Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.     

Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to alpha v beta 3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind alpha 5 beta 1 integrin. The activity of human tumstatin is mediated by alpha v beta 3 integrin, whereas the activity of human endostatin is mediated by alpha 5 beta 1 integrin. Additionally, although human tumstatin binding to alpha v beta 3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to alpha 5 beta 1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis.     

CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells

CD97, a membrane protein expressed at high levels on inflammatory cells and some carcinomas, is a member of the adhesion G protein-coupled receptor family, whose members have bipartite structures consisting of an extracellular peptide containing adhesion motifs noncovalently coupled to a class B 7-transmembrane domain. CD97alpha, the extracellular domain of CD97, contains 3 to 5 fibrillin class 1 epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) tripeptide, and a mucin stalk. We show here that CD97alpha promotes angiogenesis in vivo as demonstrated with purified protein in a directed in vivo angiogenesis assay (DIVAA) and by enhanced vascularization of developing tumors expressing CD97. These data suggest that CD97 can contribute to angiogenesis associated with inflammation and tumor progression. Strong integrin alpha5beta1 interactions with CD97 have been identified, but alpha v beta3 also contributes to cell attachment. Furthermore, soluble CD97 acts as a potent chemoattractant for migration and invasion of human umbilical vein endothelial cells (HUVECs), and this function is integrin dependent. CD97 EGF-like repeat 4 is known to bind chondroitin sulfate. It was found that coengagement of alpha5beta1 and chondroitotin sulfate proteoglycan by CD97 synergistically initiates endothelial cell invasion. Integrin alpha5beta1 is the first high-affinity cellular counterreceptor that has been identified for a member within this family of adhesion receptors.     

Interaction of alpha9beta1 integrin with thrombospondin-1 promotes angiogenesis

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.     

A novel integrin alpha5beta1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

BACKGROUND: Connective tissue growth factor (CCN2) is upregulated in pancreatic fibrosis and desmoplastic pancreatic tumours. CCN2 interacts with integrin alpha5beta1 on pancreatic stellate cells (PSC) in which it stimulates fibrogenesis, adhesion, migration, and proliferation. AIM: To determine the structural domain(s) in CCN2 that interact with integrin alpha5beta1 to regulation PSC functions. METHODS: Primary activated rat PSC were tested for their adherence to isoforms of CCN2 comprising modules 1-4 (CCN2(1-4)), modules 3-4 (CCN2(3-4)), module 3 alone (CCN2(3)), or module 4 alone (CCN2(4)). Adhesion studies were performed in the presence of EDTA, divalent cations, anti-integrin alpha5beta1 antibodies, CCN2 synthetic peptides, or heparin, or after pretreatment of the cells with heparinase, chondroitinase, or sodium chlorate. CCN2 integrin alpha5beta1 binding was analysed in cell free systems. The ability of CCN2(1-4), CCN2(3-4), or CCN2(4) to stimulate PSC migration was evaluated in the presence of anti-integrin alpha5beta1 or heparin. RESULTS: PSC adhesion was stimulated by CCN2(1-4), CCN2(3-4), or CCN2(4) and supported by Mg2+ but not Ca2+. CCN2(4) supported PSC adhesion or migration were blocked by anti-integrin alpha5beta1 antibodies or by treatment of cells with heparinase or sodium chlorate. A direct interaction between CCN2(4) and integrin alpha5beta1 was demonstrated in cell free assays. The sequence GVCTDGR in module 4 mediated the binding between CCN2(4) and integrin alpha5beta1 as well as CCN2(4) mediated PSC adhesion and migration. CONCLUSIONS: A GVCTDGR sequence in module 4 of CCN2 is a novel integrin alpha5beta1 binding site that is essential for CCN2 stimulated functions in PSC and which represents a new therapeutic target in PSC mediated fibrogenesis.     

Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through alpha 5 beta 1 integrin via mitogen-activated protein Kinase pathway

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.     

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.     

Connective tissue growth factor (CCN2) in rat pancreatic stellate cell function: integrin alpha5beta1 as a novel CCN2 receptor

BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. METHODS: CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin alpha5beta1 was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin alpha5beta1 was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. RESULTS: CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor beta1 treatment. CCN2 promoter activity was stimulated by transforming growth factor beta1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin alpha5beta1-dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin alpha5beta1 production by PSCs was verified by immunoprecipitation, while direct binding between integrin alpha5beta1 and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin alpha5beta1 in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. CONCLUSIONS: PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin alpha5beta1 and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.     

Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, alpha5beta1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to alpha5beta1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.     

Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor

The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744-1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD- containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.     

Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo

We examined the function of alpha4beta1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. Alpha4beta1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by alpha4beta1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an alpha4beta1-dependent manner. Soluble alpha4beta1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1-/- mice. Antagonizing alpha4beta1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the alpha4beta1 binding sequence stimulate angiogenesis in vivo. Therefore, alpha4beta1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.     

Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells

The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.     

Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation.

Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of alphaIIbbeta3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin alpha2beta1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP-/- mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of alpha2beta1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and alpha2beta1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.     

VEGF-mediated endothelial P-selectin translocation: role of VEGF receptors and endogenous PAF synthesis

The acute increase in vascular permeability produced by vascular endothelial growth factor (VEGF-A(165)) requires activation of endothelial Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating factor (PAF) synthesis. Like PAF, VEGF-A(165) promotes translocation of P-selectin to the endothelial cell (EC) surface. However, the mechanisms involved remain unknown. By treating human umbilical vein endothelial cells (HUVECs) with VEGF analogs, we show that activation of VEGFR-1 or VEGFR-2 or both induced a rapid and transient translocation of endothelial P-selectin and neutrophil adhesion to activated ECs. The effects mediated by VEGF-A(165) and VEGF-A(121) (VEGFR-1/VEGFR-2 agonists) were blocked by a selective VEGFR-2 inhibitor, SU1498. VEGF-A(165) was twice as potent as VEGF-A(121), which can be explained by the binding capacity of VEGF-A(165) to its coreceptor neuropilin-1 (NRP-1). Indeed, treatment with NRP-1 antagonist (GST-Ex7) reduced the effect of VEGF-A(165) to the levels observed upon stimulation with VEGF-A(121). Finally, the use of selective PAF receptor antagonists reduced VEGF-A(165)-mediated P-selectin translocation. Together, these data show that maximal P-selectin translocation and subsequent neutrophil adhesion was mediated by VEGF-A(165) on the activation of VEGFR-2/NRP-1 complex and required PAF synthesis.     

Characterization of plasminogen as an adhesive ligand for integrins alphaMbeta2 (Mac-1) and alpha5beta1 (VLA-5)

Plasminogen (Pg) has been implicated in many biologic processes involving extracellular proteolysis. We investigated whether Pg, by virtue of its capacity to be deposited within the extracellular matrix, can serve as a ligand for cell surface integrins. We report here that Pg supports cell adhesion by engaging integrins alphaMbeta2 and alpha5beta1. The immobilized Glu-Pg, but not its derivatives with the N-terminal peptide lacking, plasmin and Lys-Pg, supported efficient adhesion that was abolished by anti-alphaMbeta2 and anti-alpha5beta1 integrin-specific monoclonal antibodies (mAbs). In addition, lysine binding sites of Glu-Pg contributed to cell adhesion inasmuch as tranexamic acid and epsilon-aminocaproic acid inhibited cell adhesion. The involvement of alphaMbeta2 and alpha5)beta1 in adhesion to Glu-Pg was demonstrable with blood neutrophils, U937 monocytoid cells, and genetically engineered alphaMbeta2-transfected human embryonic kidney (HEK) 293 cells. In alphaMbeta2, the alphaMI-domain is the binding site for Glu-Pg because the "I-less" form of alphaMbeta2 did not support cell adhesion and the recombinant alphaMI-domain bound Glu-Pg directly. In comparison with cell adhesion, the binding of soluble Glu-Pg to cells and the concomitant generation of plasmin activity was inhibited by anti-alpha5beta1 but not by anti-alphaMbeta2. These findings identify Glu-Pg as an adhesive ligand for integrins alphaMbeta2 and alpha5beta1 and suggest that alpha5beta1 may participate in the binding of soluble Glu-Pg and assist in its activation.     

Ligation of CD31 (PECAM-1) on endothelial cells increases adhesive function of alphavbeta3 integrin and enhances beta1 integrin-mediated adhesion of eosinophils to endothelial cells.

