Inhibition of Growth and p21 Methylation in Vascular Endothelial Cells by Homocysteine but Not Cysteine

Abstract

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10–50 μm of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21ras (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels ofS-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21ras and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21ras-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process. Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10–50 μm of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21ras (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels ofS-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21ras and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21ras-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process. Arteriosclerosis and its complications (such as myocardial infarction, stroke, and peripheral vascular disease) are the leading causes of death in developed countries (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9988) Google Scholar). Elevated blood levels of homocysteine, an intermediate metabolite of methionine, are an important and independent risk factor for arteriosclerosis (2Stampfer M.J. Malinow M.R. N. Engl. J. Med. 1995; 332: 328-329Crossref PubMed Scopus (170) Google Scholar, 3Stampfer M.J. Willett W.C. J. Am. Med. Assoc. 1993; 270: 2726-2727Crossref PubMed Scopus (77) Google Scholar, 4Stampfer M.J. Malinow M.R. Willett W.C. Newcomer L.M. Upson B. Ullmann D. Tishler P.V. Hennekens C.H. J. Am. Med. Assoc. 1992; 268: 877-881Crossref PubMed Scopus (1579) Google Scholar, 5Selhub J. Jacques P.F. Wilson P.W. Rush D. Rosenberg I.H. J. Am. Med. Assoc. 1993; 270: 2693-2698Crossref PubMed Scopus (1856) Google Scholar, 6Selhub J. Jacques P.F. Bostom A.G. D'Agostino R.B. Wilson P.W. Belanger A.J. O'Leary D.H. Wolf P.A. Schaefer E.J. Rosenberg I.H. N. Engl. J. Med. 1995; 332: 286-291Crossref PubMed Scopus (1145) Google Scholar, 7Malinow M.R. Nieto F.J. Szklo M. Chambless L.E. Bond G. Circulation. 1993; 87: 1107-1113Crossref PubMed Google Scholar). In the Framingham Study, for example, 21% of the population in this large prospective trial had homocysteine levels that would increase the risk of a heart attack by 3.4-fold (4Stampfer M.J. Malinow M.R. Willett W.C. Newcomer L.M. Upson B. Ullmann D. Tishler P.V. Hennekens C.H. J. Am. Med. Assoc. 1992; 268: 877-881Crossref PubMed Scopus (1579) Google Scholar, 5Selhub J. Jacques P.F. Wilson P.W. Rush D. Rosenberg I.H. J. Am. Med. Assoc. 1993; 270: 2693-2698Crossref PubMed Scopus (1856) Google Scholar). Despite the importance of homocysteine as a risk factor, however, the mechanisms by which it induces arteriosclerosis are not understood. Vascular endothelial cells (VEC) 1The abbreviations used are: VEC, vascular endothelial cell(s); VSMC, vascular smooth muscle cell(s); SAH,S-adenosylhomocysteine; MAP, mitogen-activated protein; HUVEC, human umbilical vein endothelial cell(s); HAEC, human aortic endothelial cell(s); HASMC, human aortic smooth muscle cell(s); EHNA, erythro-9-(2-hydroxy-3-nonyl)-adenine; SAM,S-adenosylmethionine; PAGE, polyacrylamide gel electrophoresis; ERK, extracellular signal-regulated kinase. have been implicated in the pathogenesis of arteriosclerosis (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9988) Google Scholar). VEC normally insulate the vessel wall from circulating phagocytes and platelets. In response to various stimuli and injuries, damaged VEC attract phagocytes and produce cytokines and growth factors that act on neighboring vascular smooth muscle cells (VSMC) to promote their growth. Damaged VEC also allow platelets to adhere to the vessel wall and promote the formation of thrombi. For these reasons previous studies of homocysteine have focused on its effects on VEC (8Harker L.A. Harlan J.M. Ross R. Arterioscler. Thromb. 1993; 13: 512-516Crossref PubMed Google Scholar, 9Dudman N.P. Hicks C. Wang J. Wilcken D.E. Atherosclerosis. 1991; 91: 77-83Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 10Lentz S.R. Sadler J.E. Blood. 1993; 81: 683-689Crossref PubMed Google Scholar, 11Hajjar K.A. J. Clin. Invest. 1993; 91: 2873-2879Crossref PubMed Scopus (303) Google Scholar, 12Rodgers G.M. Conn M.T. Blood. 1990; 75: 895-901Crossref PubMed Google Scholar, 13Stamler J.S. Osborne J.A. Jaraki O. Rabbani L.E. Mullins M. Singel D. Loscalzo J. J. Clin. Invest. 