Impaired Peroxisome Proliferator-activated Receptor-γ Contributes to Phenotypic Modulation of Vascular Smooth Muscle Cells during Hypertension

The phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a pivotal role in hypertension-induced vascular changes including vascular remodeling. The precise mechanisms underlying VSMC phenotypic modulation remain elusive. Here we test the role of peroxisome proliferator-activated receptor (PPAR)-γ in the VSMC phenotypic modulation during hypertension. Both spontaneously hypertensive rat (SHR) aortas and SHR-derived VSMCs exhibited reduced PPAR-γ expression and excessive VSMC phenotypic modulation identified by reduced contractile proteins, α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α), and enhanced proliferation and migration. PPAR-γ overexpression rescued the expression of α-SMA and SM22α, and inhibited the proliferation and migration in SHR-derived VSMCs. In contrast, PPAR-γ silencing exerted the opposite effect. Activating PPAR-γ using rosiglitazone in vivo up-regulated aortic α-SMA and SM22α expression and attenuated aortic remodeling in SHRs. Increased activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling was observed in SHR-derived VSMCs. PI3K inhibitor LY294002 rescued the impaired expression of contractile proteins, and inhibited proliferation and migration in VSMCs from SHRs, whereas constitutively active PI3K mutant had the opposite effect. Overexpression or silencing of PPAR-γ inhibited or excited PI3K/Akt activity, respectively. LY294002 counteracted the PPAR-γ silencing induced proliferation and migration in SHR-derived VSMCs, whereas active PI3K mutant had the opposite effect. In contrast, reduced proliferation and migration by PPAR-γ overexpression were reversed by the active PI3K mutant, and further inhibited by LY294002. We conclude that PPAR-γ inhibits VSMC phenotypic modulation through inhibiting PI3K/Akt signaling. Impaired PPAR-γ expression is responsible for VSMC phenotypic modulation during hypertension. These findings highlight an attractive therapeutic target for hypertension-related vascular disorders. The phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a pivotal role in hypertension-induced vascular changes including vascular remodeling. The precise mechanisms underlying VSMC phenotypic modulation remain elusive. Here we test the role of peroxisome proliferator-activated receptor (PPAR)-γ in the VSMC phenotypic modulation during hypertension. Both spontaneously hypertensive rat (SHR) aortas and SHR-derived VSMCs exhibited reduced PPAR-γ expression and excessive VSMC phenotypic modulation identified by reduced contractile proteins, α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α), and enhanced proliferation and migration. PPAR-γ overexpression rescued the expression of α-SMA and SM22α, and inhibited the proliferation and migration in SHR-derived VSMCs. In contrast, PPAR-γ silencing exerted the opposite effect. Activating PPAR-γ using rosiglitazone in vivo up-regulated aortic α-SMA and SM22α expression and attenuated aortic remodeling in SHRs. Increased activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling was observed in SHR-derived VSMCs. PI3K inhibitor LY294002 rescued the impaired expression of contractile proteins, and inhibited proliferation and migration in VSMCs from SHRs, whereas constitutively active PI3K mutant had the opposite effect. Overexpression or silencing of PPAR-γ inhibited or excited PI3K/Akt activity, respectively. LY294002 counteracted the PPAR-γ silencing induced proliferation and migration in SHR-derived VSMCs, whereas active PI3K mutant had the opposite effect. In contrast, reduced proliferation and migration by PPAR-γ overexpression were reversed by the active PI3K mutant, and further inhibited by LY294002. We conclude that PPAR-γ inhibits VSMC