Heme oxygenase-1 ⁄ p21 WAF1 mediates peroxisome proliferator-activated receptor-c signaling inhibition of proliferation of rat pulmonary artery smooth muscle cells Manxiang Li 1 , Zongfang Li 2 , Xiuzhen Sun 1 , Lan Yang 3 , Ping Fang 1 , Yun Liu 1 , Wei Li 1 , Jing Xu 1 , Jiamei Lu 1 , Minxing Xie 1 and Dexin Zhang 1 1 Department of Respiratory Medicine, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, China 2 Department of General Surgery, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, China 3 Department of Respiratory Medicine, The First Affiliated Hospital of Medical College, Xi’an Jiaotong University, China Introduction Peroxisome proliferator-activated receptors (PPARs) are a group of ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARs form heterodimers with retinoid X receptors, binding to specific PPAR-responsive elements and governing the expression of relevant genes [1]. Three subclasses of PPARs have been identified: PPARa, PPARb ⁄ d, and PPARc [1]. PPARc is expressed predominantly in adipocytes, activated macrophages, vascular smooth muscle cells, and vascular endothelial cells [2]. PPARc is activated by several natural ligands, such as 15-deoxy-D12,14-prostaglandin J 2 , 9-hydroxyoctade- cadienoic acid, 3-hydroxyoctadecadienoic acid, 12-hy- droxyeicosatetaenoic acid, 15-hydroxyeicosatetaenoic acid, and nitro lipids [3]. It is also activated by syn- thetic ligands such as thiazolidinediones, e.g. troglitazone Keywords heme oxygenase-1 (HO-1); p21 WAF1 ; proliferator-activated receptor-c (PPARc); pulmonary artery smooth muscle cells; rosiglitazone Correspondence M. Li or Z. Li, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, No. 157, West 5th Road, Xi’an, Shaanxi, China 710004 Fax: +86 29 87679463 Tel: +86 29 85520128 E-mail: manxiangli@hotmail.com or lzf2568@mail.xjtu.edu.cn (Received 13 November 2009, revised 22 December 2009, accepted 15 January 2010) doi:10.1111/j.1742-4658.2010.07581.x Activation of peroxisome proliferator-activated receptor (PPAR)-c sup- presses proliferation of rat pulmonary artery smooth muscle cells (PASMCs), and therefore ameliorates the development of pulmonary hypertension in animal models. However, the molecular mechanisms under- lying this effect remain largely unknown. This study addressed this issue. The PPARc agonist rosiglitazone dose-dependently stimulated heme oxygenase (HO)-1 expression in PASMCs, 5 lm rosiglitazone inducing a 12.1-fold increase in the HO-1 protein level. Cells pre-exposed to rosiglitaz- one showed a dose-dependent reduction in proliferation in response to serotonin; this was abolished by pretransfection of cells with sequence- specific small interfering RNA against HO-1. In addition, rosiglitazone stimulated p21 WAF1 expression in PASMCs, a 2.34-fold increase in the p21 WAF1 protein level being achieved with 5 lm rosiglitazone; again, this effect was blocked by knockdown of HO-1. Like loss of HO-1, loss of p21 WAF1 through siRNA transfection also reversed the inhibitory effect of rosiglitazone on PASMC proliferation triggered by serotonin. Taken together, our findings suggest that activation of PPARc induces HO-1 expression, and that this in turn stimulates p21 WAF1 expression to suppress PASMC proliferation. Our study also indicates that rosiglitazone, a medi- cine widely used in the treatment of type 2 diabetes mellitus, has potential benefits for patients with pulmonary hypertension. Abbreviations 5-HT, 5-hydroxytryptamine; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, heme oxygenase; PASMC, pulmonary artery smooth muscle cell; PPAR, peroxisome proliferator-activated receptor; siRNA, small interfering RNA. FEBS Journal 277 (2010) 1543–1550 ª 2010 The Authors Journal compilation ª 2010 FEBS 1543 and rosiglitazone, which have been commonly used in the treatment of type 2 diabetes mellitus [2,3]. Reduced expression of PPARc has been recently reported to