Regulation of Integrin αvβ3-mediated Endothelial Cell Migration and Angiogenesis by Integrin α5β1 and Protein Kinase A
2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês
10.1074/jbc.m003668200
ISSN1083-351X
AutoresSemi Kim, M. Harris, Judith A. Varner,
Tópico(s)Platelet Disorders and Treatments
ResumoRecent studies indicate that angiogenesis depends, in part, on ligation of integrin α5β1 by fibronectin. Evidence is now provided that integrin α5β1 regulates the function of integrin αvβ3 on endothelial cells during their migration in vitro or angiogenesisin vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin α5β1, which potentiates αvβ3-mediated migration on vitronectin without influencing αvβ3-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-α5β1 restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or α5β1 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 α5β1 antagonists. Therefore, αvβ3-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin α5β1. Recent studies indicate that angiogenesis depends, in part, on ligation of integrin α5β1 by fibronectin. Evidence is now provided that integrin α5β1 regulates the function of integrin αvβ3 on endothelial cells during their migration in vitro or angiogenesisin vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin α5β1, which potentiates αvβ3-mediated migration on vitronectin without influencing αvβ3-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-α5β1 restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or α5β1 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 α5β1 antagonists. Therefore, αvβ3-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin α5β1. basic fibroblast growth factor protein kinase A bovine serum albumin human umbilical vein endothelial cell chorioallantoic membrane The growth of new blood vessels, angiogenesis, contributes to the adverse effects of solid tumor cancer, arthritis, psoriasis, and blindness. Angiogenesis is a complex biological process that depends on growth factors and the extracellular matrix (1Breier G. Risau W. Trends Cell Biol. 1996; 6: 454-456Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 2Breier G. Damert A. Plate K.H. Risau W. Thromb. Haemost. 1997; 78: 678-683Crossref PubMed Scopus (123) Google Scholar, 3Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar, 4Stromblad S. Cheresh D.A. Chem. Biol. 1996; 3: 881-885Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 5Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4866) Google Scholar, 6Varner J.A. Exs. 1997; 79: 361-390PubMed Google Scholar). A potential role for the extracellular matrix protein fibronectin in vascular development was initially suggested by studies demonstrating the presence of oncofetal fibronectin around blood vessels during development and wound healing and in tumor tissue (7Castellani P. Viale G. Dorcaratto A. Nicolo G. Kaczmarek J. Querze G. Zardi L. Int. J. Cancer. 1994; 59: 612-618Crossref PubMed Scopus (279) Google Scholar, 8Clark R.A.F. DellaPelle P. Manseua E. Lanigan J.M. Dvorak H.F. Colvin R.B. J. Invest. Dermatol. 1982; 79: 269-276Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 9Kaczmarek J. Castellani P. Nicolo G. Spina B. Allemanni G. Zardi L. Int. J. Cancer. 1994; 58: 11-16Crossref Scopus (139) Google Scholar, 10Neri D. Carnemolla B. Nissim A. Blaza E. Leprini A. Querze G. Pina A. Tarli L. Halin C. Neri P. Zardi L. Winter G. Nat. Biotech. 1997; 15: 1271-1275Crossref PubMed Scopus (286) Google Scholar). Interestingly, mice lacking fibronectin die early in development from a collection of defects, which includes an improperly formed vasculature (11George E.L. Georges E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar). Additionally, mice lacking the fibronectin receptor α5β1, one of several fibronectin receptors (12Pytela R. Pierschbacher M.D. Ruoslahti E. Cell. 1985; 40: 191-198Abstract Full Text PDF PubMed Scopus (688) Google Scholar), also die early in development and exhibit some vascular and cardiac defects (13Yang J.T. Rayburn H. Hynes R.O. Development. 