phenotypic modulation through inhibiting PI3K/Akt signaling. Impaired PPAR-γ expression is responsible for VSMC phenotypic modulation during hypertension. These findings highlight an attractive therapeutic target for hypertension-related vascular disorders. IntroductionHypertension and hypertension-induced vascular remodeling underlie numerous cardiovascular disorders. Activated vascular smooth muscle cells (VSMCs) 2The abbreviations used are: VSMCvascular smooth muscle cellα-SMAα-smooth muscle actinSM22αsmooth muscle 22αWKYWistar-KyotoSHRspontaneously hypertensive ratSBPsystolic blood pressureIMTintima-media thickeningPI3Kphosphoinositide 3-kinasesiRNAsmall interfering RNAPPAR-γperoxisome proliferator-activated receptor-γMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePtdIns-4,5-P2phosphatidylinositol 4,5-phosphatescrscrambled sequenceovoverexpressionkdknockdown. are essential contributors to this vascular remodeling (1Hayashi K. Naiki T. J. Mech. Behav. Biomed. Mater. 2009; 2: 3-19Crossref PubMed Scopus (112) Google Scholar). Unlike other muscle cells, VSMCs do not terminally differentiate. They can switch from a differentiated phenotype (also known as contractile or quiescent phenotype) to a dedifferentiated phenotype (also known as synthetic or activated phenotype) in response to vascular injury. In this process, VSMCs regain their proliferative and migratory capacities, secrete matrix proteins, and down-regulate smooth muscle contractile proteins, such as α-smooth muscle actin (α-SMA), smooth muscle 22α (SM22α), smooth muscle myosin heavy chain, and calponin (2Owens G.K. Kumar M.S. Wamhoff B.R. Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2535) Google Scholar). Although this phenotypic modulation is undoubtedly required for vascular repair, inappropriate modulation is associated with increased vascular resistance and aggravates vascular injury. However, despite intensive research efforts, the precise mechanisms underlying VSMC phenotypic modulation during hypertension remain unclear.The peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor (3Kliewer S.A. Xu H.E. Lambert M.H. Willson T.M. Recent Prog. Horm. Res. 2001; 56: 239-263Crossref PubMed Scopus (360) Google Scholar). It regulates various genes involved in glucose homeostasis, lipid metabolism, and adipocyte differentiation via binding to the PPAR response elements in target genes, and therefore participates in modulation of several diseases such as diabetes, obesity, and atherosclerosis (4Marx N. Duez H. Fruchart J.C. Staels B. Circ. Res. 2004; 94: 1168-1178Crossref PubMed Scopus (457) Google Scholar, 5Hsueh W.A. Bruemmer D. Hypertension. 2004; 43: 297-305Crossref PubMed Scopus (127) Google Scholar). Convincing evidence indicates the role of PPAR-γ in hypertension. For example, individuals with a dominant-negative PPAR-γ mutation present with early onset hypertension and elements of metabolic syndrome (6Barroso I. Gurnell M. Crowley V.E. Agostini M. Schwabe J.W. Soos M.A. Maslen G.L. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1146) Google Scholar).PPAR-γ is expressed in VSMCs in vivo and in vitro (7Law R.E. Goetze S. Xi X.P. Jackson S. Kawano Y. Demer L. Fishbein M.C. Meehan W.P. Hsueh W.A. Circulation. 2000; 101: 1311-1318Crossref PubMed Scopus (419) Google Scholar) and exerts an important role in the regulation of VSMC viability. In spontaneously hypertensive rat (SHR)-derived VSMCs, PPAR-γ overexpression or treatment with the PPAR-γ agonist thiazolidinedione retards VSMC growth to the level of non-hypertensive rat VSMCs (8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar, 9Bruemmer D. Law R.E. Am. J. Med. 2003; 115: 87S-92SAbstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, PPAR-γ inhibits the VSMC proliferation induced by platelet-derived growth factor and angiotensin II (7Law R.E. Goetze S. Xi X.P. Jackson S. Kawano Y. Demer L. Fishbein M.C. Meehan W.P. Hsueh W.A. Circulation. 