3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors Attenuate Vascular Smooth Muscle Proliferation by Preventing Rho GTPase-induced Down-regulation of p27

The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway. Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1–10 μm), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 ± 5, 50 ± 3, and 48 ± 3%, respectively. This correlated with a 20-fold increase in p27 Kip1 without changes in p16, p21 Waf1, or p53 levels compared with PDGF alone. Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho. Up-regulation of p27 Kip1 and inhibition of Rho but not Ras membrane translocation by Sim were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate. Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27 Kip1 and inhibited retinoblastoma hyperphosphorylation. In contrast, activation of Rho byEscherichia coli cytotoxic necrotizing factor-1 decreased p27 Kip1 and increased SMC DNA synthesis. These findings indicate that the down-regulation of p27 Kip1 by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall. The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway. Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1–10 μm), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 ± 5, 50 ± 3, and 48 ± 3%, respectively. This correlated with a 20-fold increase in p27 Kip1 without changes in p16, p21 Waf1, or p53 levels compared with PDGF alone. Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho. Up-regulation of p27 Kip1 and inhibition of Rho but not Ras membrane translocation by Sim were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate. Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27 Kip1 and inhibited retinoblastoma hyperphosphorylation. In contrast, activation of Rho byEscherichia coli cytotoxic necrotizing factor-1 decreased p27 Kip1 and increased SMC DNA synthesis. These findings indicate that the down-regulation of p27 Kip1 by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall. Vascular proliferative diseases such as atherosclerosis, post-angioplasty re-stenosis, and transplant arteriosclerosis are characterized by vascular smooth muscle cell (SMC) 1The abbreviations used are: SMC, smooth muscle cell; PDGF, platelet-derived growth factor; Rb, retinoblastoma; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; FPP, farensylpyrophosphate; GGPP, geranylgeranylpyrophosphate; CNF-1, cytotoxic necrotizing factor-1; FCS, fetal calf serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Cdk, cyclin-dependent kinase. DNA synthesis (1Braun-Dullaeus R.C. Mann M.J. Dzau V.J. Circulation. 1998; 98: 82-89Crossref PubMed Scopus (304) Google Scholar). The entry and progression of SMC into the cell cycle is stimulated by growth factors derived from inflammatory cells, platelets, and the vascular wall (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4976) Google Scholar). Although these growth factors which include basic fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor-β1, angiotensin II, and insulin-like growth factor utilize distinct signaling pathways to promote SMC DNA synthesis, these signaling pathways, however, must converge upon common regulators of the cell cycle (3Lukas J. Bartkove J. Bartek J. Mol. Cell. Biol. 1996; 16: 6917-6925Crossref PubMed Scopus (294) Google Scholar). These regulators include the cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors. Indeed, gene therapy with Cdk inhibitors or treatment with a neutralizing antibody to PDGF inhibits neointimal smooth muscle DNA synthesis after balloon angioplasty (1Braun-Dullaeus R.C. Mann M.J. Dzau V.J. Circulation. 1998; 98: 82-89Crossref PubMed Scopus (304) Google Scholar, 4Ferns G.A. Eines E.W. Sprugel K.H. Montani A.S. Reidy M.A. Ross R. Science. 1991; 253: 1129-1132Crossref PubMed Scopus (946) Google Scholar). The transition through the cell cycle is regulated by the expression and activity of cell cycle checkpoint proteins comprising of cyclins and Cdks (5Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). These in turn are regulated by the family of Cdk inhibitor proteins, such as p16, p21 Waf1 and p27 Kip1. The final common pathway leading to G0/G1/S transition is the hyperphosphorylation of the retinoblastoma gene product (Rb), which functions as a molecular switch dedicating the cell to DNA replication. Hyperphosphorylation of Rb results in the release of the transcription factor E2F, which induces the expression of genes required for the progression through S, G2, and M phases (5Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). Despite recent advances in the