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• W2000951003 abstract "The anti-atherogenic effects of apolipoprotein (apo) E have been attributed to its ability to reduce plasma cholesterol level and to limit foam cell formation. The purpose of this study was to ascertain if apoE also may have cytostatic functions that could attenuate vascular occlusive diseases. Purified apoE inhibited smooth muscle cell migration directed to platelet-derived growth factor (PDGF) or oxidized LDL (oxLDL) (p < 0.0001). The purified apoE also suppressed PDGF- and oxLDL-induced smooth muscle cell proliferation (p < 0.001). These apoE inhibitory effects were not because of suppression of PDGF binding to its receptors on the smooth muscle cells, but was correlated with a significant reduction in agonist-stimulated mitogen-activated protein kinase activity (p < 0.01). ApoE also inhibited PDGF-induced cyclin D1 mRNA expression, suggesting that the apoE effect was mediated by growth arrest at the G0 to G1 phase. Taken together, these results suggest that apoE has cytostatic functions in the vessel wall and may protect against vascular diseases through inhibition of cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation. The anti-atherogenic effects of apolipoprotein (apo) E have been attributed to its ability to reduce plasma cholesterol level and to limit foam cell formation. The purpose of this study was to ascertain if apoE also may have cytostatic functions that could attenuate vascular occlusive diseases. Purified apoE inhibited smooth muscle cell migration directed to platelet-derived growth factor (PDGF) or oxidized LDL (oxLDL) (p < 0.0001). The purified apoE also suppressed PDGF- and oxLDL-induced smooth muscle cell proliferation (p < 0.001). These apoE inhibitory effects were not because of suppression of PDGF binding to its receptors on the smooth muscle cells, but was correlated with a significant reduction in agonist-stimulated mitogen-activated protein kinase activity (p < 0.01). ApoE also inhibited PDGF-induced cyclin D1 mRNA expression, suggesting that the apoE effect was mediated by growth arrest at the G0 to G1 phase. Taken together, these results suggest that apoE has cytostatic functions in the vessel wall and may protect against vascular diseases through inhibition of cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation. Research in the past two decades has clearly established that apolipoprotein (apo) 1The abbreviations used are: apoapolipoproteinVLDLvery low density lipoproteinsIDLintermediate density lipoproteinsLDLlow density lipoproteinsoxLDLoxidized low density lipoproteinsPDGFplatelet-derived growth factorPDGF-BBhomodimer of PDGF B polypeptideMAP kinasemitogen-activated protein kinaseDMEMDulbecco's modified Eagle's mediumPHAS-Iinsulin-regulated phosphorylated heat- and acid-stable proteinFBSfetal bovine serumHPFhigh power fieldsMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium saltLRPlow density lipoprotein receptor-related protein.1The abbreviations used are: apoapolipoproteinVLDLvery low density lipoproteinsIDLintermediate density lipoproteinsLDLlow density lipoproteinsoxLDLoxidized low density lipoproteinsPDGFplatelet-derived growth factorPDGF-BBhomodimer of PDGF B polypeptideMAP kinasemitogen-activated protein kinaseDMEMDulbecco's modified Eagle's mediumPHAS-Iinsulin-regulated phosphorylated heat- and acid-stable proteinFBSfetal bovine serumHPFhigh power fieldsMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium saltLRPlow density lipoprotein receptor-related protein. E protects against vascular diseases (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3376) Google Scholar). Early studies suggest that the protective effect may be related to the ability of apoE to promote cholesterol efflux from peripheral cells and to mediate the clearance of cholesterol-enriched lipoproteins from circulation (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3376) Google Scholar). Transgenic mice overexpressing rat apoE displayed marked resistance to diet-induced hypercholesterolemia and did not develop atherosclerosis (2Shimano H. Yamada N. Katsuki M. Yamamoto K. Gotoda T. Harada K. Shimada M. Yazaki Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1750-1754Crossref PubMed Scopus (119) Google Scholar, 3Shimano H. Yamada N. Katsuki M. Yamamoto K. Gotoda T. Harada K. Shimada M. Yazaki Y. J. Clin. Invest. 