2000; 101: 1311-1318Crossref PubMed Scopus (419) Google Scholar, 10Hattori Y. Akimoto K. Kasai K. Biochem. Biophys. Res. Commun. 2000; 273: 1144-1149Crossref PubMed Scopus (45) Google Scholar). It is also reported that PPAR-γ can suppress the VSMC invasion (11Goetze S. Kintscher U. Kim S. Meehan W.P. Kaneshiro K. Collins A.R. Fleck E. Hsueh W.A. Law R.E. J. Cardiovasc. Pharmacol. 2001; 38: 909-921Crossref PubMed Scopus (52) Google Scholar) and migration induced by matrix metalloprotease (12Galis Z.S. Muszynski M. Sukhova G.K. Simon-Morrissey E. Unemori E.N. Lark M.W. Amento E. Libby P. Circ. Res. 1994; 75: 181-189Crossref PubMed Scopus (613) Google Scholar, 13Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar). Considering that phenotypic modulation is a prerequisite for VSMCs to regain the proliferative and migratory capacity (14Sung H.J. Eskin S.G. Sakurai Y. Yee A. Kataoka N. McIntire L.V. Ann. Biomed. Eng. 2005; 33: 1546-1554Crossref PubMed Scopus (47) Google Scholar), we postulate that PPAR-γ can negatively regulate the phenotypic modulation of VSMCs.Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a pivotal role in the regulation of cellular growth, apoptosis, and metabolism (15Kondapaka S.B. Zarnowski M. Yver D.R. Sausville E.A. Cushman S.W. Clin. Cancer Res. 2004; 10: 7192-7198Crossref PubMed Scopus (54) Google Scholar, 16Choi K.H. Kim J.E. Song N.R. Son J.E. Hwang M.K. Byun S. Kim J.H. Lee K.W. Lee H.J. Cardiovasc. Res. 2010; 859: 836-844Crossref Scopus (86) Google Scholar). Activated PI3K phosphorylates Akt and induces the expression of transcriptional factors involved in multiple processes. The PI3K/Akt signaling is reportedly required for VSMC migration and proliferation, absence of Akt impairs VSMC proliferation and migration (17Fernández-Hernando C. József L. Jenkins D. Di Lorenzo A. Sessa W.C. Arterioscler. Thromb. Vasc. Biol. 2009; 29: 2033-2040Crossref PubMed Scopus (122) Google Scholar). A previous study (18Goetze S. Eilers F. Bungenstock A. Kintscher U. Stawowy P. Blaschke F. Graf K. Law R.E. Fleck E. Gräfe M. Biochem. Biophys. Res. Commun. 2002; 293: 1431-1437Crossref PubMed Scopus (135) Google Scholar) indicated the link between PI3K/Akt signaling and PPAR-γ. PPAR-γ activation can inhibit the Akt phosphorylation induced by vascular endothelial growth factor in endothelial cells (18Goetze S. Eilers F. Bungenstock A. Kintscher U. Stawowy P. Blaschke F. Graf K. Law R.E. Fleck E. Gräfe M. Biochem. Biophys. Res. Commun. 2002; 293: 1431-1437Crossref PubMed Scopus (135) Google Scholar). However, neither the role of PPAR-γ and PI3K/Akt signaling nor their exact interaction in VSMC phenotypic modulation during hypertension is fully understood. In the present study, we test the hypothesis that PPAR-γ plays an important role in inhibiting VSMC phenotypic modulation through negatively regulating the activity of PI3K/Akt signaling and thus participates in VSMC phenotypic modulation during hypertension.DISCUSSIONHere we found that during hypertension, VSMCs exhibit impaired PPAR-γ expression, which is related to excessive phenotypic modulation. PPAR-γ overexpression can inhibit the phenotypic modulation by rescuing the contractile proteins and inhibiting proliferation and migration in SHR-derived VSMCs. Activation of PPAR-γ with rosiglitazone can ameliorate the hypertension-induced aortic remodeling. PI3K/Akt signaling is negatively modulated by PPAR-γ. Impaired PPAR-γ expression in VSMCs accounts for enhanced PI3K/Akt signaling activity, and contributes to the excessive phenotypic modulation during hypertension.Hypertension can significantly impact the vascular system. VSMCs are susceptible to hypertensive stress, and undergo phenotypic switch from contractile to synthetic