We determined the role of the heterophilic interaction of alphavbeta3 integrin on endothelial cells with CD31 on leukocytes in mediating leukocyte-endothelial cell interactions. Preincubation of interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) with anti-CD31 monoclonal antibodies (MoAbs) enhanced eosinophil adhesion to the IL-4-stimulated HUVECs, and the endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs was prevented by anti-vascular cell adhesion molecule-1 (VCAM-1) MoAb and anti-very late activation antigen-4 (VLA-4) MoAb, but not by anti-intercellular adhesion molecule-1 (ICAM-1) MoAb, anti-lymphocyte function-associated antigen-1 (LFA-1) MoAb, anti-P-selectin MoAb, or anti-E-selectin MoAb. CD31 stimulation of HUVECs increased the adhesive function of alphavbeta3 integrin to its ligand RGD peptide, the binding of which reached a maximum at 10 minutes after the stimulation, and the CD31-induced alphavbeta3 integrin activation on HUVECs was inhibited by inhibitors of protein kinase C and phosphatidylinositol 3 kinase (PI3-kinase). Furthermore, anti-alphavbeta3 integrin MoAb and RGD peptide as well as soluble CD31 inhibited endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs. However, anti-alphavbeta3 integrin MoAb had no significant inhibitory effect on the eosinophil adhesion to IL-4-stimulated or unstimulated HUVECs without CD31 stimulation of HUVECs. Finally, CD31 stimulation of eosinophils increased the adhesive function of alpha4beta1 integrin (VLA-4) to its ligand fibronectin and their adhesion to IL-4-stimulated HUVECs in a VLA-4-dependent manner. These results indicate that CD31-mediated inside-out signaling activates alphavbeta3 integrin on endothelial cells, that the heterophilic alphavbeta3 integrin/CD31 interaction induces beta1 integrin-mediated adhesion of eosinophils to endothelial cells, and that the heterophilic interaction itself is not significantly involved in firm adhesion of eosinophils to endothelial cells.     

The integrin, alpha6beta1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the migration of human hepatocarcinoma cells.

Expression of the integrin, alpha6beta1, a receptor for laminins, is associated with the progression of hepatocellular carcinoma (HCC). The approach to investigating the alpha6beta1 integrin signaling in HCC cells was to express a deletion mutant of the beta4 integrin cytoplasmic domain (beta4-Deltacyt) in 2 HCC cell lines, HepG2 and Huh7. Expression of this mutant prevents formation of the alpha6beta1 heterodimer. As expected, adhesion of both the HepG2/beta4-Deltacyt and Huh7/beta4-Deltacyt transfectants to laminin, but not to collagen, was reduced compared with the mock transfectants. However, migration of the beta4-Deltacyt transfectants toward both collagen and laminin was inhibited, suggesting a role for alpha6beta1 in the signaling of migration. Migration of HCC cells requires mitogen-activated protein (MAP) kinase. The adhesion of the beta4-Deltacyt transfectants to collagen resulted in a substantial reduction in MAP kinase activation in comparison with the mock transfectants, although their ability to activate MAP kinase in response to epidermal growth factor (EGF) stimulation was not impaired. In addition, matrix adhesion of the beta4-Deltacyt transfectants did not stimulate the tyrosine phosphorylation of focal adhesion kinase (FAK), and this defect correlated with reduced binding of adaptor protein Grb2 to FAK. These results suggest that FAK tyrosine phosphorylation is dependent on alpha6beta1 expression, and that FAK-Grb2 association plays a central role in alpha6beta1-mediated activation of MAP kinase. Moreover, the expression of alpha6beta1 in HCC cells is necessary for FAK/MAP kinase-dependent migration.     

Plasminogen activator inhibitor type 1 promotes fibrosarcoma cell migration by modifying cellular attachment to vitronectin via alpha(v)beta(5) integrin

Both urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) are associated with a poor prognosis in cancer patients. We demonstrate that PAI-1 inhibits human fibrosarcoma cell (HT-1080) adhesion to vitronectin (Vn) via alpha (v)beta (5) integrin, and stimulates cell migration from Vn toward collagen type IV (Col). The cells attached more strongly to Vn and Col than to fibronectin (Fn), whereas PAI-1 interfered with cell attachment to Vn only. An integrin antagonist, RGD peptide, and anti-alpha (v)beta (5) integrin antibodies, which similarly inhibited cell attachment to Vn, also stimulated cell migration from Vn toward Col. u-PA did not modify cell attachment directly, but reversed the PAI-1-mediated inhibitory effect on cell adhesion to Vn, and its stimulatory effect on cell migration from Vn toward Col. Thus HT-1080 cell migration appears to be modified by u-PA and PAI-1, altering cell adhesion to Vn via alpha (v)beta (5) integrin. This may be related to their tumor-promoting effect.     

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G(919-1207) to support endothelial cell adhesion. In particular, endothelial cell adhesion to G(919-1207) is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the alphavbeta3 integrin in angiogenesis.     

Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with cells expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein. A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affinity chromatography of 125I-labeled CHO cell membrane proteins, using IGFBP-1 coupled to agarose, identified the alpha 5 beta 1 integrin (fibronectin receptor) as the only cell surface molecule capable of binding IGFBP-1 in an RGD-dependent manner. Furthermore, wild-type IGFBP-1, but not the WGD mutant form, could be coprecipitated from CHO cells with an antibody directed against the alpha 5 integrin subunit. These studies demonstrate that IGFBP-1 stimulates CHO cell migration and binds to the alpha 5 beta 1 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the alpha 5 beta 1 integrin.