1992; 90: 2084-2091Crossref PubMed Scopus (82) Google Scholar). In contrast, mice with targeted disruption of the apoE gene developed spontaneous atherosclerosis even under basal low fat/low cholesterol dietary conditions (4Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1836) Google Scholar, 5Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1869) Google Scholar, 6Reddick R.L. Zhang S.H. Maeda N. Arterioscler. Thromb. 1994; 14: 141-147Crossref PubMed Scopus (549) Google Scholar, 7Zhang S.H. Reddick R.L. Burkey B. Maeda N. J. Clin. Invest. 1994; 94: 937-945Crossref PubMed Scopus (208) Google Scholar). Atherosclerosis in apoE-null mice could be prevented by increasing circulating apoE level through recombinant adenovirus-mediated apoE gene transfer to the liver (8Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer H.B. J. Clin. Invest. 1995; 96: 1612-1620Crossref PubMed Scopus (123) Google Scholar). The decrease in atherosclerosis in this model was accompanied by decreased total cholesterol and VLDL/IDL levels (8Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer H.B. J. Clin. Invest. 1995; 96: 1612-1620Crossref PubMed Scopus (123) Google Scholar). apolipoprotein very low density lipoproteins intermediate density lipoproteins low density lipoproteins oxidized low density lipoproteins platelet-derived growth factor homodimer of PDGF B polypeptide mitogen-activated protein kinase Dulbecco's modified Eagle's medium insulin-regulated phosphorylated heat- and acid-stable protein fetal bovine serum high power fields 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium salt low density lipoprotein receptor-related protein. apolipoprotein very low density lipoproteins intermediate density lipoproteins low density lipoproteins oxidized low density lipoproteins platelet-derived growth factor homodimer of PDGF B polypeptide mitogen-activated protein kinase Dulbecco's modified Eagle's medium insulin-regulated phosphorylated heat- and acid-stable protein fetal bovine serum high power fields 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl)-2H-tetrazolium salt low density lipoprotein receptor-related protein. The relationship between apoE and cholesterol transport suggests that it prevents atherosclerosis by lowering of plasma cholesterol level. However, atherosclerosis was found to be more severe in chow-fed apoE-null mice than in cholesterol-fed apoE(+/+) mice, despite the relative similar plasma cholesterol level in the two groups (7Zhang S.H. Reddick R.L. Burkey B. Maeda N. J. Clin. Invest. 1994; 94: 937-945Crossref PubMed Scopus (208) Google Scholar). Additionally, transgenic expression of apoE in the arterial wall inhibited atheroma formation and severity without affecting plasma cholesterol level and lipoprotein profile in cholesterol-fed C57BL/6 mice (9Shimano H. Ohsuga J. Shimada M. Namba Y. Gotoda T. Harada K. Katsuki M. Yazaki Y. Yamada N. J. Clin. Invest. 1995; 95: 469-476Crossref PubMed Scopus (128) Google Scholar). Although these two studies characterized atherosclerotic lesions based solely on macrophage foam cell deposition, it is noteworthy that increased number of intimal smooth muscle cells was observed in stenotic lesion area of the external carotid artery (10Seo H.S. Lombardi D.M. Polinsky P. Powell-Braxton L. Bunting S. Schwartz S.M. Rosenfeld M.E. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3593-3601Crossref PubMed Scopus (77) Google Scholar) and fibroproliferative atherosclerotic plaques of chow-fed apoE-null mice (11Dansky H.M. Charlton S.A. Harper M.M. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4642-4646Crossref PubMed Scopus (305) Google Scholar). Taken together, these results suggest that apoE may have direct impact on vascular occlusive diseases in a manner in addition to, and independent of, its property as a cholesterol-transporting apolipoprotein. Recently, apoE gene polymorphism has been shown to correlate with risk of restenosis after balloon angioplasty (12van Bockxmeer F.M. Mamotte C.D.S. Gibbons F.R. Taylor R.R. Atherosclerosis. 1994; 110: 195-202Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Because lipid deposition and foam cell formation is a late event in restenosis, this correlation adds support to the hypothesis that apoE may have cell regulatory functions in the vessel wall. A prominent vascular abnormality observed in both spontaneous atherosclerosis and restenosis after angioplasty is the migration of vascular smooth muscle cells from the medial layer of the blood vessel to its intima, followed by proliferation of the intimal smooth muscle cells. These processes are regulated by growth factors released from cells within the injured artery or from circulating cells in plasma. Potent mitogens and chemoattractants that participate in vascular disease include PDGF and oxLDL. These reagents direct migration of vascular smooth muscle cells from the media to the intima of the vessel and induce proliferation of the intimal smooth muscle cells (13Bornfeldt K. Raines E. Nakano T. Graves L. Krebs E. Ross R. J. Clin. Invest. 