understanding of cell cycle regulation in proliferative vascular diseases such as atherosclerosis and post-angioplasty restenosis, therapy is still lacking which can effectively prevent SMC DNA synthesis. Large clinical trials have shown that inhibition of cholesterol biosynthesis by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins improve clinical outcomes in patients with atherosclerosis. For example, treatment with HMG-CoA reductase inhibitors reduces post-angioplasty re-stenosis, coronary bypass occlusions (6Shepherd J. Cobbe S.M. Ford I. Isles C.G. Lorimer A.R. MacFarlane P.W. McKillop J.H. Packard C.J. N. Engl. J. Med. 1995; 333: 1301-1307Crossref PubMed Scopus (7477) Google Scholar, 7Cholesterol and Recurrent Events Trial Investigators N. Engl. J. Med. 1996; 335: 1001-1009Crossref PubMed Scopus (7200) Google Scholar), and transplant arteriosclerosis (8Wenke K. Meiser B. Thiery J. Nagel D. von Scheidt W. Steinbeck G. Seidel D. Reichart B. Circulation. 1997; 96: 1398-1402Crossref PubMed Scopus (446) Google Scholar). Although HMG-CoA reductase inhibitors have been shown to inhibit SMC proliferation in vitro (9Corsini A. Mazzotti M. Raiteri M. Soma M.R. Gabbiani G. Fumagalli R. Paoletti R. Atherosclerosis. 1993; 101: 117-125Abstract Full Text PDF PubMed Scopus (211) Google Scholar), the mechanism(s) by which they inhibit cell growth is not known. The HMG-CoA reductase inhibitors not only inhibit cholesterol synthesis, but also, inhibit the synthesis of important isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Both FPP and GGPP are important lipid attachments for the post-translational modification of a variety of proteins, including Ras and Rho GTP-binding proteins (10Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4548) Google Scholar, 11Casey P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (729) Google Scholar). We and others (12Laufs U. La Fata V. Plutzky J. Liao J.K. Circulation. 1998; 97: 1129-1135Crossref PubMed Scopus (1717) Google Scholar, 13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar) have recently shown that some of the direct effects of HMG-CoA reductase inhibitors on the vascular wall are mediated by inhibition of isoprenoid but not cholesterol synthesis. The purpose of this study, therefore, is to determine the role and mechanism of isoprenoid intermediates in regulating cell cycle progression in human vascular SMC. All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Mevastatin (compactin), farnesylpyrophosphate, geranylgeranylpyrophosphate, and l-mevalonate were purchased from Sigma. Low density lipoprotein, FTI-276, and GGTI-286 were obtained from Calbiochem (San Diego, CA). Simvastatin was obtained from Merck Sharp and Dohme, Inc. Mevastatin and simvastatin were chemically activated by alkaline hydrolysis prior to use as described previously (13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar). PDGF-BB was purchased from Genzyme (Boston, MA). α-32PATP, 3Hthymidine, 3Hgeranylgeranyl pyrophosphate, and 3Hfarnesylpyrophosphate were supplied by NEN Life Science Products Inc. The antibody detection kit (enhanced chemiluminescence) and the nylon nucleic acid (Hybond) and protein (polyvinylidene difluoride) transfer membranes were purchased from Amersham Corp. (Arlington Heights, IL). The Clostridium botulinum C3 transferase was obtained from List Biological Laboratories, Inc. (Campbell, CA). Recombinant Escherichia coli cytotoxic necrotizing factor (CNF)-1 and RhoA mutants were kindly provided by K. Aktories (University of Freiberg, Germany) and W. Moolenaar (Netherlands Cancer Institute, Netherlands), respectively. Human vascular SMC were isolated from outgrowths of tunica media explants derived from human aortic and saphenous vein tissues as described previously (14Shin W.S. Hong Y-H. Peng H-B. De Caterina R. Libby P. Liao J.K. J. Biol. Chem. 1996; 271: 11317-11324Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Cells of two to four passages were grown in culture medium containing Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 10% fetal calf serum (FCS) (Hyclone, Logan, UT), and antibiotic mixture of penicillin (100 units/ml)/streptomycin (100 μg/ml)/Fungizone (1.25 μg/ml). The cells were characterized by phase-contrast microscopy and staining for SMC-specific α-actin. Confluent SMC were rendered quiescent by incubation in 0.4% FCS for 48 h before experiments. Cellular viability was determined by cell count, morphology, and trypan blue exclusion. For measurement of isoprenoid incorporation, SMC were treated with simvastatin (10 μm) for 24 h before addition of 50 μCi/ml 