phenotype. Reportedly, SHR-derived VSMCs display an exaggerated growth rate, abnormal contact inhibition, and accelerated entry into the cell cycle S phase (8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar). In the present study, we observed impaired α-SMA and SM22α expression, and enhanced proliferative and migratory capacity in SHR-derived VSMCs, which indicated the VSMC phenotypic modulation during hypertension. Although even normal cultured VSMCs are reported to undergo phenotypic changes by expressing fewer contractile markers and more synthetic phenotype (30Sobue K. Hayashi K. Nishida W. Mol. Cell. Biochem. 1999; 190: 105-118Crossref PubMed Google Scholar), we indeed revealed more obvious phenotypic changes in SHR-derived VSMCs than in WKY-derived VSMCs, which indicated the excessive VSMC phenotypic modulation in hypertension.Activated VSMCs contribute to thickened vessel wall and vascular remodeling through accumulation in intima and medium (1Hayashi K. Naiki T. J. Mech. Behav. Biomed. Mater. 2009; 2: 3-19Crossref PubMed Scopus (112) Google Scholar, 31Wang Z. Rao P.J. Shillcutt S.D. Newman W.H. Neurosci. Lett. 2005; 373: 38-41Crossref PubMed Scopus (22) Google Scholar). As expected, we observed aortic hypertrophic remodeling during hypertension, characterized by intima-media thickening and lumen loss. This finding confirmed the involvement of VSMC phenotypic modulation in hypertension-induced vascular remodeling.It is well established that PPAR-γ participates in the modulation of multiple diseases including diabetes, atherosclerosis, and hypertension, and plays a critical role in VSMC viability (6Barroso I. Gurnell M. Crowley V.E. Agostini M. Schwabe J.W. Soos M.A. Maslen G.L. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1146) Google Scholar, 7Law R.E. Goetze S. Xi X.P. Jackson S. Kawano Y. Demer L. Fishbein M.C. Meehan W.P. Hsueh W.A. Circulation. 2000; 101: 1311-1318Crossref PubMed Scopus (419) Google Scholar, 8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar, 9Bruemmer D. Law R.E. Am. J. Med. 2003; 115: 87S-92SAbstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 10Hattori Y. Akimoto K. Kasai K. Biochem. Biophys. Res. Commun. 2000; 273: 1144-1149Crossref PubMed Scopus (45) Google Scholar, 11Goetze S. Kintscher U. Kim S. Meehan W.P. Kaneshiro K. Collins A.R. Fleck E. Hsueh W.A. Law R.E. J. Cardiovasc. Pharmacol. 2001; 38: 909-921Crossref PubMed Scopus (52) Google Scholar, 12Galis Z.S. Muszynski M. Sukhova G.K. Simon-Morrissey E. Unemori E.N. Lark M.W. Amento E. Libby P. Circ. Res. 1994; 75: 181-189Crossref PubMed Scopus (613) Google Scholar, 13Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar). Here we hypothesize that PPAR-γ participates in the regulation of VSMC phenotypic modulation, and therefore is associated with increased VSMC phenotypic modulation during hypertension. There are controversies surrounding the specifics of PPAR-γ expression during hypertension. Diep and Schiffrin (25Diep Q.N. Schiffrin E.L. Hypertension. 2001; 38: 249-254Crossref PubMed Scopus (77) Google Scholar) reported higher PPAR-γ levels in SHR mesenteric arteries compared with WKYs, but no difference between SHR and WKY aortas. In contrast, Xiong et al. (8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar) observed reduced PPAR-γ protein levels in the SHR aorta, mesenteric arteries, and SHR-derived VSMCs, despite a high mRNA level. The explanation for these conflicting results is unclear. Given that Diep and Schiffrin (25Diep Q.N. Schiffrin E.L. Hypertension. 