1993; 119: 1093-1105Crossref PubMed Google Scholar, 14Goh K.L. Yang J.T. Hynes R.O. Development. 1997; 124: 4309-4319PubMed Google Scholar). This circumstantial evidence suggests potential roles for fibronectin and its receptor, integrin α5β1, in angiogenesis. We have recently shown that integrin α5β1and its ligand fibronectin are expressed at significantly increased levels in neovessels induced by growth factors or solid tumors (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Notably, antibody, peptide, and small molecule antagonists of integrin α5β1 and fibronectin block growth factor- and tumor-induced angiogenesis (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Interestingly, antagonists of another fibronectin-binding integrin, αvβ3, also inhibit angiogenesis (16Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2749) Google Scholar, 17Brooks P.C. Montgomery A.M.P. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2184) Google Scholar, 18Brooks P.C. Stromblad S. Klemke R. Visscher D. Sarkar F.H. Cheresh D.A. J. Clin. Invest. 1995; 96: 1815-1822Crossref PubMed Scopus (757) Google Scholar, 19Brooks P.C. Montgomery A.M.P. Cheresh D.A. Methods Mol. Biol. 1999; 129: 257-269PubMed Google Scholar, 20Carron C.P. Meyer D.M. Pegg J.A. Engleman V.W. Nickols M.A. Settle S.L. Westlin W.F. Ruminski P.G. Nichols G.A. Cancer Res. 1998; 58: 1930-1955PubMed Google Scholar, 21Drake C.J. Cheresh D.A. Little C.D. J. Cell Sci. 1995; 108: 2655-2661Crossref PubMed Google Scholar, 22Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar). Antagonists of both of these integrins substantially block angiogenesis induced by basic fibroblast growth factor (bFGF)1 but not by vascular endothelial growth factor, suggesting that these integrins regulate similar pathways of angiogenesis (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 22Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar). Since antagonists of both integrins independently and substantially block the same pathway of angiogenesis, it is possible that these two integrins interact during angiogenesis. Previous studies have indicated that the occupancy of one integrin by its ligand can inhibit the functions of other integrins (23Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (222) Google Scholar, 24Pacifici R. Roman J. Kimble R. Civitelli R. Brownfield C.M. Bizzarri C. J. Immunol. 1994; 153: 2222-2233PubMed Google Scholar, 25Diaz-Gonzales F. Forsyth J. Steiner B. Ginsberg M.H. Mol. Biol. Cell. 1996; 7: 1939-1951Crossref PubMed Scopus (155) Google Scholar, 26Imhof B. Weerasinghe D. Brown E.J. Lindberg F.P. Hammel P. Piali L. Dessing M. Giesler R. Eur. J. Immunol. 1997; 27: 3242-3252Crossref PubMed Scopus (55) Google Scholar, 27Simon K.O. Nutt E.M. Abraham D.G. Rodan G.A. Duong L. J. Biol. Chem. 1997; 272: 29380-29389Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 28Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar). These studies have indicated that cross-talk between integrins regulates such properties as integrin-mediated cell migration (23Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (222) Google Scholar, 24Pacifici R. Roman J. Kimble R. Civitelli R. Brownfield C.M. Bizzarri C. J. Immunol. 1994; 153: 2222-2233PubMed Google Scholar, 26Imhof B. Weerasinghe D. Brown E.J. Lindberg F.P. Hammel P. Piali L. Dessing M. Giesler R. Eur. J. Immunol. 