1994; 93: 1266-1274Crossref PubMed Scopus (381) Google Scholar, 14Kundra V. Escobedo J.A. Kazlauskas A. Kim H.K. Rhee S.G. Williams L.T. Zetter B.R. Nature. 1994; 367: 474-476Crossref PubMed Scopus (399) Google Scholar). The mechanism of cell activation is reported to be mediated via the activation of mitogen-activated protein (MAP) kinase (15Graf K. Xi X.P. Yang D. Fleck E. Hsueh W.A. Law R.E. Hypertension. 1997; 29: 334-339Crossref PubMed Google Scholar). Because apoE has been shown to inhibit mitogen-stimulated lymphocyte proliferation through suppression of early cell signaling events (16Hui D.Y. Harmony J.A.K. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 11775-11781Abstract Full Text PDF PubMed Google Scholar), the current study is undertaken to explore the possibility that apoE may protect against vascular diseases by inhibiting vascular smooth muscle cell migration and proliferation. Human recombinant PDGF-BB, FBS, and Dulbecco's modified Eagle's medium (DMEM) were purchased from Life Technologies, Inc. (Gaithersburg, MD). [Methyl-3H]thymidine, [(γ-32P]ATP, [α-32P]dCTP, and125I-PDGF-BB were obtained from NEN Life Science Products. Activated MAP kinase, insulin-regulated phosphorylated heat- and acid-stable protein (PHAS-I), the MAP kinase assay kit, and Quikhyb™ hybridization solution were products of Stratagene (La Jolla, CA). The Cell Titer 96™ AQueous cell proliferation assay kit was purchased from Promega, Inc. (Madison, WI). The embryonic rat aortic smooth muscle-derived A7r5 cells were obtained from American Type Culture Collection (Manassas, VA). Human coronary artery smooth muscle cells were purchased from Clonetics (San Diego, CA). Transwell chambers with gelatin-treated polycarbonate membranes were obtained from Corning Coster Corp. (Cambridge, MA). The A7r5 cells were grown in monolayer cultures on plastic tissue culture-treated multiple well cluster plates in DMEM containing 10% FBS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mm glutamine. Cells were incubated at 37 °C in 95% air, 5% CO2 atmosphere and were used for experiments between passages 3–12. Human coronary artery smooth muscle cells were grown in smooth muscle cell growth medium provided by the supplier. Cells were used for experiments between passages 5 and 8. In all experiments, the cells were made quiescent by incubating for 48 h in the presence of 0.4% FBS prior to use. Human apoE was isolated from fresh plasma obtained from normal healthy volunteers as detailed by Rall et al. (17Rall S.C. Weisgraber K.H. Mahley R.W. Methods Enzymol. 1986; 128: 273-287Crossref PubMed Scopus (94) Google Scholar). The purity of the apoE preparation was verified based on a single band withMr = 34,000 in SDS-polyacrylamide gels. Human LDL (d = 1.019–1.063 gm/ml) was isolated by sequential ultracentrifugation and was stored in saline-EDTA solution (18Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6479) Google Scholar). Oxidative modification was undertaken by dialyzing the LDL against saline without EDTA and then incubating the lipoproteins with 5 μmol/L copper sulfate for 48 h at 37 °C. The oxidative modification was confirmed by 37 °C electrophoretic mobility on agarose gels. The migration of smooth muscle cells was examined according to the procedure described by Lawet al. (19Law R.E. Meehan W.P. Xi X.P. Graf K. Wuthrich D.A. Coats W. Faxon D. Hsueh W.A. J. Clin. Invest. 