2001; 38: 249-254Crossref PubMed Scopus (77) Google Scholar) detected PPAR-γ protein in whole vessels instead of VSMCs, the results might not reflect the exact level of PPAR-γ in VSMCs. In the present study, we observed a significant reduction in PPAR-γ expression in the SHR aorta and SHR-derived VSMCs compared with non-hypertensive rats. We speculated that this impaired PPAR-γ expression was associated with the increased phenotypic modulation of VSMCs in hypertension.To address this, we manipulated PPAR-γ expression and evaluated the phenotypic modulation of cultured VSMCs. It was found that PPAR-γ overexpression inhibited the proliferation and migration, and rescued the impaired expression of α-SMA and SM22α in SHR-derived VSMCs, whereas PPAR-γ silencing exerted the opposite effect. Furthermore, PPAR-γ silencing in WKY-derived VSMCs led to reduced α-SMA and SM22α expression, and enhanced proliferation and migration. In vivo experiments revealed that PPAR-γ activator rosiglitazone improved the impaired expression of α-SMA and SM22α in SHR aortas. Here we provide the first evidence that PPAR-γ negatively regulates VSMC phenotypic modulation through inhibiting proliferation and migration, and preserving the contractile proteins in cultured VSMCs. The impaired PPAR-γ expression in VSMCs is, at least partly, responsible for the enhanced VSMC phenotypic modulation during hypertension, which in turn contributes to morphological vascular remodeling. As expected, up-regulating PPAR-γ expression with rosiglitazone in vivo ameliorated aortic remodeling by increasing the internal diameter and reducing IMT, which was in line with previous studies (32Ogihara T. Rakugi H. Ikegami H. Mikami H. Masuo K. Am. J. Hypertens. 1995; 8: 316-320Crossref PubMed Scopus (271) Google Scholar).Rosiglitazone treatment also reduced the SBP in SHRs, indicating its potential therapeutic effect on hypertension. It is reported that the decreased endothelin-1 level and amelioration of endothelial function are associated with the effect of rosiglitazone on blood pressure (28Potenza M.A. Marasciulo F.L. Tarquinio M. Quon M.J. Montagnani M. Diabetes. 2006; 55: 3594-3603Crossref PubMed Scopus (73) Google Scholar). However, due to the lack of direct manipulation of PPAR-γ expression in vivo in the present study, the exact role of PPAR-γ in rosiglitazone-mediated SBP regulation is still not clarified. Rosiglitazone did not show any effects on reducing body weight in SHRs.To explore the molecular mechanism underlying the inhibition of PPAR-γ on VSMCs phenotypic modulation, we tested the role of PI3K/Akt signaling in this process. The PI3K/Akt signaling pathway is required in multiple cellular processes, including migration and proliferation of numerous cell types (33Morales-Ruiz M. Fulton D. Sowa G. Languino L.R. Fujio Y. Walsh K. Sessa W.C. Circ. Res. 2000; 86: 892-896Crossref PubMed Scopus (346) Google Scholar, 34Dimmeler S. Dernbach E. Zeiher A.M. FEBS Lett. 2000; 477: 258-262Crossref PubMed Scopus (309) Google Scholar). Recently, Hegner et al. (35Hegner B. Lange M. Kusch A. Essin K. Sezer O. Schulze-Lohoff E. Luft F.C. Gollasch M. Dragun D. Arterioscler. Thromb. Vasc. Biol. 2009; 29: 232-238Crossref PubMed Scopus (35) Google Scholar) found that Akt phosphorylation down-regulated VSMC markers in dedifferentiated mesenchymal progenitor cells, whereas inhibition of PI3K induced the VSMC contractile phenotype, with simultaneous calponin, α-SMA, and SM22α expression. In the present study, we observed enhanced PI3K activity and phosphorylation of Akt in SHR-derived VSMCs compared with cells from WKYs. The PI3K inhibitor LY294002 reduced the capacity of proliferation and migration, and rescued the expression of α-SMA and SM22α in SHR-derived VSMCs. In contrast, the active PI3K mutant exerted the opposite effect. Moreover, the active PI3K mutant promoted a synthetic phenotype in WKY-derived VSMCs identified by impaired α-SMA and SM22α expression and elevated proliferation and migration. These findings provide the evidence that enhanced PI3K/Akt signaling is an important contributor to VSMC phenotypic modulation during