p53 Mediates the Accelerated Onset of Senescence of Endothelial Progenitor Cells in Diabetes
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m509293200
ISSN1083-351X
AutoresArturo Rosso, Antonina Balsamo, Roberto Gambino, Patrizia Dentelli, Rita Falcioni, Maurizio Cassader, Luigi Pegoraro, Gianfranco Pagano, Maria Felice Brizzi,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoAdverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes. Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes. An elevated plasma level of low density lipoprotein (LDL) 2The abbreviations used are: LDL, low density lipoprotein; BSA, bovine serum albumin; CDK, cyclin-dependent kinase; CML, carboxymethyllysine; DCF-DA, 5,6-carboxy-22,72-dichlorofluorescein-diacetate; dmLDL, dense, small LDL; DPI, diphenylene iodonium; EPC, endothelial precursor cells; FACS, fluorescence-activated cell sorter; β-gal, β-galactosidase; MnSOD, manganese superoxide dismutase; NAC, N-acetylcysteine; nLDL, native (normal) LDL; ox-dmLDL, oxidized dense, small LDL; Rb, retinoblastoma; ROS, reactive oxygen species; SA-β-gal, senescence-associated β-galactosidase; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; AGE, advanced glycated end products. represents the major risk factor of premature atherosclerosis in diabetes (1.Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar, 2.Penn M.S. Chisolm G.M. 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Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar). p21 belongs to the cyclin-dependent kinase (CDK) inhibitors that, in concert with various tumor suppressor proteins, such as p53 and pRb, induce inhibition of DNA replication and control antiproliferative programs (8.Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). However, p21 together with the tumor suppressor proteins p53 and pRb is not only an important mediator of quiescence-like growth arrest but also of senescence (9.Itahana K. Campisi J. Dimri G.P. Biogerontology. 2004; 5: 1-10Crossref PubMed Scopus (275) Google Scholar, 10.Campisi J. Cell. 2005; 120: 513-522Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar, 11.Lombard D.B. Chua K.F. Mostoslavski R. Franco G. Gostissa M. Alt F.W. 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Acad. Sci. U. S. A. 2000; 97: 4291-4296Crossref PubMed Scopus (384) Google Scholar). Moreover, senescent endothelial cells have been detected in atherosclerotic, but not in nonatherosclerotic, vessels (17.Miyauchi H. Minamino T. Tateno K. Kunieda T. Toko H. Komuro I. EMBO J. 2004; 23: 212-220Crossref PubMed Scopus (267) Google Scholar). Vasculature remodeling does not rely exclusively on proliferation of resident endothelial cells but also involves circulating progenitor cells (EPC) (18.Hibbert B. Olsen S. O'Brien E. Trends Cardiovasc. Med. 2003; 13: 322-326Crossref PubMed Scopus (49) Google Scholar, 19.Dimmeler S. Zeiher A.M. J. Mol. Med. 2004; 82: 671-677Crossref PubMed Scopus (262) Google Scholar). Recent works demonstrated that in patients with cardiovascular risk factors, including diabetes, the number of EPC that can be isolated from peripheral blood is reduced, and their function is impaired (20.Hill J.M. Zalos G. Halcox J.P. Schenke W.H. Waclawiw M.A. Quyyumi A.A. Finkel T. N. Engl. J. Med. 2003; 348: 593-600Crossref PubMed Scopus (3146) Google Scholar). Moreover, despite culturing EPC under normoglycemic conditions, functional differences between EPC recovered from diabetic patients and normal subjects exist (20.Hill J.M. Zalos G. Halcox J.P. Schenke W.H. Waclawiw M.A. Quyyumi A.A. Finkel T. N. Engl. J. Med. 2003; 348: 593-600Crossref PubMed Scopus (3146) Google Scholar). Therefore the reduced number and the impaired function of EPC in diabetes can rely on adverse metabolic stress factors, different from hyperglycemia (21.Loomans C.J. de Koning E.J. Staal F.J. Rookmaaker M.B. Verseyden C. de Boer H.C. Verhaar M.C. Braam B. Rabelink T.J. van Zonneveld A.J. Diabetes. 