1996; 98: 1897-1905Crossref PubMed Scopus (461) Google Scholar). Briefly, the cells were suspended in DMEM with 0.4% FBS at a concentration of 2 × 105 cells/ml. The cells were preincubated with 0, 25, or 50 μg/ml apoE for 30 min at 37 °C, and 0.1-ml aliquots of the cell suspension (2 × 104 cells) were added to the top chamber of gelatin-treated Transwell polycarbonate membrane with 8-μm pores in 24-well plates. The lower Transwell compartment contained 0.6 ml of DMEM, 0.4% FBS, with or without PDGF-BB or oxLDL. Incubation was continued for 4 h at 37 °C, after which the adherent cells were washed extensively, fixed with methanol, and then stained with hematoxylin. The number of cells that migrated to the lower surface of each filter was counted in different high power fields (HPFs) at a magnification of 320. Cells were plated at a cell density of 5 × 103 cells/well in 96-well tissue culture dishes and then incubated for 48 h in DMEM with 0.4% FBS to synchronize cells at the quiescent state. The cells were incubated for 20 h with PDGF-BB or oxLDL in the presence or absence of apoE. One μCi of [methyl-3H]thymidine was added to the culture medium and the incubation was continued for 4 h at 37 °C. Cells were washed twice with phosphate-buffered saline followed by incubation at 4 °C in 25% trichloroacetic acid for 20 min. The plates were washed three times with cold 25% trichloroacetic acid followed by the addition of 0.25 n NaOH. Radioactivity in the cell lysate was quantitated by liquid scintillation counting. Smooth muscle cell proliferation was also assessed based on the ability of the cells to convert MTS into formazan, using the AQueous Cell Proliferation Assay Kit. In these experiments, 5 × 103 quiescent A7r5 or human coronary artery smooth muscle cells in 96-well tissue culture dishes were incubated for 24 h with or without PDGF-BB or oxLDL and in the presence or absence of apoE prior to the addition of 20 μl of MTS mixed with the electron-coupling reagent phenazine methosulfate. Incubation was continued for 1 h at 37 °C. Formazan formation was measured based on increased absorbance at 490 nm. Quiescent A7r5 cells plated in 24-well tissue culture dishes were pre-incubated for 3 h at 4 °C with Hanks' balanced salt solution containing 0.2% bovine serum albumin, with or without addition of 25 or 50 μg/ml apoE or 200 ng/ml PDGF-BB. At the end of this incubation period, 120 pmol/liter of 125I-PDGF-BB was added to each well, and incubation was continued for 3 h at 4 °C. The cells were washed three times with cold Hanks' solution and then solubilized with 1 ml of solution containing 1% Triton X-100 and 0.1% bovine serum albumin. The radioactivity associated with the cell lysate was quantitated in a gamma counter. Specific binding was determined as the total amount of 125I-PDGF-BB bound to the cells minus the nonspecific binding observed in the presence of excess unlabeled PDGF-BB. A cDNA probe for rat cyclin D1 was prepared by reverse transcription-polymerase chain reaction amplification of mRNA isolated from proliferating cells. The forward oligonucleotide primer (5′-CTGACTGCCGAGAAGTTGTGCATC-3′) and the reverse primer (5′-CTGGCTCCTTTCTCTTCGCGATG-3′) were designed based on the published sequence of rat cyclin D1 cDNA (20Bianchi S. Fabiani S. Muratori M. Arnold A. Sakaguchi K. Miki T. Brandi M.L. Biochem. Biophys. Res. Commun. 1994; 204: 691-700Crossref PubMed Scopus (26) Google Scholar). The 591-base pair amplification product was used for hybridization with 10 μg of cellular RNA extracted using the guanidine thiocyanate-phenol-chloroform method (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63147) Google Scholar). Hybridization was carried out for 3 h at 65 °C with [α-32P]dCTP-labeled cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase cDNA probes in Quikhyb™ solutions containing denatured salmon sperm DNA. The membrane was washed three times with 1× SSC buffer (1.5 mmsodium citrate, 15 mm NaCl, and 0.1% SDS) followed by another wash with 0.1 × SSC. Hybridization signal was analyzed using the Scion Image PC program. The relative level of cyclin D1 mRNA was determined based on the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA in each sample. The smooth muscle cells were plated at a density of 1.5 × 105 cells in 6-well plates and then incubated for 5 min at 37 °C in the presence or absence of PDGF-BB, oxLDL, and apoE. Cell lysate was prepared by adding 200 μl of buffer containing 25 mm HEPES, pH 7.5, 0.2 mm phenylmethylsulfonyl fluoride, 0.05% 2-mercaptoethanol, and 1% Triton X-100. The cell lysate was mixed with 40 μl of kinase buffer containing 25 mm HEPES, pH 7.5, 10 mm magnesium acetate, 50 mm ATP, and 2 μl of [(α-32P]ATP (1.0 mCi/ml). The reaction was initiated by adding 10 μl of PHAS-I, and incubation was continued for 10 min at 30 °C. The phosphorylation reaction was terminated by adding 5 μl of 0.25 m phosphoric acid. Ten μl of each reaction mixture was spotted onto chromatography paper, washed with 75 mm phosphoric acid, rinsed in 95% ethanol, and then subjected to liquid scintillation counting. The apoE effect on cell proliferation was assessed by two-factorial ANOVA. All