hypertension.Growing evidence supports the link between PPAR-γ and the PI3K/Akt signaling pathway. For example, inhibition of PI3K blocks the PPAR-γ-dependent phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 Ser-298, extracellular signal-regulated kinase (ERK) 1/2 activation, and the epithelial to mesenchymal modulation (29Chen L. Necela B.M. Su W. Yanagisawa M. Anastasiadis P.Z. Fields A.P. Thompson E.A. J. Biol. Chem. 2006; 281: 24575-24587Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). PPAR-γ ligand can markedly inhibit the enhancement of PI3K activity induced by lipopolysaccharide/interferon-γ in astrocytes (36Giri S. Rattan R. Singh A.K. Singh I. J. Immunol. 2004; 173: 5196-5208Crossref PubMed Scopus (126) Google Scholar). In endothelial cells, PPAR-γ activation inhibits Akt phosphorylation induced by vascular endothelial growth factor (8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar). Furthermore, PI3K is required for the effects of the PPAR-γ agonist pioglitazone on H2O2-induced endothelial progenitor cell apoptosis (37Gensch C. Clever Y.P. Werner C. Hanhoun M. Böhm M. Laufs U. Atherosclerosis. 2007; 192: 67-74Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).Based on previous studies, we proposed that down-regulation of PI3K/Akt activity may be an important mechanism for PPAR-γ to inhibit VSMC phenotypic modulation. Our results showed that PPAR-γ overexpression reduced, whereas PPAR-γ silencing enhanced the PI3K activity and p-Akt expression in both WKY and SHR-derived VSMCs, indicating the inhibitory effect of PPAR-γ on PI3K/Akt signaling activity. We next observed the proliferation and migration in SHR-derived VSMCs with manipulation of both PPAR-γ and PI3K. Our results showed that LY294002 impeded the elevated proliferation and migration induced by PPAR-γ silencing, whereas the active PI3K mutant had the opposite effect. In contrast, reduced proliferation and migration by PPAR-γ overexpression were reversed by the active PI3K mutant, and further inhibited by LY294002. These findings indicate that PI3K/Akt signaling is inhibited by PPAR-γ, which might be an important mechanism for PPAR-γ to inhibit the VSMC phenotypic modulation. Impaired PPAR-γ in SHR-derived VSMCs is, at least partly, responsible for the elevated PI3K/Akt activity observed in hypertension, and therefore accounts for the increased VSMC modulation from contractile to synthetic phenotype.There are some limitations to our study. First, multiple mechanisms are reportedly involved in PPAR-γ-mediated regulation of VSMC action. PPAR-γ can inhibit VSMC proliferation through down-regulating inflammatory genes (38Takata Y. Kitami Y. Yang Z.H. Nakamura M. Okura T. Hiwada K. Circ. Res. 2002; 91: 427-433Crossref PubMed Scopus (120) Google Scholar), preventing G1/S phase transition (39Wakino S. Kintscher U. Kim S. Yin F. Hsueh W.A. Law R.E. J. Biol. Chem. 2000; 275: 22435-22441Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), and inhibiting telomerase activity (40Ogawa D. Nomiyama T. Nakamachi T. Heywood E.B. Stone J.F. Berger J.P. Law R.E. Bruemmer D. Circ. Res. 2006; 98: e50-e59Crossref PubMed Scopus (74) Google Scholar). Obviously, the suppression of PI3K/Akt is one of these mechanisms. However, we provide convincing evidence that the suppression of PI3K/Akt signaling mediates the PPAR-γ-induced inhibition of VSMC phenotypic modulation, which indicates the important role of PI3K/Akt signaling in the effect of PPAR-γ during hypertension. Second, we realize that some effects of PPAR-γ agonists on VSMCs are PPAR-γ independent. For example, troglitazone can suppress angiotensin II-induced VSMC activation through a PPAR-γ-independent pathway (10Hattori Y. Akimoto K. Kasai K. Biochem. Biophys. Res. Commun. 