3HGGPP or 3HFPP, respectively. After 24 h, cells were washed four times with PBS. Aliquots of the cell lysates were counted in a liquid scintillation counter (Beckmann LS1800). Cell lysates were separated on SDS-PAGE as described (13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar). Gels were stained with Coomassie Blue to visualize protein loading and fixed according to the manufacturer's protocol (NEN Life Science Products). After vaccuum drying, autoradiography was performed for 4 weeks at −80 °C. Cellular lysates from membrane- and cytoplasma-enriched fractions were prepared and separated on SDS-PAGE as described (13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar, 15Spiecker M. Peng H.B. Liao J.K. J. Biol. Chem. 1997; 272: 30969-30974Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The presence of Gαi2 and Rel A (p65) were used as specific markers of membrane and cytoplasmic fractions, respectively, as described previously (15Spiecker M. Peng H.B. Liao J.K. J. Biol. Chem. 1997; 272: 30969-30974Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Preliminary studies using our high/low speed ultracentrifugation through sucrose gradient showed that the high-speed membrane fractions were relatively pure (i.e. presence of Gαi2 and of Rel was performed using Rb and p21 Waf1 p27 p53 Ras RhoA and to was using a antibody or antibody and the kit was performed at and the were by SMC were grown to and by incubation in Dulbecco's modified Eagle's medium containing 0.4% fetal calf serum (FCS) for 48 The media was with growth medium modified Eagle's 10% FCS containing and the reagents were For using and these reagents were h before with After 48 h, (10 was and the cells were 24 Cells were washed 4 times in and in was determined using a liquid scintillation counter The cyclin-dependent was performed as described (15Spiecker M. Peng H.B. Liao J.K. J. Biol. Chem. 1997; 272: 30969-30974Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). were prepared with a containing The -4, and -6 were with of the human antibody from of lysates for h at 4 °C. The was with protein and washed times with and with The was with a protein as a in of containing (10 (10 μm), of (10 (10 (10 μm), μm), (10 and The was by the addition of times SDS-PAGE and for were separated on SDS-PAGE and autoradiography of the gel was performed at −80 °C. SMC were on washed into Cells were with of and RhoA using (Life Technologies, the manufacturer's Both a 48 h after cells were fixed with in and with in PBS. Cells were washed and with and Kip1 antibody in serum at 4 °C. After with serum and were used as in serum and at 4 for h in the Cells were washed times with and using the was using were from from and were by the of W. of Scholar). All are as ± compared with and experiments. and were to determine the of changes in A was for pure human SMC were by phase-contrast microscopy and staining for SMC-specific not was effects of μm), simvastatin μm), FPP, GGPP, C3 or on of μm) or simvastatin (Sim, μm) were of For measurement of isoprenoid incorporation, SMC were treated with simvastatin (10 μm) for 24 h before addition of 50 μCi/ml 3HGGPP or 50 μCi/ml SDS-PAGE and scintillation of cell lysates after 24 h that ± of 3HGGPP and ± of were by SMC. SDS-PAGE showed in the cell lysates including at cell lysates showed of only at not These findings suggest that GGPP and FPP are by SMC our results were obtained using and determine the of isoprenoid synthesis to SMC cell cycle cells were treated with of Sim μm) for 24 incorporation, Sim inhibited SMC DNA synthesis by ± which was reversed by with μm) however, effect on This in by Sim correlated with a in Rb hyperphosphorylation by ± effects were with HMG-CoA reductase inhibitor, not Inhibition of SMC DNA synthesis was of cholesterol cells were in 10% fetal calf serum addition of density not the effects of Sim on These findings suggest that isoprenoid synthesis is important in PDGF-induced SMC DNA synthesis. The entry into and progression of the cell cycle is regulated by of cyclins and Cdks (5Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). determine can Cdk we the effect of Sim on PDGF-induced and protein as for Cdk, we that quiescent SMC have or -4, and -6 activity with PDGF 24 increased -4, and -6 with Sim μm) inhibited PDGF-induced -4, and -6 activity by 80 ± 5, ± 3, and 48 ± 3%, respectively. inhibition of isoprenoid synthesis Cdk activity with the with The Cdk inhibitors such as p16, p21 Waf1 and p27 Kip1 to and inhibit the activation of The gene p53 regulates cell cycle in by the expression of p21 Waf1 (1Braun-Dullaeus R.C. Mann M.J. Dzau V.J. Circulation. 