other data were analyzed for statistical significance using one-way ANOVA. A p value of less than 0.05 was considered to be statistically significant. Using the cell migration assay described under “Experimental Procedures,” 6.3 ± 2.6 quiescent A7r5 cells were found to be present in each HPF, indicating that 4.2% of the total amount of plated cells migrated to the lower chamber under basal conditions. The addition of PDGF enhanced A7r5 cell migration 4-fold, with 25.8 ± 6.1 cells observed in each HPF, representing 17.1% of the total amount of plated cells migrated to the lower chamber (Fig.1 A). Human apoE, at 25 μg/ml, inhibited the PDGF-induced A7r5 cell migration to the same level as that observed when the cells were incubated without PDGF (Fig.1 A). Significantly, at 50 μg/ml of apoE, the number of rat aortic smooth muscle cells migrated to the lower chamber was lower than that observed with unstimulated cells (Fig. 1 A). These concentrations of apoE have no effect on cell viability as determined by dye exclusion assay. Similar results were observed when experiments were performed with human coronary artery smooth muscle cells instead of the rat A7r5 smooth muscle cells (Fig. 1 B). Thus, these experiments demonstrated a role of apoE in inhibiting smooth muscle cell migration with or without growth factor stimulation. Incubation of quiescent rat A7r5 aortic smooth muscle cells with PDGF resulted in a 2- to 2.5-fold induction of [3H]thymidine incorporation into their DNA in comparison with cells incubated without the growth factor (Fig.2). The addition of 25 and 50 μg/ml of human apoE to the culture media significantly reduced the PDGF-induced [3H]thymidine incorporation into DNA (p< 0.001, Fig. 2). To confirm that apoE inhibition of PDGF-induced [3H]thymidine incorporation into DNA is a reflection of its ability to inhibit cell proliferation, a direct cell proliferation assay was performed. The results showed that PDGF stimulated the proliferation of both rat embryonic smooth muscle cells and human coronary artery smooth muscle cells in a time-dependent manner (Fig. 3). The addition of apoE effectively inhibited the PDGF-stimulated smooth muscle cell proliferation (p < 0.001, Fig. 3).Figure 3Effect of apoE on PDGF-induced proliferation of aortic smooth muscle cells. Serum-starved rat A7r5 smooth muscle cells (panel A) or human coronary artery smooth muscle cells (panel B) were incubated in 96-well plates (5 × 103 cells/well) in DMEM without PDGF and apoE (■), or with 10 ng/ml PDGF-BB in the absence (•) or presence (○) of 50 μg/ml apoE. Cell proliferation was determined based on formation of formazan from MTS and phenazine methosulfate as detected by absorbance at 490 nm. Data represent the mean value ± S.D. from five different determinations. *, p < 0.001versus no apoE control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The mechanism by which apoE inhibits smooth muscle cell response to PDGF stimulation was investigated initially by determining if apoE interferes with PDGF binding to its receptor on smooth muscle cells. The A7r5 cells were used for these experiments. Results showed that the smooth muscle cells displayed high affinity binding with 125I-labeled PDGF-BB. Excess unlabeled PDGF competitively inhibited 125I-PDGF binding to the A7r5 cells. In contrast, the addition of 50 μg/ml human apoE had no effect on 125I-PDGF binding to the A7r5 cell surface (Fig.4). To ascertain the possibility that apoE may interfere with early events in PDGF-induced smooth muscle cell proliferation and migration, experiments were performed to explore the effect of apoE on cyclin D1 gene expression, a prerequisite step for cell transition from the G0 to G1 phase in the cell cycle (22Won K.A. Xiong Y. Beach D. Gilman M.Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9910-9914Crossref PubMed Scopus (267) Google Scholar, 23Cocks B.G. Vairo G. Bodrug S.E. Hamilton J.A. J. Biol. Chem. 1992; 267: 12307-12310Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from A7r5 cells incubated with PDGF in the presence or absence of apoE for 6 h, a time period which was reported to result in maximal cyclin D1 mRNA expression after growth factor-induced proliferation of vascular smooth muscle cells (24Fukumoto S. Nishizawa Y. Hosoi M. Koyama H. Yamakawa K. Ohno S. Morii H. J. Biol. Chem. 1997; 272: 13816-13822Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Northern blot hybridization of cellular RNA with a rat cyclin D1 cDNA probe revealed a 2.5-fold