2000; 273: 1144-1149Crossref PubMed Scopus (45) Google Scholar). Therefore, we used a genetic approach in vitro as a complementation of rosiglitazone to identify the effect of PPAR-γ on VSMC phenotypic modulation. Third, given that PPAR-γ can also exert a protective effect on VSMCs by improving insulin resistance and dyslipidemia, we propose that the inhibition of phenotypic modulation is one important mechanism for PPAR-γ to protect VSMCs against hypertensive injury.Taken together, our results provide the first evidence that PPAR-γ inhibits VSMC phenotypic modulation by suppressing PI3K/Akt signaling. During hypertension, impaired PPAR-γ expression in VSMCs accounts for the enhanced activity of PI3K/Akt signaling, and ultimately contributes to excessive VSMC switching from the contractile to synthetic phenotype. These novel findings provide further insight into the mechanism of VSMC phenotypic modulation during hypertension, and offer PPAR-γ as a new target for the prevention and treatment of hypertension-associated vascular disorders. IntroductionHypertension and hypertension-induced vascular remodeling underlie numerous cardiovascular disorders. Activated vascular smooth muscle cells (VSMCs) 2The abbreviations used are: VSMCvascular smooth muscle cellα-SMAα-smooth muscle actinSM22αsmooth muscle 22αWKYWistar-KyotoSHRspontaneously hypertensive ratSBPsystolic blood pressureIMTintima-media thickeningPI3Kphosphoinositide 3-kinasesiRNAsmall interfering RNAPPAR-γperoxisome proliferator-activated receptor-γMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePtdIns-4,5-P2phosphatidylinositol 4,5-phosphatescrscrambled sequenceovoverexpressionkdknockdown. are essential contributors to this vascular remodeling (1Hayashi K. Naiki T. J. Mech. Behav. Biomed. Mater. 2009; 2: 3-19Crossref PubMed Scopus (112) Google Scholar). Unlike other muscle cells, VSMCs do not terminally differentiate. They can switch from a differentiated phenotype (also known as contractile or quiescent phenotype) to a dedifferentiated phenotype (also known as synthetic or activated phenotype) in response to vascular injury. In this process, VSMCs regain their proliferative and migratory capacities, secrete matrix proteins, and down-regulate smooth muscle contractile proteins, such as α-smooth muscle actin (α-SMA), smooth muscle 22α (SM22α), smooth muscle myosin heavy chain, and calponin (2Owens G.K. Kumar M.S. Wamhoff B.R. Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2535) Google Scholar). Although this phenotypic modulation is undoubtedly required for vascular repair, inappropriate modulation is associated with increased vascular resistance and aggravates vascular injury. However, despite intensive research efforts, the precise mechanisms underlying VSMC phenotypic modulation during hypertension remain unclear.The peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor (3Kliewer S.A. Xu H.E. Lambert M.H. Willson T.M. Recent Prog. Horm. Res. 2001; 56: 239-263Crossref PubMed Scopus (360) Google Scholar). It regulates various genes involved in glucose homeostasis, lipid metabolism, and adipocyte differentiation via binding to the PPAR response elements in target genes, and therefore participates in modulation of several diseases such as diabetes, obesity, and atherosclerosis (4Marx N. Duez H. Fruchart J.C. Staels B. Circ. Res. 2004; 94: 1168-1178Crossref PubMed Scopus (457) Google Scholar, 5Hsueh W.A. Bruemmer D. Hypertension. 2004; 43: 297-305Crossref PubMed Scopus (127) Google Scholar). Convincing evidence indicates the role of PPAR-γ in hypertension. For example, individuals with a dominant-negative PPAR-γ mutation present with early onset hypertension and elements of metabolic syndrome (6Barroso I. Gurnell M. Crowley V.E. Agostini M. Schwabe J.W. Soos M.A. Maslen G.L. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1146) Google Scholar).PPAR-γ is expressed in VSMCs in vivo and in vitro (7Law R.E. Goetze S. Xi X.P. Jackson S. Kawano Y. Demer L. Fishbein M.C. Meehan W.P. Hsueh W.A. Circulation. 