1993; 91: 308-318Crossref PubMed Scopus (832) Google Scholar). High concentrations of homocysteine (greater than 1 mm) damage VEC, inhibit thrombomodulin secretion and protein C activation, reduce the number of cellular binding sites for tissue plasminogen activator, and impair endothelium-derived vasorelaxation (8Harker L.A. Harlan J.M. Ross R. Arterioscler. Thromb. 1993; 13: 512-516Crossref PubMed Google Scholar, 9Dudman N.P. Hicks C. Wang J. Wilcken D.E. Atherosclerosis. 1991; 91: 77-83Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 10Lentz S.R. Sadler J.E. Blood. 1993; 81: 683-689Crossref PubMed Google Scholar, 11Hajjar K.A. J. Clin. Invest. 1993; 91: 2873-2879Crossref PubMed Scopus (303) Google Scholar, 12Rodgers G.M. Conn M.T. Blood. 1990; 75: 895-901Crossref PubMed Google Scholar, 13Stamler J.S. Osborne J.A. Jaraki O. Rabbani L.E. Mullins M. Singel D. Loscalzo J. J. Clin. Invest. 1993; 91: 308-318Crossref PubMed Scopus (832) Google Scholar). Unfortunately, it has been difficult to correlate these in vitro observations with clinical observations because the levels of homocysteine studied usually exceed by about 100-fold the levels found in the population at risk. In addition, most (if not all) of the biological effects of homocysteine can be mimicked by cysteine or other sulfhydryl-containing agents (9Dudman N.P. Hicks C. Wang J. Wilcken D.E. Atherosclerosis. 1991; 91: 77-83Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 14Harpel P.C. Chang V.T. Borth W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10193-10197Crossref PubMed Scopus (267) Google Scholar). Therefore a mechanism unique to homocysteine has yet to be identified. Cellular methylation, which regulates gene expression (15Bird A.P. Taggart M.H. Nicholls R.D. Higgs D.R. EMBO J. 1987; 6: 999-1004Crossref PubMed Scopus (149) Google Scholar, 16Cedar H. Cell. 1988; 53: 3-4Abstract Full Text PDF PubMed Scopus (730) Google Scholar) and modifies protein function (17Clarke S. Curr. Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (200) Google Scholar, 18Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar, 19Parish C.A. Rando R.R. Biochemistry. 1994; 33: 9986-9991Crossref PubMed Scopus (36) Google Scholar, 20Philips M.R. Pillinger M.H. Staud R. Volker C. Rosenfeld M.G. Weissmann G. Stock J.B. Science. 1993; 259: 977-980Crossref PubMed Scopus (183) Google Scholar), cannot occur without the action of methyltransferase (21Clarke S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (793) Google Scholar). Homocysteine (but not cysteine) is efficiently converted to a potent inhibitor of methyltransferase,S-adenosylhomocysteine (SAH), in the presence of adenosine (22Bergmann S. Shatrov V. Ratter F. Schiemann S. Schulze-Osthoff K. Lehmann V. J. Immunol. 1994; 153: 1736-1743PubMed Google Scholar, 23Barber J.R. Clarke S. J. Biol. Chem. 1984; 259: 7115-7122Abstract Full Text PDF PubMed Google Scholar, 24Paik W.K. Farooqui J.Z. Kim S. Adv. Enzyme Regul. 1980; 19: 471-486Crossref PubMed Scopus (5) Google Scholar). To elucidate the role of homocysteine in the development of arteriosclerosis, we studied its effect at low doses on VEC proliferation and p21ras methylation. We found that clinically relevant concentrations of homocysteine but not cysteine inhibited proliferation of VEC in a cell type-specific manner. This inhibition of VEC growth was associated with an increase in the level of SAH, a reduction in p21ras methylation and membrane association, and a reduction in mitogen-activated protein (MAP) kinase activity. Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) (Clonetics Corp, San Diego, CA) were grown in M199 medium (JRH Biosciences, Lenexa, KS) containing 20% fetal calf serum (Hyclone Laboratories), 50 μg/ml endothelial cell growth supplement (Collaborative Biomedical), and 50 μg/ml heparin (Sigma). Human aortic smooth muscle cells (HASMC) (Clonetics Corp, San Diego, CA) were grown in M199 containing 20% fetal calf serum. Porcine aortic endothelial cells were grown in Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS) containing 10% fetal calf serum. The culture medium was supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells from passages 6–8 were used in the experiments. Cells were plated onto 24-well plates and grown to 70–80% confluence. The cells were incubated with fresh medium containing 50 μm adenosine and 10 μm erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) for 24 h and exposed to l-cysteine,l-cystine, dl-homocysteine, orl-homocystine. Cells were metabolically labeled with 1 μCi/ml [methyl-3H]thymidine (20 Ci/mmol, NEN Life Science Products) for the last 3 h. After labeling, the cells were washed with phosphate-buffered saline, fixed with cold 10% trichloroacetic acid, and then washed with 95% ethanol. Incorporated [methyl-3H]thymidine was extracted in 0.2 nNaOH and measured in a liquid scintillation counter (25Tsai J.