increase in cyclin D1 mRNA level when cells were challenged with PDGF without apoE (Fig.5). However, the addition of apoE to the culture media in addition to PDGF reduced the growth factor-induced increase in cyclin D1 mRNA by >50% (Fig. 5). An early event important for PDGF stimulation of smooth muscle cells is the activation of MAP kinase (25Pazin M.J. Williams L.T. Trends Biochem. Sci. 1992; 17: 374-378Abstract Full Text PDF PubMed Scopus (145) Google Scholar). Lysate obtained from cells incubated for 5 min with 10 ng/ml of PDGF-BB displayed a 2- to 3-fold increase in MAP kinase activity compared with extracts obtained from unstimulated cells (Fig.6). However, when 50 μg/ml of human apoE was added simultaneously with PDGF to the cell culture medium, a significant reduction in PDGF-induced MAP kinase activity was observed (p < 0.01, Fig. 6). In addition to apoE effects on PDGF-stimulated smooth muscle cell functions, the impact of apoE on oxLDL-stimulated smooth muscle cell response was also explored. Results shown in Fig.7 indicated that apoE also inhibited the chemoattractive and mitogenic properties of oxLDL on smooth muscle cells (26Autrio I. Jaakkola O. Solakivi T. Nikkari T. FEBS Lett. 1990; 277: 247-249Crossref PubMed Scopus (69) Google Scholar, 27Chatterjee S. Mol. Cell. Biochem. 1992; 111: 143-147Crossref PubMed Scopus (110) Google Scholar). This inhibition was also mediated through suppression of the MAP kinase pathway (Fig. 7). Pathological studies indicated that narrowing of the coronary vessels in atherosclerosis and restenosis is related to neointimal hyperplasia, with abnormal proliferation and migration of vascular smooth muscle cells from the tunica media to the intima (28Giraldo A.A. Esposo O.M. Meis J.M. Arch. Pathol. Lab. Med. 1985; 109: 173-175PubMed Google Scholar, 29Austin G.E. Ratliff M.B. Hollman J. Tabei S. Phillips D.F. J. Am. Coll. Cardiol. 1985; 6: 369-375Crossref PubMed Scopus (485) Google Scholar). The current hypothesis suggests that injury to the vascular endothelium exposes the underlying smooth muscle cells to mitogenic growth factors, thereby inducing their phenotypic conversion from the contractile nonproliferating phenotype to that of a secretory proliferating phenotype. Platelet-derived growth factor and oxLDL have been implicated to play a major role in activating both processes of smooth muscle cell migration and proliferation (30Grotendorst G.R. Chang T. Seppa H.E.J. Kleinman H.K. Martin G.R. J. Cell Physiol. 1982; 113: 261-266Crossref PubMed Scopus (232) Google Scholar, 31Jawien A. Bowen-Pope D.F. Lindner V. Schwartz S.M. Clowes A.W. J. Clin. Invest. 1992; 89: 507-511Crossref PubMed Scopus (593) Google Scholar, 32Jackson C.L. Raines E. Ross R. Reidy M.A. Arterioscler. Thromb. 1993; 13: 1218-1226Crossref PubMed Scopus (229) Google Scholar). The mechanism of cell activation is related to the stimulation of MAP kinase activity (33Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Abstract Full Text PDF PubMed Google Scholar,34Graves L.M. Bornfeldt K.E. Sidhu J.S. Argast G.M. Raines E.W. Ross R. Leslie C.C. Krebs E.G. J. Biol. Chem. 1996; 271: 505-511Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), which plays critical intermediary roles in mediating signal transduction from the membrane to the nucleus (35Seth A. Gonzalez A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 24796-24804Abstract Full Text PDF PubMed Google Scholar). The induction of cell proliferation by MAP kinase has been shown to be a direct result of increased transcription of many immediate early genes (35Seth A. Gonzalez A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1992; 267: 24796-24804Abstract Full Text PDF PubMed Google Scholar, 36Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar), including cyclin D1 that is required for cell transition from the G0 to the G1 phase in the cell cycle (37Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar,38Abieu A. Lorca T. Labbe J.C. Morin N. Keyse S. Doree M. J. Cell Sci. 1996; 109: 239-246PubMed Google Scholar). A possible role of apoE in modulation of vascular smooth muscle cell growth was suggested by the observation of its synthesis in quiescent but not actively proliferating vascular smooth muscle cells (39Majack R.A. Castle C.K. Goodman L.V. Weisgraber K.H. Mahley R.W. Shooter E.M. Gebicke-Haerter P.J. J. Cell Biol. 1988; 107: 1207-1213Crossref PubMed Scopus (44) Google Scholar). Results described in this manuscript provided the first experimental evidence to