1998; 98: 82-89Crossref PubMed Scopus (304) Google Scholar). determine inhibition of isoprenoid synthesis can the expression of Cdk we investigated the effects of Sim μm) on p16, p21 Waf1, p27 and p53 levels in SMC stimulated with PDGF for and with PDGF we that Sim not the levels of and p21 Waf1 In contrast, with PDGF decreased p27 Kip1 by 80 ± ± 3, and ± after and h, respectively. with Sim μm) reversed the down-regulation of p27 Kip1 by PDGF in 20-fold increase in p27 Kip1 after 24 h compared to that of Although p53 levels in a after PDGF the levels were not in the presence or of These findings indicate that inhibition of isoprenoid synthesis by Sim p27 and suggest that p27 Kip1 may in the inhibition of The of the Ras and Rho family have been shown to entry into the cell cycle M.R. Nature. PubMed Scopus Google Scholar, R. C.J. 1995; Google Scholar). Ras and Rho are modified by which is for membrane localization and P.J. Science. 1995; 268: 221-225Crossref PubMed Scopus (729) Google Scholar). We have previously shown that Ras and Rho are required for GTPase activity (13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar). with PDGF 24 increased the of RhoA by ± without the of RhoA in smooth muscle cells Inhibition of isoprenoid synthesis by Sim μm) reversed and PDGF-induced RhoA membrane in the presence of PDGF, Sim increased the expression of RhoA to for in RhoA Indeed, with GGPP, but not FPP reversed the effect of Sim on RhoA membrane the expression of Ras in SMC membranes was not by treatment with PDGF or Sim after 24 h In cell however, treatment with Sim in the of a molecular studies have that Ras on SDS-PAGE Ras K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, 13Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar). Indeed, the to Ras in SMC treated with PDGF and Sim is in the presence of FPP, but not These findings that inhibition of by Sim PDGF-induced membrane localization of but not determine which isoprenoid p27 PDGF SMC were treated with Sim μm) in the presence or of GGPP μm) and FPP with GGPP, but not FPP, reversed the of p27 Kip1 by Sim Since Rho is a for we investigated inhibition of Rho p27 Kip1. of SMC with botulinum C3 transferase 24 which Rho by K. J. 1997; Scholar), the PDGF-induced down-regulation of p27 Kip1 determine changes in p27 Kip1 are specific to inhibition of we SMC with RhoA and a dominant-negative RhoA Both of these RhoA a The as by and is overexpression of in PDGF-induced SMC not p27 Kip1 expression to cells (i.e. cells without In contrast, SMC the dominant-negative N19RhoA as by for showed increased p27 Kip1 these findings indicate that RhoA regulates p27 Kip1 expression and suggest that Sim p27 Kip1 by Rho membrane localization and determine the effects of Rho on cell cycle we investigated the effects of PDGF Sim μm), GGPP μm), FPP μm), or C3 transferase on Rb hyperphosphorylation in SMC after 24 with GGPP, and to a FPP, reversed the effect of Sim on PDGF-induced Rb hyperphosphorylation in SMC of Rho by C3 transferase decreased Rb hyperphosphorylation by 80 ± This in Rb hyperphosphorylation by C3 transferase was not reversed by GGPP or FPP not SMC was treated coli which is to Rho by Rho hydrolysis G. P. M. J. Mann M. Aktories K. Nature. 1997; PubMed Scopus Google Scholar, G. M. P. P. Nature. 1997; PubMed Scopus Google Scholar), Rb in the of fetal calf serum in the presence of 10% fetal calf serum in the culture Rb hyperphosphorylation by with treatment with PDGF was inhibited in the presence of Sim ± with GGPP μm) reversed the effect of Sim ± incorporation, compared with as FPP μm) only reversed the effects of Sim ± incorporation, compared with with density not the effects of simvastatin with increased by ± with C3 transferase by ± for compared with determine the that and to PDGF-induced SMC DNA synthesis, we investigated the effects of inhibitors of and on PDGF-induced The inhibitor μm) decreased PDGF by ± the inhibitor decreased PDGF by ± We have shown that the and the protein that mediates PDGF-induced cell cycle progression by the expression of the Cdk inhibitor p27 Kip1 and the activity of and hyperphosphorylation of with the HMG-CoA reductase inhibitor, Rho and membrane inhibits Cdk activity and Rb and SMC DNA synthesis. Indeed, Rb hyperphosphorylation and DNA synthesis are and for or PDGF-induced SMC DNA synthesis and are with in p27 Kip1 These findings indicate important role of Rho in and are with studies that p27 Kip1 mediates cell cycle Science. 