-C. Perrella M.A. Yoshizumi M. Hsieh C.-M. Haber E. Schlegel R. Lee M.-E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6369-6373Crossref PubMed Scopus (762) Google Scholar, 26Tsai J.-C. Wang H. Perrella M.A. Yoshizumi M. Sibinga N.E.S. Tan L.C. Haber E. Chang T.-H. Schlegel R. Lee M.-E. J. Clin. Invest. 1996; 97: 146-153Crossref PubMed Scopus (194) Google Scholar). HAEC (2–5 × 105 cells/cm2) were plated onto six-well plates and cultured to 25–30% confluence. The cells were then cultured with fresh medium containing 50 μm adenosine and 10 μm EHNA and exposed to l-cysteine,l-cystine, dl-homocysteine,l-homocystine, or l-homocysteine thiolactone. Medium and chemicals were changed every other day. Cells were harvested by trypsinization, and cell number was determined in triplicate in a hemocytometer (Coulter Electronics, Hialeah, FL) at 2-day intervals. HAEC (2–5 × 105 cells/cm2) were plated onto 100-mm dishes and cultured to 70–80% confluence. Thereafter the cells were cultured in fresh medium containing 50 μm adenosine and 10 μm EHNA plus 50 μmdl-homocysteine or l-cysteine. After 24 h the cells were harvested, washed twice with phosphate-buffered saline/0.1% and fixed in at The fixed cells were with units/ml) at for 1 h and to cell of DNA as K. Wang H. Lee Lee Haber E. J. Clin. Invest. 1996; PubMed Scopus Google Scholar). HAEC and were plated onto six-well plates and grown to 70–80% confluence. The cells were then cultured with fresh medium containing 50 μm adenosine and 10 μm EHNA or and labeled metabolically with NEN Life Science 50 for h. Thereafter cells were extracted twice with of 1 The levels of and were determined by as by and H. EMBO J. 1991; 10: PubMed Scopus Google Scholar, H. E. 1993; PubMed Scopus Google Scholar). In the had been the was and in of was then to plates × 10 J. by and 3 were used as the and were also to the as a and their was determined by to The of and was measured on a the HAEC and at 70–80% were cultured in fresh medium containing 50 μm adenosine and 10 μm EHNA plus 50 μmdl-homocysteine or for 24 h. Cells of were metabolically labeled with μCi/ml Ci/mmol, NEN Life Science Products) or Ci/mmol, NEN Life Science Products) for the last h. Cells were extracted with 1 of 1 μm and 1 μm (100 was with of and by for 3 h. We p21ras protein from the gel by as a methylation of p21ras was measured by of protein in a phase as S. Stock J. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar) with gel were with of 1 in a This was a scintillation containing of the scintillation for 24 h at from protein was measured in a liquid scintillation The phase of was determined in a in which 10 of was to of as of the in the was to the scintillation these HAEC at 70–80% were cultured in fresh medium containing 50 μm adenosine and 10 μm EHNA plus 50 μmdl-homocysteine or for 24 h. The cells were labeled metabolically with μCi/ml Ci/mmol, NEN Life Science Products) for the last h. cell were harvested with of were harvested with of 1 10 and 1 × inhibitor and as J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar) with After a on were and by The was then to for The and were by for in a in the phase were then with to which no had been (100 was with of and then by p21ras was measured on a the HAEC and at 70–80% were cultured in fresh medium containing 50 and 10 μm EHNA plus 50 μmdl-homocysteine or for 24 h. Cells were extracted with 1 of (20 1 1 and MAP kinase activity was measured by a kinase Lee M. M.R. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). (20 were by with protein (Sigma). After the gel was washed twice with 50 for 1 washed with 50 for 1 by with for 1 in 50 and then incubated for 1 h in kinase 10 kinase and of The gel was washed with trichloroacetic and MAP kinase activity was determined in a the of for 24 h with 50 μm adenosine and 10 μm EHNA, an adenosine inhibitor to the of adenosine in had effect on and cell growth not in the presence of adenosine and EHNA, homocysteine and decreased in a as decreased DNA synthesis by than 50% in In cysteine had effect on DNA synthesis in This effect was not because homocysteine also inhibited in HAEC and aortic endothelial cells Homocysteine had no effect on in or in human or not that this effect is cell To in cell number homocysteine-induced in DNA we the number of HAEC in the presence of adenosine and EHNA and addition of homocysteine or with their effect on homocysteine, and homocysteine but not cysteine or decreased cell number in a and Although the effect of μm homocysteine was not a in cell number in the presence of 50 μm homocysteine was as as We then HAEC to 24 h addition of homocysteine to its effect on the cell cycle. is in and the of cells in phase of the cell independent is in 3 B. Homocysteine but not cysteine the number of cells in the G1 phase from to and decreased the number in the phase from to and In homocysteine had no effect on the number of cells in the homocysteine to the cell in G1 or at the In the presence of adenosine and homocysteine increase the level of SAH, a potent inhibitor of (22Bergmann S. Shatrov V. Ratter F. Schiemann S. Schulze-Osthoff K. Lehmann V. J. Immunol. 1994; 153: 1736-1743PubMed Google J.R. Clarke S. J. Biol. Chem. 1984; 259: 7115-7122Abstract Full Text PDF PubMed Google Scholar, J. Med. Chem. 1980; PubMed Scopus Google Scholar, W.K. Kim S. Scholar). The of to a of cellular methylation S. 1994; PubMed Scopus Google Scholar, F. G. Life Sci. 1990; PubMed Scopus Google Scholar, Biochem. 1995; PubMed Scopus Google Scholar). To the effect of homocysteine on the in vascular we metabolically labeled or HAEC and with and from the cell by gel is in the from independent Homocysteine (but not cysteine) the level of in HAEC and the by had effect on the in that homocysteine-induced in the cellular may inhibition of VEC growth by the effect of homocysteine on VEC and growth may be explained by its ability to dramatically increase the in HAEC but not The p21ras is for cell growth. on its ability to with the cell which is by a of that prenylation and carboxyl methylation of the p21ras C (17Clarke S. Curr. Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (200) Google Scholar, 18Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar, 20Philips M.R. Pillinger M.H. Staud R. Volker C. Rosenfeld M.G. Weissmann G. Stock J.B. Science. 1993; 259: 977-980Crossref PubMed Scopus (183) Google Scholar, M.R. Staud R. Pillinger M. A. Volker C. Stock J.B. Weissmann G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). To inhibition of VEC growth is associated with in p21ras methylation and membrane association, we labeled or HAEC and with or After the with an of and the by we measured carboxyl methylation of p21ras p21ras protein used as by of protein in a phase S. Stock J. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar). p21ras carboxyl methylation was inhibited by 50% in HAEC for 24 h with homocysteine but not in HAEC with cysteine In homocysteine not p21ras carboxyl methylation in an with that homocysteine had no effect on DNA synthesis and the in We then determined hypomethylation of p21ras its ability to with the cell of HAEC for 24 with homocysteine or cysteine) cell or membrane were with the and then by In HAEC cell homocysteine not the of p21ras protein In HAEC membrane however, homocysteine decreased membrane-associated by Because membrane of and to the of and MAP homocysteine-induced in the membrane of to in MAP kinase activity. Therefore we studied the effect of homocysteine on MAP kinase activity by an Homocysteine but not cysteine decreased the activity of extracellular signal-regulated kinase MAP kinase in by 66% in HAEC In homocysteine to activity in Because is important for cellular proliferation Curr. Opin. Cell Biol. 1993; 5: PubMed Scopus Google Scholar, J.S. Science. 1992; PubMed Scopus Google Scholar), these are with that homocysteine decreased DNA synthesis in HAEC but not In the had activity than activity in than in In homocysteine had no effect on of in that the reduction in membrane in and the in activity in are in activity by serum in but not and were incubated on dishes for 24 h with 50 μm adenosine and 10 μm EHNA plus 50 μmdl-homocysteine in To the cells we the serum to for 24 reduction of the would have to VEC and to for Cells were with or without serum for was and the MAP kinase was as for To this is the that a biological response can be by clinically relevant levels of homocysteine but not The growth effect of homocysteine on VEC at concentrations that the blood homocysteine levels observed in 21% of the in the Framingham (4Stampfer M.J. Malinow M.R. Willett W.C. Newcomer L.M. Upson B. Ullmann D. Tishler P.V. Hennekens C.H. J. Am. Med. Assoc. 