document apoE inhibition of agonist-induced smooth muscle cell migration and proliferation. This inhibition was shown to be due to apoE suppression of growth factor-stimulated MAP kinase activity and cyclin D1 gene expression in aortic smooth muscle cells. These in vitro experiments implicated a cytostatic function for apoE in the vessel wall and suggested an additional mechanism by which apoE protects against vascular occlusive diseases. The results of this study add to a growing list of mechanisms by which apoE is anti-atherogenic. First, apoE reduces hyperlipidemia by mediating cholesterol clearance from circulation (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3376) Google Scholar). Second, apoE suppresses foam cell formation by mediating cholesterol efflux from peripheral cells (40Kruth H.S. Skarlatos S.I. Gaynor P.M. Gamble W. J. Biol. Chem. 1994; 269: 24511-24518Abstract Full Text PDF PubMed Google Scholar, 41Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar). Third, apoE suppresses lipoprotein oxidation, thereby reducing oxidation-induced endothelial toxicity (42Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (799) Google Scholar). Fourth, apoE inhibits lymphocyte proliferation (16Hui D.Y. Harmony J.A.K. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 11775-11781Abstract Full Text PDF PubMed Google Scholar), thus may limit inflammatory response in the arterial wall. Fifth, apoE inhibits agonist-induced platelet aggregation (43Riddell D.R. Graham A. Owen J.S. J. Biol. Chem. 1997; 272: 89-95Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), which will retard the progression of atherosclerotic lesion; and sixth, apoE suppresses growth factor and oxLDL-induced smooth muscle cell migration and proliferation (this study). Taken together, these apoE activities may explain the versatility of this apolipoprotein in conferring protection against various forms of vascular diseases, including cholesterol-induced atherosclerosis (2Shimano H. Yamada N. Katsuki M. Yamamoto K. Gotoda T. Harada K. Shimada M. Yazaki Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1750-1754Crossref PubMed Scopus (119) Google Scholar), angioplasty- or injury-induced restenosis (10Seo H.S. Lombardi D.M. Polinsky P. Powell-Braxton L. Bunting S. Schwartz S.M. Rosenfeld M.E. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3593-3601Crossref PubMed Scopus (77) Google Scholar, 12van Bockxmeer F.M. Mamotte C.D.S. Gibbons F.R. Taylor R.R. Atherosclerosis. 1994; 110: 195-202Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 44De Geest B. Zhao Z. Collen D. Holvoet P. Circulation. 1997; 96: 4349-4356Crossref PubMed Scopus (90) Google Scholar), and transplant-induced accelerated arteriosclerosis (46Russell P.S. Chase C.M. Colvin R.B. Am. J. Pathol. 1996; 149: 91-99PubMed Google Scholar). In addition to its cell regulatory functions in the cardiovascular system, apoE has also been shown to modulate normal physiology and pathophysiology of other cells and tissues. For example, apoE suppresses steroidogenesis in adrenals and ovaries (47Reyland M.E. Gwynne J.T. Forgez P. Prack M.M. Williams D.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2375-2379Crossref PubMed Scopus (39) Google Scholar, 48Dyer C.A. Curtiss L.K. J. Biol. Chem. 1988; 263: 10965-10973Abstract Full Text PDF PubMed Google Scholar, 49Reyland M.E. Williams D.L. J. Biol. Chem. 1991; 266: 21099-21104Abstract Full Text PDF PubMed Google Scholar). ApoE is also expressed in the brain, where the apoE4 allele is correlated with an increased risk for late onset Alzheimer's disease (50Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3693) Google Scholar). The relationship between apoE genotype and Alzheimer's disease is related to apoE interaction with β-amyloid peptide and with τ in regulation of fibrillogenesis (50Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3693) Google Scholar, 51Strittmatter W.J. Saunders A.M. Goedert M. Weisgraber K.H. Dong L.M. Jakes R. Huang D.Y. Pericak-Vance M. Schmechel D. Rose A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11183-11186Crossref PubMed Scopus (460) Google Scholar). The ability of different apoE isoforms to regulate neurite outgrowth from dorsal root ganglion neurons (52Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Science. 1994; 264: 850-852Crossref PubMed Scopus (732) Google Scholar) and to induce neurotoxicity (53LaFerla F.M. Troncoso J.C. Strickland D.K. Kawas C.H. Jay G. J. Clin. Invest. 