1997; 27: 3242-3252Crossref PubMed Scopus (55) Google Scholar, 27Simon K.O. Nutt E.M. Abraham D.G. Rodan G.A. Duong L. J. Biol. Chem. 1997; 272: 29380-29389Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 28Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar) and ligand binding (25Diaz-Gonzales F. Forsyth J. Steiner B. Ginsberg M.H. Mol. Biol. Cell. 1996; 7: 1939-1951Crossref PubMed Scopus (155) Google Scholar). For example, ligation of integrin αvβ3 has been shown to inhibit cell migration and phagocytosis mediated by integrin α5β1 (23Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (222) Google Scholar). Interactions between individual integrins are known as cross-talk. Integrin cross-talk appears to depend on the actions of signal transduction molecules (23Blystone S.D. Graham I.L. Lindberg F.P. Brown E.J. J. Cell Biol. 1994; 127: 1129-1137Crossref PubMed Scopus (222) Google Scholar, 24Pacifici R. Roman J. Kimble R. Civitelli R. Brownfield C.M. Bizzarri C. J. Immunol. 1994; 153: 2222-2233PubMed Google Scholar, 28Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar). For example, integrin αvβ3 suppression of α5β1-mediated migration is dependent on calcium/calmodulin-dependent protein kinase II (28Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar). In this report, we provide evidence that integrin α5β1 regulates the functions of integrin αvβ3 in vitro and in vivo. Antagonists of integrin α5β1suppress αvβ3-mediated focal contact formation and cell migration without inhibiting cellular attachment. This inhibition of αvβ3 function is dependent on the activation of protein kinase A (PKA) and can be blocked by inhibitors of PKA. Culture media and reagents were from Irvine Scientific (Irvine, CA). HUVECS were maintained in M199 medium containing sodium bicarbonate, HEPES, heparin, endothelial cell growth supplement, 20% fetal bovine serum, and gentamycin. Fibronectin and collagen were from Collaborative Biomedical Products (Bedford, MA). Human 120-kDa chymotryptic fragments of fibronectin were purchased from Chemicon (Temecula, CA). Vitronectin was purified by denaturation/heparin affinity column standard procedures. LM609 (29Cheresh D.A. Spiro R.C. J. Biol. Chem. 1987; 262: 17703-17711Abstract Full Text PDF PubMed Google Scholar) and W6/32 (30Brodsky F.M. Parham P. J. Immunol. 1992; 128: 129-135Google Scholar) were provided by Dr. David Cheresh. AP3 was provided by Dr. Peter Newman (31Newman P.J. Allen R.W. Kahn R.A. Kunicki T.J. Blood. 1985; 65: 227-232Crossref PubMed Google Scholar). Anti-α5β1 function blocking antibodies (JBS5) and anti-α2β1antibodies were generously provided by Chemicon. Anti-vinculin was purchased from Seratec (Washington, DC). Anti-paxillin was purchased from Transduction Laboratories (San Diego, CA). Anti-fibronectin cell-binding peptide monoclonal antibody (784A2A6) and anti-fibronectin N-terminal peptide monoclonal antibody were from Chemicon. Cross-absorbed secondary antibodies were purchased fromBIOSOURCE International (Camarillo, CA). OCT embedding medium was obtained from Baxter (McGraw Park, IL). Integrin α5β1 nonpeptide small molecule antagonist SJ749 had the following structure: (S)-2-[(2,4 6-trimethylphenyl) sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]carbonylamino]propionic acid. Control nonpeptide small molecule XU065 had the following structure: 3-[(3-[(4-amidinophenyl)oxy]isoxazol-5-yl]carboxamido]-2(S)(butoxycarbonylamino)propionic acid methyl ester. Ten-day-old chicken eggs were purchased from McIntyre Poultry (Ramona, CA). Basic fibroblast growth factor was purchased from Genzyme, Inc. (Cambridge, MA). PKA assay kits were purchased from Life Technologies, Inc. Round 1-mm-thick glass coverslips were coated with 1 μg/ml fibronectin, vitronectin, collagen, or poly-l-lysine for 1 h at room temperature, then blocked with 3% bovine serum albumin for 2 h at 37 °C. HUVECS were removed from culture dishes by trypsizination, washed in serum-free culture medium, and resuspended in Hanks' balanced salt solution supplemented with 10 mmHEPES, pH 7.4, 2 mm MgCl2, 2 mmCaCl2, 0.2 mm MnCl2, 1% BSA. Cells were then incubated for 1 h at 37 °C on coated coverslips. Cells were incubated in the presence of 25 μg/ml anti-α5β1, anti-αvβ3, anti-αvβ5, control antibodies, or no antibodies for 60 min. Coverslips