be associated with the development of pulmonary hyper- tension [4,5]. On the other hand, activation of PPARc has been shown to inhibit the proliferation of pulmo- nary vascular smooth muscle cells [1,4] and the devel- opment of pulmonary hypertension in animal models [1,6]. However, the mechanisms by which activation of PPARc inhibits the proliferation of pulmonary vascu- lar smooth muscle cells, a pivotal point in pulmonary vascular remodeling and consequent development of pulmonary hypertension, are still largely unknown. Heme oxygenase (HO) is the rate-limiting enzyme of heme catabolism. Three isoforms of HO have been identified: HO-1, HO-2, and HO-3 [7]. HO-1 is an inducible isoform of HO, and its induction has been shown to be cytoprotective [8]. HO catalyzes the breakdown of heme into iron, biliverdin, and carbon monoxide [9]. Both biliverdin and carbon monoxide have been found to dilate the vasculature and to inhi- bit the proliferation of vascular smooth muscle cells [10]. Induction of HO-1 by either genetic approaches or pharmacological intervention has been shown to be effective in preventing or treating pulmonary hyperten- sion in animal models [11,12]. A recent study has sug- gested that activation of PPARc induces expression of HO-1 in human umbilical vein endothelial cells and human umbilical artery or vein smooth muscle cells [13]. However, it is still unknown whether activation of PPARc also stimulates HO-1 expression in pulmonary artery smooth muscle cells (PASMCs) (vascular smooth muscle cells showing some differences from systemic vascular smooth muscle cells). If so, whether and how HO-1 induction further inhibits proliferation of PASMCs are still unclear, especially stimulated with several mitogenic agonists involved in the pathogenesis of pulmonary hypertension, such as serotonin [5-hydroxytryptamine (5-HT)] and endothelin-1 [14,15]. Vascular smooth muscle cells are normally quies- cent, and remain in the G 1 ⁄ G 0 phase of the cell cycle. However, upon stimulation, cells exit the G 1 ⁄ G 0 phase and start to divide [16]. Cell cycle progression is precisely controlled by the activity of a series of cyclin- dependent kinases (CDKs), which are activated by cyclin binding and negatively regulated by CDK inhibitors. P21 WAF1 is one of several important CDK inhibitors [17]. We hypothesized that activation of PPARc could induce the expression of HO-1, and that this in turn could upregulate the expression of p21 WAF1 , leading to suppression of PASMC prolife- ration. To test our hypothesis, we isolated and cultured primary PASMCs, and determined the impact of activation of PPARc on the expression of HO-1 and p21 WAF1 . We also explored whether these responses modulate cell proliferation induced by 5-HT. Results Effect of PPARc agonist on HO-1 expression Activation of PPARc by rosiglitazone has been shown to induce the expression of HO-1 in several types of mammalian cells, including non-PASMCs; however, this effect has not been reported in PASMCs to date. To examine this effect in pulmonary vascular smooth mus- cle cells, we treated PASMCs with various concentra- tions of rosiglitazone for 24 h, and analyzed the expression of HO-1 using western blotting. As shown in Fig. 1, cells treated with rosiglitazone displayed a dose- dependent increase in HO-1 expression. As compared with control cells, 5 lm rosiglitazone caused a 12.1-fold increase in protein expression of HO-1 (P < 0.01), sug- gesting that activation of PPARc specifically and effec- tively mediates HO-1 induction in PASMCs. Role of HO-1 in PPARc agonist suppression of proliferation of PASMCs HO-1 has been found to be highly effective against pulmonary hypertension, through vasodilating, inhibit- ing the inflammatory response, and suppressing the proliferation of PASMCs. At the same time, activation HO-1 GAPDH Con Rosiglitazone 0 5 10 15 20 ** ** ** HO-1/GAPDH Fold over control (AU) Fig. 1. The PPARc agonist rosiglitazone induces HO-1 expression. Primary cultured PASMCs were stimulated with different concen- trations of rosiglitazone for 24 h. The expression of HO-1 was determined using immune blotting. GAPDH was used as loading control. Representative western blotting and quantification of bands are shown (n = 3 in each group). **P < 0.01 versus control (Con). PPARc inhibition of cell proliferation M. Li et al. 1544 FEBS Journal 277 (2010) 1543–1550 ª 2010 The Authors Journal compilation ª 2010 FEBS of PPARc has also been shown to inhibit the prolifera- tion of PASMCs and thus to ameliorate the develop- ment of pulmonary hypertension. It is therefore interesting to know whether induction of HO-1 medi- ates the protective effect of PPARc against PASMC proliferation. To test this, we applied serotonin to stimulate PASMC proliferation, and then examined whether knockdown of HO-1 by small interfering RNA (siRNA) attenuated the effect of PPARc agonist on cell proliferation. Figure 2A shows that PASMCs stimulated with 5-HT (1 lm for 24 h) exhibited 4.21- fold increase in DNA synthesis as assessed by [ 3 H]thy- midine incorporation assay (P < 0.01 as compared with control), and pretreatment of cells with the PPARc agonist rosiglitazone for 12 h dose-depen- dently suppressed 5-HT-induced cell proliferation. At 5 lm, rosiglitazone fully inhibited 5-HT-triggered DNA synthesis in cells (Fig. 2A). Figure 2B shows that sequence-specific HO-1 siRNA transfection for 72 h reduced basal HO-1 expression by 91% (P < 0.01 ver- sus control), whereas nontargeting siRNA transfection did not change the HO-1 level. More importantly, we found that prior HO-1 knockdown by siRNA abol- ished the inhibitory effect of rosiglitazone on the pro- liferation of PASMCs induced by 5-HT (Fig. 2C), whereas HO-1 knockdown alone did not impact on basal or 5-HT-stimulated DNA synthesis in cells. Our study indicates that induction of HO-1 mediates the suppressive effect of PPARc agonist on PASMC proliferation. Role of p21 WAF1 in HO-1-mediated suppression of proliferation of PASMCs Recent studies have revealed that an antiproliferative effect of HO-1 on non-PASMC pulmonary vascular smooth muscle cells and other types of cells is associ- ated with upregulation of the CDK inhibitor p21 WAF1 , which is involved in negative regulation of cellular pro- liferation [18,19]. We thus determined whether increased HO-1 expression induced by PPARc agonist could, in turn, trigger upregulation of p21 WAF1 , lead- ing to an increase in its activity against PASMC prolif- eration stimulated with 5-HT. Figure 3 shows that PASMCs treated with rosiglitazone (5 lm for 24 h) displayed a 2.34-fold increase in expression of p21 WAF1 (P < 0.01 as compared with control), whereas this increase was dramatically blocked by prior knockdown of HO-1, suggesting that HO-1 induction caused by PPARc agonist is apparently involved in the upregula- tion of p21 WAF1 in PASMCs. To further confirm this observation functionally, we examined whether knock- down of p21 WAF1 by siRNA transfection could reverse the effect of PPARc agonist on suppression of PASMC proliferation. We first applied sequence-spe- cific siRNA to knock down expression of p21 WAF1 .As shown in Fig. 4A, transfection of p21 WAF1 siRNA for 72 h produced an 82% reduction in p21 WAF1 protein 0 100 200 300 400 500 5-HT 0 0 0.5 1.5 5 µM 0 + + + + 1 µM rosiglitazone DNA synthesis (% of control) ** ** # ** ## ## DNA synthesis (% of control) Con 5-HT HO-1 siRNA Rosi 5-HT Rosi 5-HT HO-1 siRNA HO-1 siRNA 5-HT HO-1 GAPDH 0 100 200 300 400 500 600 ** ## ** ## ** †† 0 0.5 1 1.5 HO-1/GAPDH fold over control (AU) ** Con HO-1 siRNA Non-targeting siRNA C B A Fig. 2. HO-1 mediates the inhibitory effect of the PPARc agonist rosiglitazone (Rosi) on PASMC proliferation. (A) Primary cultured PASMCs were stimulated 5-HT (1 l M for 24 h), and this was