1997; 100: 310-320Crossref PubMed Scopus (146) Google Scholar, 54Marques M.A. Tolar M. Crutcher K.A. Alzheimer's Res. 1997; 3: 1-6Google Scholar) also has been implicated to play a significant role in Alzheimer's disease. The ability of apoE to modulate such diverse cell functions appears to be related to its ability in modulating cell signal transduction pathways in a cell- and tissue-specific manner. For example, the suppressive effect of apoE on steroidogenesis in adrenals and ovaries is mediated through inhibition of cyclic AMP signaling mechanisms (47Reyland M.E. Gwynne J.T. Forgez P. Prack M.M. Williams D.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2375-2379Crossref PubMed Scopus (39) Google Scholar, 48Dyer C.A. Curtiss L.K. J. Biol. Chem. 1988; 263: 10965-10973Abstract Full Text PDF PubMed Google Scholar, 49Reyland M.E. Williams D.L. J. Biol. Chem. 1991; 266: 21099-21104Abstract Full Text PDF PubMed Google Scholar). In contrast, apoE has no effect on cyclic AMP pathway in lymphocytes, but its inhibition of lymphocyte proliferation was shown to be mediated by suppression of other early cell signaling events that lead to cell arrest at the G1a/G1b boundary (55Hui D.Y. Berebitsky G.L. Harmony J.A.K. J. Biol. Chem. 1979; 254: 4666-4673Abstract Full Text PDF PubMed Google Scholar, 56Hui D.Y. Harmony J.A.K. Biochem. J. 1980; 192: 91-98Crossref PubMed Scopus (41) Google Scholar, 57Mistry M.J. Clay M.A. Kelly M.E. Steiner M.A. Harmony J.A.K. Cellular Immunology. 1995; 160: 14-23Crossref PubMed Scopus (50) Google Scholar). The mechanism for apoE inhibition of smooth muscle cell proliferation and migration reported herein is different from the apoE effect on lymphocyte proliferation. The smooth muscle cell effect is mediated via inhibition of MAP kinase activity and cyclin D1 activation, resulting in the prevention of cell entry from G0 to G1 phase. Other studies revealed that apoE suppresses agonist-induced platelet aggregation by activation of nitric oxide synthesis (43Riddell D.R. Graham A. Owen J.S. J. Biol. Chem. 1997; 272: 89-95Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). ApoE also stimulates nitric-oxide synthase in human macrophages (58Vitek M.P. Snell J. Dawson H. Colton C.A. Biochem. Biophys. Res. Commun. 1997; 240: 391-394Crossref PubMed Scopus (60) Google Scholar). Interestingly, nitric oxide has been shown to inhibit migration and proliferation of vascular smooth muscle cells (59Garg U.C. Hassid A. J. Clin. Invest. 1989; 83: 1774-1777Crossref PubMed Scopus (1992) Google Scholar). Whether the effects of apoE on smooth muscle cells reported here are related to its effect on nitric oxide production remains to be determined. The diverse effects of apoE on cell signaling mechanisms may be related to its ability to bind various different receptors on the cell surface. Currently identified receptors capable of binding apoE include LDL receptor, LRP, gp330/megalin, the VLDL receptor, apoE receptor-2, LR11, and LR7/8 (60Yamamoto T. Bujo H. Curr. Opin. Lipidol. 1996; 7: 298-302Crossref PubMed Scopus (26) Google Scholar, 61Schneider W.J. Nimpf J. Bujo H. Curr. Opin. Lipidol. 1997; 8: 315-319Crossref PubMed Scopus (62) Google Scholar). Whereas ligand binding to these receptors usually results in its internalization and degradation in the lysosomes, the potential for signal transduction via ligand interaction with these receptors has not been fully appreciated. There are reports that lactoferrin, lipoprotein lipase, and Pseudomonas exotoxin A binding to LRP on macrophages resulted in a pertussis toxin-sensitive G protein-mediated increase in intracellular calcium ion and inositol triphosphate (62Misra U.K. Chu C.T.C. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar). Significant to the current study is the report that interaction of the 39-kDa receptor-associated protein or anti-LRP with LRP on vascular smooth muscle cells resulted in inhibition of cell migration (63Okada S.S. Grobmyer S.R. Barnathan E.S. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1269-1276Crossref PubMed Scopus (90) Google Scholar). Whether apoE inhibition of ligand-induced smooth muscle cell migration and proliferation is mediated by LRP, or other members of the LDL receptor gene family present on these cells, such as the VLDL receptor (45Takahashi S. Kawarabayasi Y. Nakai T. Sakai J. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9252-9256Crossref PubMed Scopus (478) Google Scholar), remains to be determined." @default.
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