1992; 268: 877-881Crossref PubMed Scopus (1579) Google Scholar, 5Selhub J. Jacques P.F. Wilson P.W. Rush D. Rosenberg I.H. J. Am. Med. Assoc. 1993; 270: 2693-2698Crossref PubMed Scopus (1856) Google Scholar). the effect of homocysteine was cell because homocysteine inhibited growth in VEC but not in or fibroblasts. is also important to that the of homocysteine, also inhibited VEC proliferation 1 and of the homocysteine in blood is in the A.J. Biol. Med. 1993; PubMed Scopus Google Scholar, M.R. Clin. Chem. 1995; PubMed Scopus Google Scholar, J. Clin. Biochem. 1995; PubMed Scopus Google Scholar). Although 10–50 μm of homocysteine inhibited VEC growth in the presence of adenosine 1 and these same levels of homocysteine had no effect in the of adenosine not Because adenosine by has effect on VEC growth, these that the effect of homocysteine is at in by its ability to reduce cellular methylation. This is by that homocysteine the levels of the in HAEC but not Although we not the effect of homocysteine is for VEC, it is that homocysteine is the cell membrane in VEC than in other cell as and fibroblasts. This also be the of an increase in the activity of in VEC, which would to levels in the presence of concentrations of adenosine and of a C is a and (17Clarke S. Curr. Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (200) Google Scholar, S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (793) Google Scholar). the function of a protein by its or its ability to with the cell For example, carboxyl methylation is for the binding of to the membrane J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar) and for the to the membrane of the protein and the protein M.R. Pillinger M.H. Staud R. Volker C. Rosenfeld M.G. Weissmann G. Stock J.B. Science. 1993; 259: 977-980Crossref PubMed Scopus (183) Google Scholar, M.R. Staud R. Pillinger M. A. Volker C. Stock J.B. Weissmann G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). of carboxyl methylation the response of to M.R. Pillinger M.H. Staud R. Volker C. Rosenfeld M.G. Weissmann G. Stock J.B. Science. 1993; 259: 977-980Crossref PubMed Scopus (183) Google Scholar, M.R. Staud R. Pillinger M. A. Volker C. Stock J.B. Weissmann G. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and carboxyl methylation the activity of the C.A. Rando R.R. Biochemistry. 1994; 33: 9986-9991Crossref PubMed Scopus (36) Google Scholar, D. F.J. Rando R.R. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). observations are with that homocysteine-induced in p21ras methylation are associated with a in membrane in HAEC and a in the activity of the MAP kinase Because the MAP kinases have been implicated in cell growth Curr. Opin. Cell Biol. 1993; 5: PubMed Scopus Google Scholar, J.S. Science. 1992; PubMed Scopus Google Scholar), the p21ras-MAP kinase pathway may represent one of the mechanisms that mediates the effect of homocysteine on VEC growth. we cannot be that homocysteine's growth effect is by the p21ras-MAP kinase are to hypomethylation of other cellular as or protein C. D. J. Biol. Chem. 1992; Full Text PDF PubMed Google H. Clarke S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), also to the growth effect of Because endothelial cell is to play a role in the development of arteriosclerosis, homocysteine-induced inhibition of proliferation and p21ras methylation in these cells may have an important effect on The effect of homocysteine on VEC methylation and growth in with previous that μm homocysteine (a level observed in with proliferation of (25Tsai J.-C. Perrella M.A. Yoshizumi M. Hsieh C.-M. Haber E. Schlegel R. Lee M.-E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6369-6373Crossref PubMed Scopus (762) Google Scholar, 26Tsai J.-C. Wang H. Perrella M.A. Yoshizumi M. Sibinga N.E.S. Tan L.C. Haber E. Chang T.-H. Schlegel R. Lee M.-E. J. Clin. Invest. 1996; 97: 146-153Crossref PubMed Scopus (194) Google Scholar) may represent an important homocysteine and arteriosclerosis. We are to and H. for We B. for and for