followed by labeling with [ 3 H]thymidine (1 lCiÆmL )1 for 12 h). Rosiglitazone was added 12 h before stimulation of cells with 5-HT. Cells were lysed, and cell-associated radioactivity was measured by liquid scintillation counting. Summary data show that rosiglitazone dose-dependently suppressed 5-HT-induced DNA synthesis (n =4 in each group). (B) Primary cultured PASMCs were transfected with HO-1 sequence-specific siRNA (HO-1 siRNA) or nontargeting con- trol siRNA for 72 h. Equal amounts of protein were loaded and probed using specific HO-1 and GAPDH (loading control) antibodies. Representative western blotting and quantification of HO-1 bands are shown. (C) Prior knockdown of HO-1 by siRNA significantly reversed the inhibitory effect of rosiglitazone on DNA synthesis in 5-HT-treated cells (n = 4 in each group). **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus 5-HT-treated cells; P < 0.01 versus rosiglitazone and 5-HT-treated cells. M. Li et al. PPARc inhibition of cell proliferation FEBS Journal 277 (2010) 1543–1550 ª 2010 The Authors Journal compilation ª 2010 FEBS 1545 level (P < 0.01 versus control), whereas nontargeting control siRNA transfection did not change the p21 WAF1 level in cells. Next, we investigated the influ- ence of the loss of p21 WAF1 on the effect of PPARc activation on suppression of cell proliferation. Fig- ure 4B indicates that knockdown of p21 WAF1 by siRNA transfection significantly reversed the inhibitory effect of PPARc agonist on PASMC proliferation induced by 5-HT. The DNA synthesis rate was increased again from a 1.4-fold increase over control in cells treated with PPARc agonist and 5-HT to a 4.04-fold increase over control in cells with p21 WAF1 siRNA silencing (despite the presence of PPARc ago- nist and 5-HT), which is similar to that in cells stimu- lated with 5-HT alone or cells treated with the combination of HO-1 siRNA transfection, PPARc agonist, and 5-HT. These results suggest that upregu- lation of p21 WAF1 by HO-1 mediates the effect of PPARc agonist in suppression of PASMC prolife- ration. Discussion In this study, we demonstrate that activation of PPARc by rosiglitazone induces significant HO-1 expression in primary cultured PASMCs, and this sub- sequently upregulates the expression of p21 WAF1 , lead- ing to inhibition of proliferation of PASMCs stimulated with 5-HT. The present study provides a P21 GAPDH HO-1 siRNA rosiglitazone Con Rosiglitazone 0 1 2 3 p21/GAPDH fold over control (AU) ** ## Fig. 3. HO-1 mediates the effect of the PPARc agonist rosiglitaz- one in upregulating p21 WAF1 expression. Primary cultured PASMCs were treated with rosiglitazone (5 l M), with or without prior knock- down of HO-1, for 24 h. Expression of p21 WAF1 was determined using immune blotting. GAPDH was used as loading control (Con). Representative western blotting and quantification of bands are shown (n = 4 in each group). **P < 0.01 versus control; ##P < 0.01 versus rosiglitazone-treated cells. P21 GAPDH 0 0.2 0.4 0.6 0.8 1 1.2 ** p21/GAPDH fold over control (AU) p21 siRNACon Non-targeting siRNA Con 5-HT Rosiglitazone 5-HT HO-1 siRNA rosiglitazone 5-HT p21 siRNA rosiglitazone 5-HT DNA synthesis (% of control) 0 100 200 300 400 500 600 ** ** †† ## ** †† B A Fig. 4. p21 WAF1 mediates the inhibitory effect of HO-1 on PASMC proliferation. (A) Primary cultured PASMCs were transfected with p21 WAF1 sequence-specific siRNA (p21 siRNA) or nontargeting control siRNA for 72 h. Equal amounts of protein were loaded and probed using specific p21 WAF1 and GAPDH (loading control) antibodies. Representative western blotting and quantification of p21 WAF1 bands are shown. (B) Primary cultured PASMCs with or without prior p21 WAF1 or HO-1 siRNA transfection were stimulated with 5-HT (1 l M for 24 h), and this was followed by labeling with [ 3 H]thymidine (1 lCiÆmL )1 for 12 h). Rosiglitazone (5 l M) was added 12 h before stimulation