Akt Mediates Cytoprotection of Endothelial Cells by Vascular Endothelial Growth Factor in an Anchorage-dependent Manner

Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival. Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival. The survival of endothelial cells is critical for angiogenesis and the maintenance of blood vessel integrity. VEGF 1The abbreviations used are: VEGF, vascular endothelial growth factor; PI3-kinase, phosphoinositide 3-kinase; HUVEC, human umbilical vein endothelial cell; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HA, hemagglutinin; CMV, cytomegalovirus. is an endothelial cell-specific mitogen that functions to induce blood vessel formation during normal development and various pathological processes (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar). VEGF biological activity is mediated through its binding to at least two endothelial cell-specific receptors, fms-like tyrosine kinase (flt-1) and fetal liver kinase (flk-1) (2de Vries C. Escobedo J.A. Ueno H. Houck K.A. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar, 3Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ulrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1764) Google Scholar). Heterozygous deletion of theVEGF gene results in the embryonic lethality due to the abnormal blood vessel development (4Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3475) Google Scholar, 5Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3062) Google Scholar). VEGF also functions in adult organisms to promote angiogenesis in ischemic tissue (6Banai S. Jaklitsch M.T. Shou M. Lazarous D.F. Scheinowitz M. Biro S. Epstein S.E. Unger E.F. Circulation. 1994; 89: 2183-2189Crossref PubMed Scopus (594) Google Scholar) and during wound healing (7Brown L.F. Yeo K.T. Berse B. Yeo T.K. Senger D.R. Dvorak H.F. Van De Water L. J. Exp. Med. 1992; 176: 1375-1379Crossref PubMed Scopus (787) Google Scholar). 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Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1422) Google Scholar, 16Yuan F. Chen Y. Dellian M. Safabakhsh N. Ferrara N. Jain R.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14765-14770Crossref PubMed Scopus (608) Google Scholar,17Benjamin L.E. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8761-8766Crossref PubMed Scopus (444) Google Scholar). Endothelial cells are also dependent on adhesion to extracellular matrix for their survival (18Meredith J.E. Fazeli B. Schwartz M.A. Mol. Biol. Cell. 1993; 4: 953-961Crossref PubMed Scopus (1405) Google Scholar). Cell adhesion to matrix is mediated by integrins and disruption of proper integrin-ligand interactions leads to a phenomenon termed anoikis, apoptosis induced by the disruption of cell-matrix interactions (19Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2788) Google Scholar). 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Chibale K. Rosenfeld M. Diefenbach B. Cheresh D.A. Goodman S.L. Bioorg. Med. Chem. 1998; 6: 1185-1208Crossref PubMed Scopus (71) Google Scholar). The serine/threonine protein kinase Akt/PKB is recognized increasingly as a key regulator of cell viability in a number of systems (24Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2222) Google Scholar, 25Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1075) Google Scholar, 26Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 710-713Crossref Scopus (980) Google Scholar, 27Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (966) Google Scholar). Activation of Akt occurs through the direct binding of the phosphoinositide products of the PI3-kinase reaction to its pleckstrin homology domain (28Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1528) Google Scholar). Phosphoinositol lipids also activate a protein kinase that phosphorylates Akt, resulting in further activation (29Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar,30Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1054) Google Scholar). Here, we examined the involvement of Akt in the survival of cultured human umbilical vein endothelial cells (HUVEC). VEGF-mediated HUVEC survival correlated with the wortmannin-sensitive induction of Akt activity. Adenovirus-mediated expression of a dominant-negative mutant of Akt reduced HUVEC viability in the presence of VEGF. Forced expression of wild-type Akt promoted cell survival in response to low doses of VEGF, whereas constitutive