Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2

Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.     

Fibroblast growth factor-2 did not restore plasminogen system activity in endothelial cells on glycated collagen

People with diabetes experience morbidity and mortality from unregulated microvascular remodeling, which may be linked to hyperglycemia. Elevated glucose leads to extracellular matrix collagen glycation, which delays endothelial capillary-like tube formation in vitro. Glucose also increases endothelial cell fibroblast growth factor-2 (FGF-2) release and extracellular matrix storage, which should increase tube formation. In this study, we determined if FGF-2 could restore plasminogen system activity and angiogenic function in endothelial cells on glycated collagen. Human umbilical vein endothelial cells cultured on native or glycated collagen substrates were stimulated with FGF-2. Plasminogen system activity, cell migration, and capillary-like tube formation were measured, along with plasminogen system protein and mRNA levels. Glycated collagen decreased endothelial cell plasminogen system activity, cell migration, and tube length. FGF-2 did not restore plasminogen system activity or tube formation in cells on glycated collagen, despite decreasing plasminogen activator inhibitor-1 (PAI-1) protein level. We now show that PAI-1 binds to glycated collagen, which may localize PAI-1 to the extracellular matrix. These data suggest that FGF-2 may not restore angiogenic functions in endothelial cells on glycated collagen due to PAI-1 bound to glycated collagen.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Hepatocyte growth factor upregulates α2β1 integrin in Madin-Darby canine kidney cells: Implications in tubulogenesis

It has been well established that hepatocyte growth factor (HGF) induces branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, focalization proteolysis, cell-cell interaction and differentiation. However, signaling pathways and proteins involved in HGF-induced tubulogenesis by MDCK cells have not been thoroughly studied. Because cell-matrix interactions play important roles in tubulogenesis, we analyzed whether HGF altered the expression of extracellular matrix receptor (alpha2, alpha3, beta1 and alphavbeta3 integrin). We found that among those proteins examined, alpha2beta1 integrin levels were enhanced by HGF. HGF-induced upregulation of alpha2beta1 integrin was mediated via upregulation of alpha2 integrin mRNA abundance. Cycloheximide blocked the HGF-induced increase in alpha2 integrin mRNA expression. To understand the signaling pathways leading to an HGF-induced increase in alpha2beta1 integrin levels, PD98059 (MEK1 inhibitor), LY294002 (PI3-kinase inhibitor), and GF109203X (PKC inhibitor) were used. We found that PD98059 blocked the HGF-induced increase in alpha2beta1 integrin expression. Furthermore, 5E8 (specific anti-alpha2beta1 integrin antibody) was employed to elucidate the potential role of HGF-induced upregulation of alpha2beta1 integrin in branching morphogenesis. 5E8 did not alter HGF-induced scattering effects but disrupted HGF-induced branching tubulogenesis in collagen gel via inhibition of cell-cell interactions and growth. Taken together, HGF upregulates alpha2beta1 integrin expression via an indirect pathway, the results of which contribute to the regulation of cell-cell interactions and cell growth during branching morphogenesis in collagen gel.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.     

Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.     

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.     

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.     

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.     

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.     

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.     

Role of autophagy in angiogenesis in aortic endothelial cells

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Identification of a novel integrin alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin alphavbeta3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin alphavbeta3. Immobilized synthetic V2 peptide supports alphavbeta3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin alphavbeta3 and abolished alphavbeta3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alphavbeta3 and impaired in alphavbeta3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds alphavbeta3 and supports alphavbeta3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alphavbeta3 binding site in soluble CCN1, whereas additional cryptic alphavbeta3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin alphavbeta3 and provide a new approach for dissecting alphavbeta3-specific CCN1 functions both in cultured cells and in the organism.