were then washed to remove unbound cells, fixed for 5 min in 3.7% paraformaldehyde and permeabilized in 0.3% Triton X-100 for 3 min. Coverslips were incubated with 1.25 μg/ml anti-paxillin antibodies, 0.2 μg/ml anti-vinculin antibodies, 1:500 dilution of anti-integrin β3 monoclonal antibodies, or 2.7 μg/ml anti-phosphotyrosine antibodies in 20 mg/ml bovine serum albumin in phosphate-buffered saline for 1 h at room temperature. Coverslips were well washed in phosphate-buffered saline and incubated in 10 μg/ml goat anti-mouse fluorescein isothiocyanate for 1 h at room temperature. Coverslips were well washed in phosphate-buffered saline, and mounted in Gelvatol, prior to confocal fluorescent microsopy. Cell adhesion assays were performed as described (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). In brief, the wells of 48-well non-tissue culture-treated culture dishes (Costar, Inc.) were coated with 5 μg/ml vitronectin, fibronectin, Del-1, or collagen for 1 h at 37 °C and blocked with 2% heat-denatured bovine serum albumin in phosphate-buffered saline for 1 h. Fifty thousand cells in 25 μg/ml anti-α5β1 function blocking antibody (JBS5), 25 μg/ml anti-αvβ3function blocking antibody (LM609), 25 μg/ml anti-α2β1 function blocking antibody, 25 μg/ml anti-β1 function blocking antibody (P4C10), or 25 μg/ml control antibody (W6/32) in adhesion buffer (Hepes-buffered Hanks' balanced salt solution containing 1% bovine serum albumin, 2 mm MgCl2, 2 mm CaCl2, and either 0 mm or 0.2 mm MnCl2) were allowed to adhere to dishes for 20 min at 37 °C. In some experiments, dibutyryl cAMP was used at 500 μm and forskolin was used at 20 μg/ml. Each experiment was performed in triplicate, with triplicate samples per condition. The data are presented as percentage of adhesion exhibited by the positive control (adhesion medium alone) ± standard error of the mean. Migration assays were performed essentially as described (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). The lower side of 8-μm pore Transwell inserts (Costar, Inc.) were coated with 5 μg/ml fibronectin, vitronectin, Del-1, collagen, or no protein for 1 h and were blocked with 2% bovine serum albumin in phosphate-buffered saline for 1 h. HUVECS (25,000) were added to the upper chamber of inserts in migration buffer (Hepes-buffered M199 medium containing 1% BSA, 1.8 mm CaCl2, 1.8 mm MgCl2, and either 0 or 0.2 mm MnCl2, as well as 25 μg/ml various antibodies or 10 μm SJ749 or XU065. In some experiments, cells were also incubated in the presence of final concentrations of 20 μm H8, 25 μmautocamtide 2, 50 nm calphostin C, or 48 nmH89, 500 μm dibutyryl cAMP, or 20 μg/ml forskolin. Some experiments were performed in the presence of 200 μg/ml cycloheximide, after a 4-h preincubation in 200 μg/ml cycloheximide. Cells were allowed to migrate from the upper to the lower chamber for 4 h at 37 °C. Non-migratory cells were removed from the upper chamber by wiping the upper surface with an absorbent tip. Cells that had migrated to the lower side of the Transwell insert were then fixed for 15 min with 3.7% paraformaldehyde for 15 min and with a 2% crystal violet solution. After extensive water washing to remove excess crystal violet, the number of cells that had migrated (to the bottom of the insert) were counted in three representative high power (200×) fields per insert. No cells were found on the culture dish in which the insert was placed. The data are presented as number of cells migrating ± standard error of the mean. Angiogenesis assays were performed as described (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Briefly, chorioallantoic membranes (CAMs) of 10-day-old embryonated chicken eggs were stimulated with cortisone acetate-pretreated filter discs saturated in 1 μg/ml bFGF or saline. Twenty-five μg of function blocking anti-α5β1 in or a control antibody (25 μl), 25 μm cyclic peptide (CRRETAWAC) or