overexpression of Akt was sufficient for endothelial cell survival in the absence of VEGF. VEGF did not promote Akt activation in suspension culture nor did it inhibit anoikis. However, expression of constitutively active Akt was sufficient for survival in the absence of attachment. These data suggest that growth factor and attachment-mediated signaling pathways converge on Akt to control endothelial cell survival under conditions of angiogenesis and vessel regression. Anti-Akt antibody was purchased from Santa-Cruz. Wortmannin was purchased from Sigma. Akt plasmids were a gift from Dr. J. Testa. Recombinant human VEGF was the 165 amino acid isoform. HUVECs were cultured in monolayers in Dulbecco's modified Eagle medium (DMEM) under conditions of serum deprivation or stimulation with VEGF as described by Spyridopouloset al. (14Spyridopoulos I. Brogi E. Kearney M. Sullivan A.B. Cetrulo C. Isner J.M. Losordo D.W. J. Mol. Cell. Cardiol. 1997; 29: 1321-1330Abstract Full Text PDF PubMed Scopus (193) Google Scholar). For suspension cultures, HUVEC monolayers were washed with phosphate-buffered saline (PBS) twice and incubated with PBS containing 0.5% EDTA. Cells were collected with centrifugation (2,000 rpm for 5 min) and washed with serum-depleted media. For suspension culture, bacterial culture dishes were precoated with 20 mg/ml bovine serum albumin overnight to prevent the cells from attaching to the substratum. Cells were washed twice with PBS and lysed with cell lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mm NaCl, 20 mm Tris-HCl, pH 7.4, 20 mm NaF, 2 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride). Lysates were precleaned with protein G-agarose for 30 min at 4 °C, and immunoprecipitated for 2 h with anti-Akt antibodies in the presence of 2 mg/ml bovine serum albumin with or without 16 μg/ml competitor peptides (Santa Cruz). Immunoprecipitates were washed twice with cell lysis buffer, once with water, and once with kinase buffer (20 mm HEPES, pH 7.2, 10 mm MgCl2, 10 mm MnCl2). Immunoprecipitated proteins were incubated in 50 μl of kinase buffer containing 2 μg of histone H2B (Roche Molecular Biochemicals) and γ-32PATP (5 μm, 10 μCi) for 30 min at room temperature. Kinase reactions were stopped by the addition of SDS sample buffer and subjected to SDS-polyacrylamide gel on Cells were on dishes at a of 4 overnight the was to containing fetal bovine serum or the of VEGF for Cells were washed with PBS, by and viability was by the J. Cell Biol. 1992; PubMed Scopus Google Scholar, S. D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). cells were for the of of culture in PBS was to the for 20 cells were washed in PBS twice and with Cells were examined with a Cell viability was also by the Cells were cultured in precoated with bovine serum albumin in the presence or absence of VEGF for was to the Cells were incubated with reaction for 2 adenovirus expressing Akt proteins to the under the control of the were to the of T. C.B. in Cell Scholar). In from was the of was with cells to for The mutant Akt and constitutively active Akt adenoviral were by the The dominant-negative mutant Akt protein by D.R. M. B. P. N. P. J. 1996; PubMed Scopus Google Scholar) and it functions in a dominant-negative T. W. H. Y. S. M. M. T. H. U. M. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). The constitutively active Akt has the to the of the Akt were in cells and by through a the bacterial gene from the D. K. H. C. A. P. Isner J.M. Walsh K. Genes Dev. 1997; 11: PubMed Scopus Google Scholar). HUVEC were for h with or at a of of a transduction not Cells were cultured under the and cells were collected and in μl of PBS containing and 30 min at °C, μl of mm 0.5% 10 mg/ml Tris-HCl, pH was Cell were incubated at for min and μl of was was by centrifugation at rpm for once with and once with the was was examined on a Cells were as described in the and cell were as described for the Akt kinase 20 μg of protein was on SDS-polyacrylamide gel gel and a with containing containing for 1 the was incubated with antibody anti-Akt antibody (Santa Akt antibody or was used for Consistent with H.-P. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar, B. H. B. K. J.M. R. Mol. Cell. 1998; 1: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. N. J. Cell Biol. 1998; PubMed Scopus Google Scholar), serum deprivation endothelial cell deprivation for h in in HUVEC viability on the and cells 4 or were to cells has been that VEGF HUVEC apoptosis induced by serum deprivation H.