2004; 53: 195-199Crossref PubMed Scopus (760) Google Scholar). Increasing evidence indicates that native oxidized LDL by inducing reactive oxygen species (ROS) production activates a cascade of molecular events that mainly contribute to accelerate atherosclerosis in diabetic patients (22.Baynes J.W. Diabetes. 1991; 40: 405-412Crossref PubMed Scopus (0) Google Scholar). In vivo small and dense LDL, because of their qualitative abnormalities, are more susceptible than natural occurring LDL to undergo oxidation and to activate the oxidative stress pathways. In this study we investigated the effects of in vitro oxidized diabetic LDL on EPC fate and in particular the molecular mechanisms activated by oxidized small and dense LDL which can contribute to EPC dysfunction in diabetes. We found that in vitro oxidized small and dense diabetic LDL accelerate the onset of senescence of EPC recovered from normal subjects via a p53-mediated signaling pathway. Moreover, we demonstrate that depletion of endogenous p53 in EPC recovered from diabetic patients prevents the accelerated onset of senescence, indicating that a p53-mediated pathway contributes to the impaired EPC function in this setting. Patients and Controls—Blood from nine type 2 diabetic patients who arrived in our patient clinic (sex, M/F 4/5; HbA1c, 6.4 ± 0.6%; age-years, 50.0 ± 5; creatinine, 1 ± 1 mg/dl; no retinopathy, no hypertension: blood pressure ≤ 140/90 mm Hg; Chol/apoB, 1.3 ± 0.1). None of them was under insulin, and all were treated only with diet (no other medicaments were used by diabetic patients). Ten blood donors were used as controls (sex, M/F 5/5; HbA1c, 5 ± 01.0%; age-years, 50.0 ± 1; creatinine, 0.7 ± 0.4 mg/dl, no retinopathy, no hypertension: blood pressure ≤ 140/90 mm Hg, Chol/apoB, 1.6 ± 0.1). The study was approved by the ethical committee, and informed consent was obtained from all subjects. Reagents—M199 medium (endotoxin tested), bovine serum albumin (BSA), Sepharose-protein A, N-acetylcysteine (NAC), diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and actinomycin D were from Sigma. SB203580 and LY294002 were from Hyclone Laboratories, Inc., Logan, Utah. Horseradish peroxidase-conjugated protein A, molecular weight markers, [α-32P]dCTP, and the chemiluminescence reagent (ECL) were from Amersham Biosciences. Endotoxin contamination of LDL preparation was tested by the Limulus amebocyte assay, and the concentration was <0.1 ng/ml. The acidic β-galactosidase staining kit was from Invitrogen. Carboxymethyllysine (CML)-BSA was prepared by incubating 50 mg/ml BSA at 37 °C for 24 h with 45 mm glyoxylic acid and 150 mm/liter sodium cyanoborohydride (NaCNBH3) in 2 ml of 0.2 m phosphate buffer, pH 7.4, followed by PD-10 column chromatography and dialysis against phosphate-buffered saline as described previously (23.Brizzi M.F. Dentelli P. Rosso A. Calvi C. Gambino R. Cassader M. Salvidio G. Deferrari G. Camusi G. Pegoraro L. Pagano G. Cavallo-Perin P. FASEB J. 2004; 11: 1249-1251Crossref Scopus (59) Google Scholar). Antisera—Monoclonal anti-p53 clone DO-1, anti-CD31, anti-p16, anti-tubulin, anti-β-actin, anti-p21, anti-RAGE, and anti-cyclin D1 antisera were obtained from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. Anti-VE-cadherin and anti-CD34 were from New England Biolabs. Anti-phosphor-Akt-Ser-473 or anti-Akt, phospho-Rb was from Cell Signaling Technology (Beverly, MA). Monoclonal anti-CD146 antibody was from BioCytex (Marseille, France). Anti-catalase antibody was from Sigma, and antibody to manganese superoxide dismutase (MnSOD) was from Upstate Biotechnology (Lake Placid, NY). Anti-LOX-1 antibody was from R&D Systems. Anti-AGE antibody was from Trans Genic Inc., Japan. Isolation, Characterization, and Oxidation of LDL—Blood from diabetic patients or healthy controls was processed according to Redgrave and Carlson (24.Redgrave T.G. Carlson L.A. J. Lipid Res. 1979; 20: 217-229Abstract Full Text PDF PubMed Google Scholar). Plasma was brought to a density of 1.10 g/ml and processed as described previously (7.Brizzi M.F. Dentelli P. Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar). The density of the LDL was measured in the tubes where the highest levels of cholesterol were found. LDL was dialyzed against 0.02 m EDTA-free phosphate buffer, pH 7.4, containing 0.15 m NaCl. LDL was adjusted to 0.2 mg/ml protein concentration. CuSO4 was added for 24 h (37 °C) at a ratio of 25 μm/mg LDL protein (25.Vedie B. Myara I. Pech M.A. Maziere J.C. Maziere C. Caprani A. Moatti N. J. Lipid Res. 1991; 32: 1359-1369Abstract Full Text PDF PubMed Google Scholar). Oxidation was stopped by the addition of 1 mm EDTA and 0.02 mm butylated hydroxytoluene. Isolation and Culture of EPC from Peripheral Blood Mononuclear Cells—ox-dmLDL or dmLDL studies were performed on EPC recovered from normal donors. Isolation of CD34+ cells was performed with the direct CD34 or CD133+ isolation kit (MINIMACS system, Miltenyi Biotec) according to the manufacturer's instruction (26.Pesce M. Orlandi A. Iachininoto M.G. Straino S. Torella A.R. Rizzuti V. Pompilio G. Bonanno G. Scambia G. Capogrossi M.C. Circ. Res. 2003; 93: 51-62Crossref PubMed Google Scholar). The purity of sorted cells was assessed by FACS analysis (26.Pesce M. Orlandi A. Iachininoto M.G. Straino S. Torella A.R. Rizzuti V. Pompilio G. Bonanno G. Scambia G. Capogrossi M.C. Circ. Res. 2003; 93: 51-62Crossref PubMed Google Scholar). The isolated EPC were cultured for 10 or 15 days on 20 μg/ml fibronectin-coated dishes in EGM endothelial growth medium containing 20% fetal calf serum and 20 ng/ml VEGF alone or in combination with 10 μg/ml dmLDL or with 10 μg/ml ox-dmLDL. FACS was used to analyze the differentiated phenotype (anti-CD34, anti-CD31, anti-CD146, or anti-VE-cadherin antibodies were used) (26.Pesce M. Orlandi A. Iachininoto M.G. Straino S. Torella A.R. Rizzuti V. Pompilio G. Bonanno G. Scambia G. Capogrossi M.C. Circ. Res. 2003; 93: 51-62Crossref PubMed Google Scholar). CD34+cells were also isolated from 9 diabetic patients (HbA1c <7%) and assayed for the presence of the senescence-associated β-galactosidase (SA-β-gal) marker (27.Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar). Silencing of Endogenous p53 and Akt by Small Interfering RNAs (siRNA)—To obtain inactivation of p53, EPC cultured with ox-dmLDL were transiently transfected by Lipofectamine PLUS™ reagent (Invitrogen) according to the vendor's instructions with the vector pSUPER retro containing p53 siRNA or a scramble p53 siRNA (control siRNA) sequences (1.5 μg) as described by Brummelkamp et al. (28.Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). The pSUPER retro containing p53 siRNA and the scramble p53 siRNA were gently provided by Dr. S. Soddu. 60 h later whole cell extracts were prepared, separated on 10% SDS-PAGE, and immunoblotted with antibody against p53. To obtain inactivation of Akt EPC cultured as above were transiently transfected with purified duplex siRNAs for Akt and for a scramble control purchased from Qiagen (Valencia, CA). Transfection was performed using Lipofectamine according to the vendor's instructions. Cell viability was evaluated at the end of the experiment. Flow Cytometry—EPC treated as indicated were fixed with 70% ethanol. After digestion with RNase, DNA was stained with propidium iodide and analyzed with a flow cytometer (FACScan, Becton Dickinson Immunocytometry Systems, San Jose, CA). In selected experiments NAC, SB203580, or LY294002 inhibitors were used. The percentage of cells in each phase of the cell cycle was determined by ModFit LT software (Verity Software House, Inc., Topsham, ME). FACS was used for analyzing VE-cadherin, CD146, or CD34 expression. Fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) was used as secondary antibody. Detection of ROS—DCF-DA (20 mm final concentration) was added to EPC in the various cultured conditions. At the times indicated the cells were subjected to flow cytometric analysis and processed as described previously (7.Brizzi M.F. Dentelli P. Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar). Tumor necrosis factor-α was used as positive control. A lucigenin-derived chemiluminescence assay was also performed. Briefly, EPC were resuspended in a glass tube containing phenol red-free basal medium and incubated in a 37 °C water bath for 10 min. 5 μm lucigenin and 250 μm NADPH were then added and immediately placed inside the luminometer (Lumat LB 9501, Berthold Technologies, Pforzheim, Germany). The lucigenin-derived chemiluminescence assay was measured for a 10-min period. EPC were then stimulated with saline (0.1% BSA in phosphate-buffered saline), ox-dmLDL, or dm-LDL as indicated, and LDLCL was monitored for 30 min. In a few experiments 30 mm/liter NAC, 30 μm/liter DPI, or 10 μm/liter LY294002 was used. Western Blot Analysis—EPC were cultured in the presence of dmLDL or ox-dmLDL in combination with VEGF as indicated. Cells were lysed (in lysis buffer including a protease inhibitor mixture (Sigma), 200 mm/liter sodium vanadate, and 20 μm/liter sodium fluoride), and the protein concentration was obtained as described previously (7.Brizzi M.F. Dentelli P. Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar, 29.Brizzi M.F. Dentelli P. Gambino R. Cabodi S. Cassader M. Castelli A. Defilippi P. Pegoraro L. Pagano G. Diabetes. 2002; 51: 3311-3317Crossref PubMed Scopus (22) Google Scholar). 50 μg of proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. In selected experiments 10 μm/liter SB203580, 10 μm/liter LY294002, or 30 mm/liter NAC was used. To evaluate the p21 half-life EPC were treated with 10 μg/ml cycloheximide for the indicated intervals. 50 μg of whole cell lysates were prepared at each time point and assayed for the expression of p21 and β-actin by Western blotting. Reaction with antibodies and detection with an enhanced chemiluminescence detection system (Amersham Biosciences) were performed as described previously (7.Brizzi M.F. Dentelli P. Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar). For dot blot experiments a sheet of nitrocellulose was clamped between the gasket and the 96-well sample template. 200 μg of LDL sample was allowed to filter through the membrane. After the antigen was immobilized, the nitrocellulose was incubated in 4% albumin blocking solution. Nitrocellulose was subsequently incubated with rabbit IgG anti-AGE at room temperature for 1.5 h and then with goat IgG anti-rabbit IgG labeled with alkaline phosphatase. Dots were visualized with the procedure contained in the Immun-Blot Assay kit (Bio-Rad). In Vitro Angiogenesis—EPC cultured with dmLDL or ox-dmLDL were prelabeled with a permanent green fluorescent dye (PKH2 from Sigma). In vitro angiogenesis was studied on Matrigel-coated surface as described by Montesano and Orci (30.Montesano R. Orci L. Cell. 1985; 42: 469-477Abstract Full Text PDF PubMed Scopus (373) Google Scholar). VEGF was added to the medium or incorporated into the Matrigel. To evaluate cell viability, trypan blue exclusion staining was used (31.Brizzi M.F. Battaglia E. Montrucchio G. Dentelli P. Del Sorbo L. Garbarono G. Pegoraro L. Camusi G. Circ. Res. 1999; 8: 785-796Crossref Scopus (86) Google Scholar). In vitro angiogenesis was expressed as the percentage ± S.D. of the tube-like structure area to the total Matrigel area. Northern Blot Analysis—Northern blot analysis was performed accordingly to standard methods (32.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). The filter was hybridized to 32P-random priming-labeled cDNA probes corresponding to cyclin D1, p21, and β-actin. EPC Senescence—Senescence was evaluated on EPC recovered from normal subjects cultured for 15 days with VEGF plus dmLDL or with ox-dmLDL or on EPC recovered from diabetic patients after 10 days of culture in EGM endothelial growth medium. Acidic β-gal activity was used for these assays (27.Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar). Briefly, EPC were washed in phosphate-buffered saline, fixed for 3 min at room temperature in 2% paraformaldehyde, washed, and incubated for 24 h at 37 °C with fresh SA-β-gal stain solution: 1 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal), 5 mm/liter potassium ferrocyanide, 5 mm/liter ferricyanide, 150 mm/liter NaCl, 2 mm/liter MgCl2, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40. SA-β-gal-positive cells were also quantified on cells cultured in the presence of NAC or LY294002 as indicated. The number of blue cells was counted manually from a total of 200 cells. Statistical Analysis—All in vitro results are representative of at least three independent experiments performed in triplicate. Densitometric analysis using a Bio-Rad GS 250 molecular imager was used to calculate the differences in the -fold induction of protein activation or expression (* and § p < 0.05, statistically significant between experimental and control values). Significance of differences between experimental and control values was calculated using analysis of variance with Newman-Keuls multicomparison test. LDL Preparation and Characterization—In all diabetic subjects the peak of cholesterol was in the LDL subfractions with a density ranging from 1.037 to 1.044 g/ml. In healthy subjects the highest level of cholesterol was found in the LDL subfractions of density ranging from 1.028 to 1.035 g/ml. In diabetic samples LDL were constantly smaller and denser than normal LDL, and this effect was confirmed by the density gradient ultracentrifugation profile (data not shown) and by a lower cholesterol: apoB ratio found in these subjects (1.6 versus 1.3). Our previous findings that unlike 8% glycated LDL, 10 and 15% glycated LDL inhibit cell cycle progression (29.Brizzi M.F. Dentelli P. Gambino R. Cabodi S. Cassader M. Castelli A. Defilippi P. Pegoraro L. Pagano G. Diabetes. 2002; 51: 3311-3317Crossref PubMed Scopus (22) Google Scholar) indicate that the level of glycation is crucial to elicit damaging signals in primary endothelial cells. To rule out the possibility that the biological effects exerted by oxidized diabetic LDL could rely on LDL glycation, AGE immunoreactivity on LDL was evaluated. To this end a dot blot assay using an anti-AGE antibody was performed. As shown in Fig. 1A no significant difference in AGE immunoreactivity was detected in LDL recovered from normal subject (HbA1c = 5%) and diabetic patients in good metabolic control (HbA1c = 7%). By contrast, AGE immunoreactivity increased in LDL recovered from patients with HbA1c above 8%. Thus, only LDL recovered from diabetic patients with HbA1c <7% were subjected to oxidation. Before in vitro oxidation lipid peroxidation of natural occurring diabetic LDL used in our study was also assayed by thiobarbituric acid reactive substances (TBARS), assay and capillary electrophoresis and compared with that of the native LDL (nLDL). Consistent with our previous results (7.Brizzi M.F. Dentelli P. Pavan M. Rosso A. De Cesaris M.G. Garbarino G. Camussi G. Pagano G. Pegoraro L. J. Clin. Invest. 2002; 109: 111-119Crossref PubMed Scopus (22) Google Scholar) we failed to detect differences in TBARS and conjugates dienes between dmLDL or nLDL (data not shown). ox-dmLDL Increases p21 Expression and Induces Growth Arrest—Cell cycle events induced by in vitro ox-dmLDL on EPC were first evaluated. The results reported in Table 1 demonstrate that the fraction of cells in S and G2/M decreases when cells are cultured with ox-dmLDL. This effect was not observed in EPC cultured in the presence of dmLDL not subjected to oxidation. Indeed, cells in S phase were reduced to 13% compared with cells cultured with VEGF alone or in combination with dmLDL (38 and 36%, respectively). ox-dmLDL were used at a dose of 10 μg/ml because higher doses such as 50 μg/ml induced an apoptotic phenotype in 10% of the cells. To dissect the molecular events leading to growth arrest, cell cycle-related proteins were analyzed. The dose-response curve shown in Fig. 1B demonstrates that treatment with increasing concentrations of ox-dmLDL is associated with an increase of p21 expression. Moreover, we failed to detect a significant difference between 10 and 50 μg/ml ox-dmLDL. On the contrary, the level of p21 is not affected by the same concentrations of dmLDL. Consistent with the increased level of p21 expression also a decreased level of cyclin D1 expression occurs (Fig. 1C). A sustained increasing level of p21 can be mediated by various mechanisms, including regulation of protein stability (8.Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). As shown in Fig. 1D this possibility is ruled out by the finding that the p21 half-life in EPC cultured in the presence of dmLDL or ox-dmLDL is similar. In addition (Fig. 1E) the level of p21 mRNA, but not that of cyclin D1, is significantly increased in EPC cultured in the presence of ox-dmLDL, and pretreatment with actinomycin D abrogates the effect of ox-dmLDL on both p21 mRNA and protein expression. These findings suggest that mechanisms other than protein accumulation account for our results.TABLE 1ox-dmLDL-mediated cell cycle events EPC cultured with 20 ng/ml VEGF, 10 μg/ml VEGF + dmLDL, or 10 or 50 μg/ml VEGF + ox-dm-LDL were washed and fixed with ethanol and processed by FACS. 0.5 μm/liter doxorubicin was used as a positive control for apoptosis.Cell cycle phasesPercentage of cellsap < 0.05 experimental groups versus VEGFVEGFox-dmLDL 10 μg/mlox-dmLDL 50 μg/mldmLDL 50 μg/mlDoxorubicinG0/G142 ± 373 ± 4ap < 0.05 experimental groups versus VEGF68 ± 4ap < 0.05 experimental groups versus VEGF45 ± 410 ± 3ap < 0.05 experimental groups versus VEGFS38 ± 217 ± 3ap < 0.05 experimental groups versus VEGF12 ± 3ap < 0.05 experimental groups versus VEGF36 ± 3/G2/M20 ± 110 ± 2ap < 0.05 experimental groups versus VEGF10 ± 2ap < 0.05 experimental groups versus VEGF19 ± 2/Apoptosis//10 ± 3/90 ± 5a p < 0.05 experimental groups versus VEGF Open table in a new tab It is known that oxidized LDL exert their effects by interacting with membrane receptor structure different from the canonical LDL receptor (33.Krieger M. Curr. Opin. Lipidol. 1997; 8: 275-280Crossref PubMed Scopus (255) Google Scholar). The possibility that ox-dmLDL affects EPC by interacting with receptors other than that of dmLDL is sustained by competition experiments (Fig. 1F). Indeed, addition of an excess of dmLDL or of LDL recovered from normal subjects (nLDL) does not change the level of p21 in EPC. On the contrary the induction of p21 expression is abrogated by blocking the receptor LOX-1 but not by blocking RAGE, indicating that p21 expression is mediated by LDL oxidation rather that by LDL glycation. p21 Expression and Growth Arrest Are Dependent on Accumulation of p53 and the Presence of Unphosphorylated Rb Protein—p21 is a well known target of p53 (34.Boulaire J. Fotedar A. Fotedar R. Pathol. Biol. 2000; 48: 190-202PubMed Google Scholar, 35.Jascur T. Brickner H. Salles-Passador I. Barbier V. El Khissiin A. Smith B. Fotedar R Fotedar A. Mol. Cell. 2005; 17: 237-249Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Thus, nuclear accumulation of p53 was first evaluated in EPC cultured in the presence of ox-dmLDL or dmLDL. Moreover, to exclude further the possibility that glycation of LDL can account for our results in selected experiments CML-BSA was used. As shown in Fig. 2A p53 accumulates in the nucleus in EPC cultured with ox-dmLDL but not with dmLDL or CML, strongly supporting the role of LDL oxidation in this event (Fig. 2B). Consistently phosphorylated Rb protein can be only detected in EPC cultured with dmLDL. To confirm the involvement of p53 in mediating this signaling pathway EPC were depleted of endogenous p53 by siRNA. As shown in Fig. 2D ox-dmLDL fails to induce both accumulation of p53 and p21 expression in EPC transfected with p53 siRNA (Fig. 2, C and D). Moreover, depletion of endogenous p53 also prevents the effect of ox-dmLDL on Rb protein (Fig. 2E) and of ox-dmLDL-induced
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