scrambled control peptide (CATAERWRC) in 25 μl, or 25 μl of saline were applied to the growth factor-saturated filter disc 24 h later. In some cases, 50 μm H8, 480 nm H89, 50 nm calphostin C, 20 μm forskolin, or 500 μm dibutyryl cAMP were added to CAMs. CAMs were harvested on the 4th day of stimulation by fixation with a drop of 3% paraformaldehyde in phosphate-buffered saline prior to excision of the stimulated area. Blood vessel branch points in the 5-mm disc were counted at a magnification of 30 under fiber optic illumination in a blinded fashion (19Brooks P.C. Montgomery A.M.P. Cheresh D.A. Methods Mol. Biol. 1999; 129: 257-269PubMed Google Scholar). At least 10 embryos were used per treatment group. Each experiment was performed a minimum of three times. Data were evaluated in terms of average number of blood vessel branch points per treatment group ± standard error of the mean. Statistical analyses were performed using Student's t test. Culture plates were coated overnight at 4 °C with 5 μg/ml poly-l-lysine, 10 μg/ml fibronectin, or 10 μg/ml vitronectin. Plates were washed and blocked in 3% bovine serum albumin for 1 h at 37 °C. Additional plates were coated with 25 μg/ml goat anti-mouse antibodies for 2 h at 37 °C, blocked in 3% bovine serum albumin for 1 h at 37 °C, and incubated in 10 μg/ml anti-α5β1 or control antibodies overnight at 4 °C. HUVECS were trypsinized and resuspended in adhesion buffer (Hanks' balanced salt solution supplemented with 10 mmHEPES, pH 7.4, 2 mm MgCl2, 2 mmCaCl2, 0.2 mm MnCl2, 1% BSA). Cells were plated on matrix protein or antibody-coated plates in medium or the presence of 25 μg/ml anti-α5β1 or control antibodies (W6/32, anti-MHC) for 60 min. Plates were washed with phosphate-buffered saline and extracted with cold extraction buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 25 μg/ml aprotinin, and 25 μg/ml leupeptin). Protein kinase A activity was assessed using a kit from Life Technologies, Inc. Briefly, equal volumes of lysate and reaction mixture containing final concentrations of 50 mm Tris, pH 7.5, 10 mm MgCl2, 0.25 mg/ml BSA, 50 μm kemptide (protein kinase A-specific substrate), 3000 Ci/mmol [γ-32P]ATP, and 100 μm ATP were combined for 5 min at 30 °C. Total PKA in each sample was measured by performing the assay in the presence of 10 μm cAMP. Background PKA activity in test and total PKA samples were determined by performing the assay in the presence of a PKA inhibitor peptide, PKI-(6–22) amide. Twenty μl samples of reaction mixtures were spotted onto phosphocellulose discs, air-dried, and washed repeatedly with 1% (v/v) phosphoric acid in water. Incorporated radioactivity was determined by scintillation counting. Activated PKA was calculated from the ratio of pmol/min test sample PKA to pmol/min total PKA. PKA activity in triplicate samples was measured and experiments were repeated at least three times. Statistical analyses were performed using Student's t test. HUVECs were detached from culture dishes by trysinization, washed, and resuspended in serum-free culture medium in the presence or absence of 200 μg/ml cycloheximide. Cells were plated on cell culture plates that had been coated with vitronectin, collagen, or fibronectin and blocked with bovine serum albumin. Cells were incubated for 4 h at 37 °C prior to lysis with boiling SDS sample buffer. Fifty micrograms of protein from each lysate was electrophoresed in 6% or 10% SDS-polyacrylamide gels. The separated proteins were electrophoretically transferred to nitrocellulose and then incubated in anti-fibronectin, anti-integrin α5 or β3 cytoplasmic tail, or anti-protein kinase A catalytic subunit antibodies, followed by horseradish peroxidase-conjugated secondary antibodies. Blots were incubated in a chemiluminescent substrate for horseradish peroxidase and then exposed to film. Recent studies show that antagonists of integrin α5β1 or αvβ3can substantially block growth factor and tumor-induced angiogenesis in chicken embryos and in human skin transplanted onto SCID mice (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 16Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2749) Google Scholar, 17Brooks P.C. Montgomery A.M.P. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2184) Google Scholar, 18Brooks P.C. Stromblad S. Klemke R. Visscher D. Sarkar F.H. Cheresh D.A. J. Clin. Invest. 1995; 96: 1815-1822Crossref PubMed Scopus (757) Google Scholar,32Christofidou-Solomidou M. Bridges M. Murphy G. Albelda S. DeLisser H.M. Am. J. Pathol. 1997; 151: 975-983PubMed Google Scholar). Furthermore, antagonists of these adhesion receptors block angiogenesis induced by bFGF but not by vascular endothelial growth factor, suggesting that these integrins may cooperate by regulating the same angiogenic pathways (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 22Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar). To evaluate the interactions of α5β1 and αvβ3in angiogenesis, experiments were designed to examine their contributions to endothelial cell migration in vitro, as endothelial cell migration is an essential feature of angiogenesis. As shown in Fig. 1 (A andB), function blocking antibodies selective for integrin α5β1 blocked both the adhesion and migration of HUVECs on fibronectin, while antibodies against integrin αvβ3 had little effect. Surprisingly, both anti-α5β1 and anti-αvβ3 inhibited endothelial cell migration on vitronectin (Fig. 1 C), yet only anti-αvβ3 blocked adhesion to vitronectin (Fig. 1 D). In contrast, anti-α5β1 had no impact on collagen-mediated cell migration, which depends on integrin α2β1 (Fig. 1 E). Similar results were observed for adult dermal microvascular endothelial cells (data not shown). Anti-α5β1 blocked αvβ3−mediated cell migration to similar extents when measured after 2, 4, or 18 h of incubation time (data not shown). Anti-α5β1 blocked αvβ3-mediated migration but not adhesion to the same extent in the absence or in the presence of high levels (200 μm) of Mn2+, a divalent cation that enhances the affinity of αvβ3 for its ligand (data not shown). Therefore, α5β1 function is required for endothelial cell migration on vitronectin and other substrates of integrin αvβ3, as well as on fibronectin. Interestingly, both antibody and small molecule antagonists of integrin α5β1 block cell migration on vitronectin (Fig. 1 F) without affecting cell attachment (Fig.1 C; Ref. 15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Thus, antibodies directed against α5β1 inhibit αvβ3-mediated cell migration by antagonizing α5β1 ligation, rather than by cross-linking cell surface α5β1 molecules. These results indicate that integrin α5β1selectively impacts αvβ3-mediated cellular migration without influencing αvβ3-dependent adhesion, suggesting that these integrins may function in a cooperative manner on the surface of endothelial cells. Endothelial cells readily secrete fibronectin during angiogenesis (8Clark R.A.F. DellaPelle P. Manseua E. Lanigan J.M. Dvorak H.F. Colvin R.B. J. Invest. Dermatol. 1982; 79: 269-276Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). In fact, endothelial cell-derived oncofetal fibronectin specifically accumulates around tumor-associated blood vessels (7Castellani P. Viale G. Dorcaratto A. Nicolo G. Kaczmarek J. Querze G. Zardi L. Int. J. Cancer. 1994; 59: 612-618Crossref PubMed Scopus (279) Google Scholar, 8Clark R.A.F. DellaPelle P. Manseua E. Lanigan J.M. Dvorak H.F. Colvin R.B. J. Invest. Dermatol. 1982; 79: 269-276Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 9Kaczmarek J. Castellani P. Nicolo G. Spina B. Allemanni G. Zardi L. Int. J. Cancer. 1994; 58: 11-16Crossref Scopus (139) Google Scholar, 10Neri D. Carnemolla B. Nissim A. Blaza E. Leprini A. Querze G. Pina A. Tarli L. Halin C. Neri P. Zardi L. Winter G. Nat. Biotech. 