-P. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar). of VEGF in HUVEC is shown in 1 A. the conditions of VEGF cell induced by serum Consistent with an with VEGF also the of endothelial cells 1 of VEGF the of from to in that were incubated in the absence of serum for The cell survival effects of VEGF were by the PI3-kinase of VEGF-mediated cell viability by was of VEGF-mediated HUVEC survival were incubated with 10 or 50 whereas the survival effects of VEGF. also the VEGF-mediated of in the VEGF the activity of Akt proteins in endothelial HUVEC were incubated in with or without VEGF The kinase activity of Akt was in immunoprecipitated with anti-Akt shown in VEGF Akt kinase activity in HUVEC Akt is by PI3-kinase in cell T.F. Datta K. A. Kaplan D.R. Tsichlis P.N. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar), we the effects of on Akt activation by VEGF. with Akt activity with anti-Akt antibody competitor the H2B signal to levels in incubated with data that HUVEC survival with Akt activity. the of Akt activity in endothelial cell adenoviral expressing wild-type dominant-negative or constitutively active Akt were were with the to from were with an adenoviral expressing which not endothelial cell viability under the conditions of anti-Akt antibodies on immunoprecipitated that and levels of protein Akt protein kinase activity was in the from cells with in the not the of VEGF 4 In kinase activity was in the with the in the presence or absence of VEGF. with high levels of Akt activity that was not by VEGF HUVEC were with to Akt is essential for VEGF-mediated survival. In the absence of growth factor, the dominant-negative Akt did not endothelial cell it the of VEGF from the HUVEC the gel that is of apoptosis 5 The was by the of VEGF in the culture media. Consistent with the data from the with the ability of VEGF to inhibit transduction of wild-type Akt endothelial cell survival with and cell control at 1 and 10 whereas in cell was in the to in the absence of VEGF. These data that expression of wild-type the of endothelial cells to VEGF survival at levels of VEGF. In to with of with promoted endothelial cell survival in the absence of growth of HUVEC that with the of to levels to that in the presence of VEGF 2 that VEGF the protein in HUVECs and that expression of is sufficient to prevent apoptosis in the absence of VEGF H.-P. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar). we used the adenoviral Akt to for the involvement of Akt activation in VEGF-mediated shown in VEGF stimulation to a induction of was by with that Akt is essential for VEGF-mediated induction of However, with was not sufficient to induce that the endothelial cell survival conferred by Akt under conditions the role of Akt in anchorage-dependent endothelial cell HUVECs were cultured in suspension under conditions in endothelial cells (19Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2788) Google Scholar) as by cell and of the of VEGF in the suspension culture on cell However, cells with the adenoviral expressing constitutively active Akt in suspension culture that constitutively active Akt endothelial cell viability under were on cells cultured under conditions Consistent with of cell with an of viability M. T. K. N. K. F. K. M. S. S. K. S. Nat. Med. 1996; PubMed Scopus Google Scholar). These data that constitutive Akt not stimulation with VEGF, is sufficient to survival in endothelial cells in the absence of matrix attachment. further the role of Akt in anchorage-dependent endothelial cell Akt levels were in HUVEC and suspension an antibody that is for Akt at and of the of Akt activation D.R. M. B. P. N. P. J. 1996; PubMed Scopus Google Scholar). Consistent with of histone activity VEGF stimulation of HUVEC monolayers the of Akt with in the of Akt protein However, VEGF stimulation on the of Akt 1 h of in suspension culture suspension culture cells levels of Akt levels of Akt protein were between cells and cells in VEGF stimulation of HUVECs in suspension for h also did not induce Akt this HUVEC is and Akt protein levels are reduced to of endothelial cells to survival and proper cell-matrix are for the formation of blood as as the maintenance of Consistent with its function as a survival factor, VEGF withdrawal is associated with vascular regression in both developing retina and tumors (13Alon T. Hemo I. Itin A. Pe'er J. Stone J. Keshet E. Nat. Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1422) Google Scholar, 16Yuan F. Chen Y. Dellian M. Safabakhsh N. Ferrara N. Jain R.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14765-14770Crossref PubMed Scopus (608) Google Scholar, L.E. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8761-8766Crossref PubMed Scopus (444) Google L.E. D. Itin A. D. Keshet E. J. Clin. Invest. PubMed Scopus Google Scholar). In this study, we the role of the Akt protein kinase in the survival functions of VEGF and matrix attachment in endothelial shown that survival in with the wortmannin-sensitive activation of the Akt The of Akt activation is by the that expression of a dominant-negative Akt mutant the cell survival of VEGF. also that expression of constitutively active Akt is sufficient to survival to endothelial the of this al. H.-P. A. J. M. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) that VEGF activates Akt and that overexpression of constitutively active Akt endothelial cells from apoptosis, whereas a dominant-negative Akt the cytoprotection conferred by VEGF. The results of two that Akt activation is essential for cytoprotection and that or gene transfer of constitutively active Akt for VEGF with to cell survival. Here, we also shown that transfer of wild-type Akt not promote endothelial cell survival in the absence of VEGF, it the survival effects of levels of VEGF. data that activation of Akt a in the signaling that endothelial cell growth it has been that proper matrix are essential for endothelial cell survival during neovascularization as cells the (20Brooks 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 (2185) Google Scholar). it was of to Akt was also in attachment-mediated endothelial cell survival VEGF and matrix survival were with to Consistent with in endothelial cells and cell (18Meredith J.E. Fazeli B. Schwartz M.A. Mol. Biol. Cell. 1993; 4: 953-961Crossref PubMed Scopus (1405) Google Scholar), HUVECs apoptosis incubated in suspension VEGF stimulation did not inhibit apoptosis under transduction of constitutively active Akt was sufficient to promote cell viability in the absence of matrix attachment. cell VEGF was unable to Akt activation of that matrix attachment is essential for signal transduction of in suspension culture, Akt protein was that in Akt protein also to this in cell However, overexpression of wild-type Akt in either the presence or absence of VEGF stimulation on endothelial cell viability in suspension culture not Therefore, it that the of Akt protein is a of the as has been in and cell C. S. G.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and that apoptosis from a of VEGF to promote Akt activation in the absence of matrix attachment. attachment to for signal transduction of has been shown that on cells growth signal transduction by the tyrosine kinase activity of the receptor B. D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), and in endothelial cells with to VEGF activation of the is that VEGF function by attachment-mediated survival signals. in the absence of VEGF cell attachment has a on the of Akt activation These data suggest that growth of Akt activation endothelial and it is that VEGF stimulation to from integrins and the extracellular the of which are also by VEGF (14Spyridopoulos I. Brogi E. Kearney M. Sullivan A.B. Cetrulo C. Isner J.M. Losordo D.W. J. Mol. Cell. Cardiol. 1997; 29: 1321-1330Abstract Full Text PDF PubMed Scopus (193) Google Scholar). In endothelial VEGF tyrosine of various signal including PI3-kinase, and (11Guo D. Jia Q. Song H.-Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Here, we shown that Akt activation and cytoprotection in endothelial cells was by a PI3-kinase that Akt acts of PI3-kinase in the VEGF signaling In VEGF mitogenic functions are through protein kinase which is not by P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (525) Google Scholar). data suggest that the and mitogenic of VEGF are by pathways in endothelial In the of this that Akt is essential in both and attachment-mediated survival in endothelial In data the of cross-talk between two signaling pathways in Akt activation with on endothelial cell survival. endothelial cell viability is essential for the maintenance of blood further of Akt the control of angiogenesis during normal development and for