1996; 271: PubMed Scopus Google Scholar, W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and that the of p27 Kip1 the growth of cells A. M. I. K. J. Biol. Chem. 1996; 272: Full Text Full Text PDF Scopus Google Scholar). however, are in to studies which suggest that PDGF-induced SMC DNA synthesis is mediated by Ras M.R. Nature. PubMed Scopus Google Scholar). The Rho GTPase family which are for post-translational modification by modification is important for and these Rho to the membrane (10Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4548) Google Scholar, A. Science. 1998; PubMed Scopus Google Scholar). The membrane translocation of or Rho the release of cytoplasmic inhibitor, Rho inhibitor, and activation of Rho through in the presence of factor 1997; PubMed Scopus Google Scholar, B. D. R.A. Nature. 1997; PubMed Scopus Google Scholar, M. M. B. Moolenaar J. Cell Biol. 1997; PubMed Scopus Google Scholar). Since of the cytoplasmic Rho proteins are activity in the presence of HMG-CoA reductase inhibitor inhibition of Rho and membrane translocation by HMG-CoA reductase inhibitors to a of Rho in the This is with our that in the presence of HMG-CoA reductase addition of GGPP, but not FPP, membrane expression of p27 Kip1 and cell cycle in addition to RhoA activity by we that with PDGF the of The mechanism by which PDGF RhoA in the however, to The of Rho in SMC DNA synthesis was by studies that inhibition of Rho by botulinum C3 transferase inhibits SMC DNA synthesis. The C3 transferase of RhoA and and in the K. J. 1997; Scholar). Although the C3 transferase may or K. J. 1997; Scholar, G. P. M. J. Mann M. Aktories K. Nature. 1997; PubMed Scopus Google Scholar), our results that the overexpression of a dominant-negative RhoA p27 are with the effects of C3 transferase on Rho. In contrast, direct activation of Rho by reversed the effects of HMG-CoA reductase inhibitors. was to and SMC cell cycle progression in the presence and of respectively. These findings indicate that Rho GTPase is a of PDGF-induced SMC DNA synthesis. results suggest that cell cycle progression is mediated by down-regulation of the Cdk inhibitor p27 Kip1. In contrast, Rho been shown to p21 Waf1 but not p27 Kip1 levels in cells C.J. Nature. 1998; PubMed Scopus Google Scholar). Indeed, HMG-CoA reductase inhibitor, been shown to increase the of p21 Waf1 in cells M.J. J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). studies with human vascular SMC, however, showed effect of HMG-CoA reductase inhibitors on p21 Waf1 or cell cycle in SMC by HMG-CoA reductase inhibitors to mediated by which are of p21 Waf1 and important gene which is in cell cycle regulation is p53 (2Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4976) Google Scholar, Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Since of p53 was not after inhibition of isoprenoid synthesis, SMC growth by HMG-CoA reductase inhibitors by which are of p53 These results are with the of changes in p21 which been shown in studies to by p53 Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In to and p21 Waf1, p27 Kip1 levels are increased by treatment with HMG-CoA reductase inhibitors and the of p27 Kip1 correlated with the inhibition of and to a and -6 Both Ras and Rho have been shown to in the regulation of growth in a variety of cell In to GGPP, addition of FPP only reversed the effects of HMG-CoA reductase inhibitors on SMC DNA synthesis by that Ras may not the of PDGF-induced SMC DNA synthesis. This may to the that after 24 h of was effect of PDGF, GGPP, or FPP on the of Ras in the These findings are with recent studies that the translocation of but not to the membrane with cell cycle progression from to in cells A. S.M. 1996; Google Scholar, M. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and that GGPP reversed the of p27 Kip1 by HMG-CoA reductase FPP or effect on p27 Kip1. a recent that Ras and Rho to p21 Waf1 and cell cycle progression in cells C.J. Nature. 1998; PubMed Scopus Google Scholar). therefore, are to determine the role of Ras in PDGF-induced SMC cell cycle clinical suggest that some of the effects of treatment with HMG-CoA reductase inhibitors may of changes in serum cholesterol levels J. for the of Circulation. 1998; 97: PubMed Scopus Google Scholar). indicate that is a direct effect of these on the wall inhibition of Rho We that Rho is a and of SMC cell cycle progression and that inhibition of Rho in SMC may have effects in vascular proliferative diseases such as atherosclerosis and post-angioplasty The mechanism(s) by which Rho p27 however, to We K. W. and P. Libby for kindly CNF-1, RhoA and human vascular smooth muscle cells, respectively. We are to for in the