, to adhere, and to grow in response to Tat in vitro. Tat angiogenic effects correlate with the expression of the alpha v beta 3 integrin that is induced by bFGF and binds the arginine-glycine-aspartic acid (RGD) region of Tat. In contrast, no correlation is observed with the expression of alpha v beta 5, which is promoted by VEGF and binds Tat basic region. Finally, KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD-binding integrins. Because alpha v beta 3 is an RGD-binding integrin that is highly expressed in primary KS lesions, where it colocalizes with extracellular Tat on vessels and spindle cells, these results suggest that alpha v beta 3 competitors may represent a new strategy for the treatment of AIDS-KS.     

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.     

The snake venom disintegrin salmosin induces apoptosis by disassembly of focal adhesions in bovine capillary endothelial cells

Salmosin, a disintegrin purified from a Korean snake (Agkistrodon halys brevicaudus) venom, interacts with integrin alpha(v)beta(3) and inhibits the proliferation of bovine capillary endothelial (BCE) cells induced by basic fibroblast growth factor (bFGF). We investigated salmosin's mechanism of inhibition of BCE cell proliferation by examining changes in the cytoskeleton and activation of integrin-mediated signaling molecules. Salmosin disassembled cortical actins at focal adhesions and induced cells to be rounded and detached, but it did not alter microtubule structures in the early stage of cells being rounded. Immunolocalization of paxillin also demonstrated that focal adhesions were disassembled by salmosin. In salmosin-treated BCE cells, focal adhesion kinase (FAK) was dephosphorylated and expression of paxillin and p130(CAS) was decreased, but PI3 kinase, ILK, and beta-catenin were not expressed in decreased amounts or modified, suggesting that salmosin inactivated FAK-dependent integrin signaling pathways. While BCE cells proliferated normally on plates coated with salmosin, cells treated with salmosin eventually underwent apoptosis. These observations strongly suggest that salmosin disorganizes focal contacts to detach cells by competing with the extracellular matrix (ECM) for direct binding to integrin alpha(v)beta(3) on the cell surface, eventually leading to apoptosis.     

Basic FGF and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells.

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) are known to alter the migratory and proliferative capacity of endothelial cells in vitro and to stimulate angiogenesis in vivo. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor expression and activity. We examined the ability of these growth factors to modulate the expression of specific integrins in human microvascular endothelial cells (MEC). Immunoprecipitation of metabolically labeled MEC showed that bFGF upregulated the biosynthesis of alpha 2, alpha 5, beta 1, and beta 3. bFGF induced an increase in the levels of mRNA for alpha 2 and beta 1. TGF-beta increased synthesis of alpha 2, alpha 5, and beta 1. These results suggest that bFGF and TGF-beta selectively alter integrin profiles and influence interactions of MEC with the extracellular matrix during neovascularization. In particular, the upregulation of the collagen/laminin receptor, alpha 2 beta 1, by bFGF may provide activated endothelial cells with an enhanced capacity to migrate through both their underlying basement membrane and the interstitial matrix.     

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Regulation of in vitro capillary tube formation by anti-integrin antibodies

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti- integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.     

Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth

We have previously hypothesized that the development of severe angioproliferative pulmonary hypertension is associated with not only initial endothelial cell (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC but also with proliferation of vascular smooth muscle cells (VSMC). We have demonstrated that EC death results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype. We postulate here that the initial apoptosis of EC induces the release of mediators that cause VSMC proliferation. We cultured EC in an artificial capillary CellMax system designed to simulate the highly efficient functions of the human capillary system. We induced apoptosis of microvascular EC using shear stress and the combined VEGF receptor (VEGFR-1 and -2) inhibitor SU-5416. Flow cytometry for the proliferation marker bromodeoxyuridine showed that serum-free medium conditioned by apoptosed EC induced proliferation of VSMC, whereas serum-free medium conditioned by nonapoptosed EC did not. We also show that medium conditioned by apoptosed EC is characterized by increased concentrations of transforming growth factor (TGF)-beta1 and VEGF compared with medium conditioned by nonapoptosed EC and that TGF-beta1 blockade prevented the proliferation of cultured VSMC. In conclusion, EC death induced by high shear stress and VEGFR blockade leads to the production of factors, in particular TGF-beta1, that activate VSMC proliferation.     

Proatherogenic flow conditions initiate endothelial apoptosis via thrombospondin-1 and the integrin-associated protein

Recently it has been shown that vascular endothelial cells (EC) are completely devoid of apoptosis if cultivated under a steady laminar flow and that apoptosis is induced by turning off the flow. An autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex has been identified as the molecular coupling device between flow and apoptosis. Lack of blood flow is a rare and mostly transient phenomenon whereas irregular flow conditions are permanently present at arterial bifurcations and sites of abnormal vessel morphology. Irregular flow conditions are established here either by the action of a cone-and-plate type flow apparatus generating a uniform turbulent flow or in a flow chamber by insertion of a local hindrance creating a zone of unsteady laminar flow with vortex formation and lowered shear stress. In both cases apoptosis is induced either throughout the entire monolayer or restricted to the locally defined area. Flow disturbance and apoptosis are coupled by the described autocrine loop of TSP-1 and the integrin/IAP receptor complex. In vivo atherosclerotic lesions occur predominantly at sites of flow irregularities, which are thought to be pro-atherogenic. Thus we propose a key role of the identified mechanosensitive apoptosis induction for the initiation of atherosclerosis.     

Localization of integrin alpha(v)beta3 and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in cutaneous and oral melanomas of dog

Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.     

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

Nicotine regulates basic fibroblastic growth factor and transforming growth factor beta1 production in endothelial cells

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.     

Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1

Endothelium extracellular matrix (ECM) interactions can provide distinct spatial and molecular signals which control cellular proliferation, migration, and differentiation. Here, we investigated the role of fibronectin (FN), a major ECM protein, on the functions of lymphatic endothelial cells (LEC). We observed that FN, the ligand for integrin alpha5beta1, selectively promoted the growth of LEC as compared with vitronectin (VN) in the presence of the ligand for vascular endothelial growth factor receptor 3 [VEGFR-3 (VEGF-C156S)]. Upon investigating the mechanisms whereby ECM components regulate VEGFR-3 signaling, we found that FN transactivated VEGFR-3 and significantly enhanced the phosphorylation of VEGFR-3 induced by VEGF-C156S as compared to VN. An enhanced association of the integrin subunit alpha5 or beta1 with VEGFR-3, after stimulation with VEGF-C156S, was observed by co-immunoprecipitation. While blockade of integrin alpha5beta1 inhibited the VEGF-C156S-induced phosphorylation of VEGFR-3, no similar effect was obtained by blocking integrin alphavbeta3. FN also protected the endothelial cells from serum deprivation-induced apoptosis. Moreover, while the specific PI3 kinase inhibitor, LY294002, abolished this FN-mediated cell survival, the MAPK kinase inhibitor, PD98059, had no significant effect. Furthermore, a dominant-negative mutant of VEGFR-3 (G857R) reduced VEGF-C156S or FN-mediated cell survival, as well as the activities of PI3 kinase/Akt. Our results indicate that integrin alpha5beta1 participates in the activation of both VEGFR-3 and its downstream PI3 kinase/Akt signaling pathway, which is essential for FN-mediated lymphatic endothelial cell survival and proliferation.