1997; 15: 1271-1275Crossref PubMed Scopus (286) Google Scholar). Recent studies suggest that antibodies directed to the cell-binding domain of fibronectin block angiogenesis (15Kim S. Bell K. Mousa S.A. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1346Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). The indirect inhibition of endothelial cell migration on vitronectin by α5β1 antagonists (Fig. 1) suggests the possibility that endothelial cell-derived fibronectin may influence cell migration on vitronectin. Indeed, cell migration on vitronectin is partially inhibited by function-blocking antibodies directed against the cell-binding domain of fibronectin, but not by antibodies directed against the N terminus of fibronectin (Fig.2 A). In contrast, anti-fibronectin antibodies (Fig. 2 B) do not block cellular migration on collagen, even though endothelial cells cultured on either vitronectin (Fig. 2 C) or collagen (data not shown) synthesize fibronectin. These results suggest that ligation of α5β1 by endothelial cell-secreted fibronectin specifically regulates αvβ3function but not that of other integrins. To determine further whether ligation of α5β1 by endothelial cell secreted fibronectin potentiates integrin αvβ3function, endothelial cells were treated with cycloheximide to inhibit protein synthesis and then evaluated for their abilities to migrate on fibronectin, vitronectin, or collagen substrates. Endothelial cell synthesis of fibronectin is inhibited by cycloheximide pretreatment of endothelial cells (Fig. 2 C). Inhibition of protein synthesis results in an overall reduction in cellular migration rates compared with untreated cells (Fig. 2, D–F). In contrast to untreated cells, the migration of cycloheximide-treated cells on vitronectin is not inhibited by anti-α5β1antibodies (Fig. 2 E). However, migration on vitronectin is inhibited to the same extent by anti-αvβ3antibodies in treated and untreated cells. In addition, in contrast to untreated cells, migration of cycloheximide-treated cells on vitronectin is refractory to anti-fibronectin antibodies (Fig.2 F). Migration on collagen is equally sensitive to anti-α2β1 and insensitive to anti-α5β1 antibodies in cycloheximide-treated and untreated cells (Fig. 2 E). Migration on fibronectin is equally sensitive to anti-α5β1 treatment in cycloheximide-treated and untreated cells (data not shown). In fact, expression levels of α5 and β3 integrins measured by immunoblotting with anti-cytoplasmic tail antibodies in treated and untreated cells remains unchanged as does expression of protein kinase A (data not shown). Taken together, these results suggest that fibronectin synthesized by HUVECS contributes to vitronectin migration based on its ability to ligate integrin α5β1. Temporal prerequisites to cellular migration are the attachment and spreading of cells on the extracellular matrix, with concomitant formation of stress fibers and focal adhesions. Since antagonists of α5β1 do not impact αvβ3-mediated cell attachment but do inhibit migration, we examined the effect of α5β1 antagonists on cell spreading and focal contact formation to establish the earliest integrin αvβ3-mediated events that are affected by inhibition of α5β1. During spreading on vitronectin (within 30 min after initial cell attachment), endothelial cells form stress fibers (Fig.3 A) as well as focal adhesions incorporating paxillin, vinculin, integrin β3 (Fig.3 B), and phosphotyrosine (data not shown). Integrin-mediated signal transduction during this time leads to the tyrosine phosphorylation of focal adhesion proteins such as paxillin and focal adhesion kinase. In the presence of integrin α5β1antagonists, endothelial cells generally appear less polarized than in control cells (Fig. 3 A). Instead, these cells acquire a rounded, pancake-like morphology. Notably, stress fiber formation i
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