of cells with 5-HT. Cells were lysed, and cell-associated radioactivity was measured by liquid scintillation counting (n = 4 in each group). **P < 0.01 versus control; ##P < 0.01 versus 5-HT-treated cells; P < 0.01 versus cells treated with rosiglitazone and 5-HT. PPARc inhibition of cell proliferation M. Li et al. 1546 FEBS Journal 277 (2010) 1543–1550 ª 2010 The Authors Journal compilation ª 2010 FEBS novel molecular mechanism by which PPARc activa- tion suppresses PASMC proliferation and therefore ameliorates the development of pulmonary hyperten- sion. It also indicates that rosiglitazone might be useful in the treatment of pulmonary hypertension. Activation of PPARc by pharmacological ligands has been shown to exert anti-inflammatory and anti- proliferative effects on a variety of cell types, and thus has potential value in the treatment of multiple diseases [2,20–22]. Recent evidence from studies with animal models indicates that the enhancing activity of PPARc attenuates the development of pulmonary hypertension [4,6,23]. Further studies suggest that acti- vation of PPARc confers protection against pulmo- nary hypertension by suppressing PASMC proliferation. Proliferation of PASMCs is a hallmark of pathogenesis of pulmonary hypertension [1,4]. How- ever, the mechanisms responsible for inhibition of PASMC proliferation by activation of PPARc are still largely unknown. Recent studies have suggested that induction of HO-1 mediates the effect of activation of PPARc against proliferation of non-PASMCs and endothelial cells [13]. In the present study, we show that the synthetic PPARc agonist rosiglitazone dose- dependently inhibited 5-HT-stimulated proliferation of PASMCs, and that this was accompanied by a dose- dependent increase in expression of HO-1. Knockdown of HO-1 abolished the inhibitory effect of PPARc agonist on PASMC proliferation, suggesting that induction of HO-1 fully mediates this effect. Our study not only confirms previous findings, but also extends this notion to the pulmonary system. Mammalian cell proliferation is controlled by a group of cell cycle protein complexes consisting of two key regulatory molecules: CDKs and cyclins [17,24]. A CDK molecule is activated by association with a cyclin, forming a CDK complex. CDKs are constitu- tively expressed in cells, whereas cyclins are synthesized at specific stages of the cell cycle [25]. The expression of a cyclin is regulated at the transcriptional and degradation level to influence CDK activity [26]. In addition, CDK activity is modulated by a group of CDK inhibitors comprising two families of proteins: inhibitor of kinase 4 ⁄ alternative reading frame and CDK inhibitor protein ⁄ kinase inhibitor protein. The inhibitor of kinase 4 ⁄ alternative reading frame family includes p16INK4a and p14arf, which bind to CDK4 and arrest the cell cycle in the G 1 phase or prevent p53 degradation, respectively [27,28]. The CDK inhibitor protein ⁄ kinase inhibitor protein family includes p21 WAF1 , p27 Kip1 , and p57 Kip2 . They halt the cell cycle in the G 1 phase by binding to, and inactivating, cyclin–CDK complexes [29,30]. The results of our study reveal that activation of PPARc increases p21 WAF1 expression, and that this effect is significantly blocked by prior knockdown of HO-1. This indicates that PPARc agonist-induced HO-1 expression mediates p21 WAF1 upregulation. We further confirmed this observation functionally by using p21 WAF1 siRNA silencing, when loss of p21 WAF1 significantly reversed the inhibitory effect of PPARc agonist on cell proli- feration. Our result is consistent with that of Pae [31], showing that curcumin-induced HO-1 expression regu- lates p21 expression in aortic smooth muscle cells. The mechanisms underlying HO-1 induction of p21 WAF1 expression may be explained by accumulation of iron and carbon monoxide, two key products of HO-1 [32]. Pulmonary hypertension and