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).     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.     

Human IL-3 stimulates endothelial cell motility and promotes in vivo new vessel formation.

Angiogenesis is a critical process for growth of new capillary blood vessels from preexisting capillaries and postcapillary venules, both in physiological and pathological conditions. Endothelial cell proliferation is a major component of angiogenesis and it is regulated by several growth factors. It has been previously shown that the human hemopoietic growth factor IL-3 (hIL-3), predominantly produced by activated T lymphocytes, stimulates both endothelial cell proliferation and functional activation. In the present study, we report that hIL-3 is able to induce directional migration and tube formation of HUVEC. The in vivo neoangiogenetic effect of hIL-3 was also demonstrated in a murine model in which Matrigel was used for the delivery of the cytokine, suggesting a role of hIL-3 in sustaining neoangiogenesis. Challenge of HUVEC with hIL-3 lead to the synthesis of platelet-activating factor (PAF), which was found to act as secondary mediator for hIL-3-mediated endothelial cell motility but not for endothelial cell proliferation. Consistent with the role of STAT5 proteins in regulating IL-3-mediated mitogenic signals, we herein report that, in hIL-3-stimulated HUVEC, the recruitment of STAT5A and STAT5B, by the beta common (betac) subunit of the IL-3R, was not affected by PAF receptor blockade.     

CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.     

Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo

Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor alphavbeta3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-alphavbeta3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-alphavbeta3-dependent pathways.     

Apolipoprotein(a) stimulates vascular endothelial cell growth and migration and signals through integrin alphaVbeta3

Elevated plasma concentrations of Lp(a) [lipoprotein(a)] are an emerging risk factor for atherothrombotic disease. Apo(a) [apolipoprotein(a)], the unique glycoprotein component of Lp(a), contains tandem repeats of a plasminogen kringle (K) IV-like domain. In the light of recent studies suggesting that apo(a)/Lp(a) affects endothelial function, we evaluated the effects of apo(a)/Lp(a) on growth and migration of cultured HUVECs (human umbilical-vein endothelial cells). Two full-length r-apo(a) [recombinant apo(a)] variants (12K and 17K), as well as Lp(a), were able to stimulate HUVEC growth and migration to a comparable extent; 17K r-apo(a) also decreased the levels of total and active transforming growth factor-beta secreted by these cells. Using additional r-apo(a) variants corresponding to deletions and/or site-directed mutants of various kringle domains in the molecule, we were able to determine that the observed effects of full-length r-apo(a) on HUVECs were dependent on the presence of a functional lysine-binding site(s) in the apo(a) molecule. With respect to signalling events elicited by apo(a) in HUVECs, we found that 17K treatment of the cells increased the phosphorylation level of FAK (focal adhesion kinase) and MAPKs (mitogen-activated protein kinases), including ERK (extracellular-signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase). In addition, we showed that LM609, the function-blocking antibody to integrin alphaVbeta3, abrogated the effects of 17K r-apo(a) and Lp(a) on HUVECs. Taken together, the results of the present study suggest that the apo(a) component of Lp(a) signals through integrin alphaVbeta3 to activate endothelial cells.     

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.     

Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression

Monocytes play an important role in collateral vessel formation (arteriogenesis) by attaching to activated endothelium and by invading the walls of innate collateral vessels where they produce growth factors. Previous studies have demonstrated that this process can be promoted by several chemokines and growth factors. In this study we examined the interaction between monocytes and endothelium under stimulation of the angiogenic agent vascular endothelial growth factor (VEGF). We report here the novel finding that VEGF stimulates the expression of the alphaL-, alphaM- and beta2-integrin monomers. In functional assays and by using neutralizing antibodies it was shown that VEGF stimulates adhesion of monocytes to human umbilical vein endothelial cells (HUVEC), and increased transmigration through endothelial monolayers is dependent on interaction of monocyte beta2-integrins with its endothelial counter ligand ICAM-1. Based on these in vitro data we hypothesize that the positive effect of VEGF on arteriogenesis may involve monocyte activation.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Betaig-h3 supports keratinocyte adhesion,migration, and proliferation through alpha3beta1 integrin

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.     

Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration

Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.     

Cross-talk of integrin alpha3beta1 and tissue factor in cell migration

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.     

Platelet endothelial aggregation receptor-1 regulates bovine muscle satellite cell migration and differentiation via integrin beta-1 and focal adhesion kinase

PEAR1 is highly expressed at bovine MDSC differentiation. However, its biological function remains unclear. Western blotting results showed that PEAR1 increased between day 0 and day 2 of cell differentiation and decreased from day 3. Moreover, scratch test showed that wound healing rate increased after PEAR1 overexpression and decreased upon its suppression. Meanwhile, we found that, upon PEAR1 induction, both the expression of the focal adhesion-associated and MyoG, and the myotube fusion rate increased. However, when PEAR1 was suppressed, opposite results were obtained. Immunoprecipitation revealed an association between PEAR1 and ITGB1. Notably, inhibition of FAK and ITGB1 repressed cell differentiation. In conclusion, our study indicated that PEAR1 is involved in the regulation of bovine MDSC migration and differentiation.     

TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen

The TEM8 gene is selectively expressed in tumor versus normal blood vessels, though its function in endothelial cell biology is not known. Towards the goal of clarifying this function, we tested whether TEM8 overexpression, or blocking TEM8's function with a dominant negative protein, would modulate endothelial cell activities. We found that TEM8-expressing endothelial cells migrated at a rate 3-fold greater than control cells in a monolayer denudation assay. Also, the addition of recombinant TEM8 extracellular domain (TEM8-ED) specifically inhibited both chemokinetic and chemotactic migration on collagen in the denudation and Boyden chamber assays, respectively. The TEM8-ED binds preferentially to collagen, and TEM8 expression enhanced endothelial adhesion to collagen 3-fold; the latter response was antagonized by the TEM8-ED. Consistent with the TEM8-ED acting as a dominant negative inhibitor of endogenously expressed protein were data showing that the TEM8-ED had no effect on the activation of beta1 integrin. TEM8 protein is present in human umbilical vein in situ and is expressed in low passage HUVEC in vitro. TEM8 protein expression in HUVEC was increased 5-fold by the initiation of tube formation, correlating expression of TEM8 with the angiogenic response. Taken together, these results indicate that TEM8 plays a positive role in endothelial cell activities related to angiogenesis.     

NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin

The NG2 proteoglycan is expressed by microvascular pericytes in newly formed blood vessels. We have used in vitro and in vivo models to investigate the role of NG2 in cross-talk between pericytes and endothelial cells (EC). Binding of soluble NG2 to the EC surface induces cell motility and multicellular network formation in vitro and stimulates corneal angiogenesis in vivo. Biochemical data demonstrate the involvement of both galectin-3 and alpha3beta1 integrin in the EC response to NG2 and show that NG2, galectin-3, and alpha3beta1 form a complex on the cell surface. Transmembrane signaling via alpha3beta1 is responsible for EC motility and morphogenesis in this system. Galectin-3-dependent oligomerization may potentiate NG2-mediated activation of alpha3beta1. In conjunction with recent studies demonstrating the early involvement of pericytes in angiogenesis, these data suggest that pericyte-derived NG2 is an important factor in promoting EC migration and morphogenesis during the early stages of neovascularization.     

Sphingosylphosphorylcholine induces endothelial cell migration and morphogenesis

Sphingosylphosphorylcholine (SPC) is one of the biologically active phospholipids that may act as extracellular messengers. Particularly important is the role of these lipids in the angiogenic response, a complex process involving endothelial cell migration, proliferation, and morphologic differentiation. Here we demonstrate that SPC and its hydrolytic product, sphingosine, induce chemotactic migration of human and bovine endothelial cells. The response is approximately equal to that elicited by vascular endothelial cell growth factor. The effect of SPC and sphingosine was associated with a rapid down-regulation of Edg1, a sphingosine 1-phosphate (SPP)-specific receptor involved in endothelial cell chemotaxis. Both SPC and sphingosine induced differentiation of endothelial cells into capillary-like structures in vitro. Thus, SPC and sphingosine join SPP among the biologically active lipids with angiogenic potential. Since neuronal abnormalities accompany pathological accumulation of SPC in brain tissue, it is possible that SPC is a modulator of angiogenesis in neural tissue upon its release from brain cells following trauma or neoplastic growth.     

5-hydroxytryptamine induces mast cell adhesion and migration

The neurotransmitter serotonin (5-hydroxytryptamine (5-HT)) is implicated in enhancing inflammatory reactions of skin, lung, and gastrointestinal tract. To determine whether 5-HT acts, in part, through mast cells (MC), we first established that mouse bone marrow-derived MC (mBMMC) and human CD34(+)-derived MC (huMC) expressed mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mouse and human MC degranulation, adhesion, and chemotaxis. We found no evidence that 5-HT degranulates MC or modulates IgE-dependent activation. 5-HT did induce mBMMC and huMC adherence to fibronectin; and immature and mature mBMMC and huMC migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin, we identified 5-HT(1A) as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT(1A) receptor knockout mouse (5-HT(1A)R(-/-)) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT(1A)R(+/+) mice, but not in 5-HT(1A)R(-/-) mice. These studies are the first to demonstrate an effect of 5-HT on MC. Furthermore, both mouse and human MC respond to 5-HT through the 5-HT(1A) receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.     

Heparanase induces endothelial cell migration via protein kinase B/Akt activation

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor.     

Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration.

Interactions between integrins and tyrosine kinase receptors can modulate a variety of cell functions. We observed a cooperative interaction between the beta(1) integrin and vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4) that appeared to be required for cell migration. By using VEGFR-3-transfected 293 cells (293/VEGFR-3) or primary dermal microvascular endothelial cells (DMEC), we found that stimulation with either soluble or immobilized extracellular matrix (ECM) proteins, collagen or fibronectin (FN), resulted in the increased tyrosine phosphorylation of VEGFR-3 in the absence of a cognate ligand. This increased tyrosine phosphorylation of VEGFR-3 was diminished by pretreatment with a blocking antibody against the beta(1) integrin. Cross-linking with anti-beta(1) integrin antibody induced a similar degree of tyrosine phosphorylation of VEGFR-3. Stimulation with collagen or FN induced an association between beta(1) integrin and VEGFR-3 in both 293/VEGFR-3 and primary DMEC cells. Collagen or FN-induced tyrosine phosphorylation of VEGFR-3 was inhibited by treatment with cytochalasin D, an inhibitor of actin polymerization. Collagen or FN was able to induce the migration of 293/VEGFR-3 or DMEC cells to a limited extent. However, migration was dramatically enhanced when a gradient of the cognate ligand, VEGF-D, was added. VEGF-D failed to induce cell migration in the absence of ECM proteins. Introducing a mutation at the kinase domain of VEGFR-3 or treatment with blocking antibody against either VEGFR-3 or beta(1) integrin inhibited cell migration induced by ECM and VEGF-D, indicating that signals from both beta(1) integrin and VEGFR-3 are required for this cell function.