2000; 101: 1311-1318Crossref PubMed Scopus (419) Google Scholar) and exerts an important role in the regulation of VSMC viability. In spontaneously hypertensive rat (SHR)-derived VSMCs, PPAR-γ overexpression or treatment with the PPAR-γ agonist thiazolidinedione retards VSMC growth to the level of non-hypertensive rat VSMCs (8Xiong C. Mou Y. Zhang J. Fu M. Chen Y.E. Akinbami M.A. Cui T. Life Sci. 2005; 77: 3037-3048Crossref PubMed Scopus (14) Google Scholar, 9Bruemmer D. Law R.E. Am. J. Med. 2003; 115: 87S-92SAbstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, PPAR-γ inhibits the VSMC proliferation induced by platelet-derived growth factor and angiotensin II (7Law R.E. Goetze S. Xi X.P. Jackson S. Kawano Y. Demer L. Fishbein M.C. Meehan W.P. Hsueh W.A. Circulation. 2000; 101: 1311-1318Crossref PubMed Scopus (419) Google Scholar, 10Hattori Y. Akimoto K. Kasai K. Biochem. Biophys. Res. Commun. 2000; 273: 1144-1149Crossref PubMed Scopus (45) Google Scholar). It is also reported that PPAR-γ can suppress the VSMC invasion (11Goetze S. Kintscher U. Kim S. Meehan W.P. Kaneshiro K. Collins A.R. Fleck E. Hsueh W.A. Law R.E. J. Cardiovasc. Pharmacol. 2001; 38: 909-921Crossref PubMed Scopus (52) Google Scholar) and migration induced by matrix metalloprotease (12Galis Z.S. Muszynski M. Sukhova G.K. Simon-Morrissey E. Unemori E.N. Lark M.W. Amento E. Libby P. Circ. Res. 1994; 75: 181-189Crossref PubMed Scopus (613) Google Scholar, 13Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar). Considering that phenotypic modulation is a prerequisite for VSMCs to regain the proliferative and migratory capacity (14Sung H.J. Eskin S.G. Sakurai Y. Yee A. Kataoka N. McIntire L.V. Ann. Biomed. Eng. 2005; 33: 1546-1554Crossref PubMed Scopus (47) Google Scholar), we postulate that PPAR-γ can negatively regulate the phenotypic modulation of VSMCs.Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a pivotal role in the regulation of cellular growth, apoptosis, and metabolism (15Kondapaka S.B. Zarnowski M. Yver D.R. Sausville E.A. Cushman S.W. Clin. Cancer Res. 2004; 10: 7192-7198Crossref PubMed Scopus (54) Google Scholar, 16Choi K.H. Kim J.E. Song N.R. Son J.E. Hwang M.K. Byun S. Kim J.H. Lee K.W. Lee H.J. Cardiovasc. Res. 2010; 859: 836-844Crossref Scopus (86) Google Scholar). Activated PI3K phosphorylates Akt and induces the expression of transcriptional factors involved in multiple processes. The PI3K/Akt signaling is reportedly required for VSMC migration and proliferation, absence of Akt impairs VSMC proliferation and migration (17Fernández-Hernando C. József L. Jenkins D. Di Lorenzo A. Sessa W.C. Arterioscler. Thromb. Vasc. Biol. 2009; 29: 2033-2040Crossref PubMed Scopus (122) Google Scholar). A previous study (18Goetze S. Eilers F. Bungenstock A. Kintscher U. Stawowy P. Blaschke F. Graf K. Law R.E. Fleck E. Gräfe M. Biochem. Biophys. Res. Commun. 2002; 293: 1431-1437Crossref PubMed Scopus (135) Google Scholar) indicated the link between PI3K/Akt signaling and PPAR-γ. PPAR-γ activation can inhibit the Akt phosphorylation induced by vascular endothelial growth factor in endothelial cells (18Goetze S. Eilers F. Bungenstock A. Kintscher U. Stawowy P. Blaschke F. Graf K. Law R.E. Fleck E. Gräfe M. Biochem. Biophys. Res. Commun. 2002; 293: 1431-1437Crossref PubMed Scopus (135) Google Scholar). However, neither the role of PPAR-γ and PI3K/Akt signaling nor their exact interaction in VSMC phenotypic modulation during hypertension is fully understood. In the present study, we test the hypothesis that PPAR-γ plays an important role in inhibiting VSMC phenotypic modulation through negatively regulating the activity of PI3K/Akt signaling and thus participates in VSMC phenotypic modulation during hypertension.