consequent cor pulmo- nale, particularly secondary to chronic obstructive pul- monary disease, are common clinical conditions and some of the major causes of hospitalization and death in patients with chronic obstructive pulmonary disease [33,34]. Increased pulmonary vascular resistance caused by pulmonary vasoconstriction and vascular remodel- ing (prominent with vascular smooth muscle cell pro- liferation) is the major basis for the development of pulmonary hypertension [35,36]. Most drugs currently used in the treatment of pulmonary hypertension are vasodilators; few are aimed effectively against pulmo- nary vascular remodeling [37,38], which is considered to be a more critical mechanism for chronic pulmonary hypertension [39]. Therefore, putative candidates to modulate vascular remodeling have important poten- tial applications in the treatment of pulmonary hyper- tension. Rosiglitazone is a wildly used medicine with beneficial effects in the long-term treatment of diabetic mellitus [40]. Accumulated clinical experience and the safety record of rosiglitazone suggest that this may be an important chronic therapeutic approach for human pulmonary hypertensive disease. Experimental procedures Cell preparation and culture Primary smooth muscle cells from pulmonary arteries were prepared from Sprague–Dawley rats (125–250 g) by the method reported by Golovina et al. [41]. Isolated arterial rings were incubated in Hanks’ balanced salt solution con- taining 1.5 mg ÆmL )1 collagenase II (Worthington, Lake- wood, NJ, USA) for 20 min. After incubation, a thin layer of the adventitia was carefully stripped off with fine forceps, and the endothelium was removed by gently scratching the intima surface with a surgical blade. The remaining smooth muscle was then digested with 2.0 mgÆmL )1 collagenase II and 0.5 mgÆmL )1 elastase IV M. Li et al. PPARc inhibition of cell proliferation FEBS Journal 277 (2010) 1543–1550 ª 2010 The Authors Journal compilation ª 2010 FEBS 1547 (Sigma, St Louis, MO, USA) for 45 min at 37 °C. The cells were plated onto 10 cm Petri dishes containing DMEM (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum, 100 UÆmL )1 penicillin, and 100 lgÆmL )1 strepto- mycin, and cultured in a 37 °C ⁄ 5% CO 2 humidified incuba- tor. Cells were passaged by trypsinization, using 0.05% trypsin ⁄ EDTA (Invitrogen). All experiments were per- formed using cells between passages 4 and 8. To test the purity of smooth muscle cells, cells were stained with 4¢,6¢-diamidino-2-phenylindole (Invitrogen) and fluorescein isothiocyanate-labeled antibody against smooth muscle a-actin (Sigma), for nucleus and smooth muscle actin, respectively. Fluorescence microscope images indicated that cells contained more than 93% smooth muscle cells (data not shown here). Before each experiment, cells were incubated in 0.5% fetal bovine serum ⁄ DMEM for 24 h to minimize serum-induced effects on the cells. 5-HT (Sigma) was used to stimulate the proliferation of PASMCs. Rosig- litazone (Cayman Chemical Co., Ann Arbor, MI, USA) was used to stimulate PPARc activation. siRNA transfection To silence the expression of HO-1 and p21 WAF1 protein, PASMCs were transfected with sequence-specific or nontar- geting control siRNA (Dharmacon, Lafayette, CO, USA), using Lipofectamine 2000 reagent (Invitrogen). Briefly, cells were cultured up to 30–40% confluence, and siRNA and Lipofectamine were diluted in serum-free DMEM sepa- rately and incubated for 5 min at room temperature. siRNA was mixed with Lipofectamine and incubated at room temperature for 20 min. Then, the complex of siRNA and Lipofectamine was added to cells, and culture was maintained for 72 h at 37 °C and 5% CO 2 in a humidified incubator. Cells were transfected for 24 h before the prepa- ration of the [ 3 H]thymidine incorporation assay. The effect of protein silencing was analyzed using western blot. Immunoblotting Cells were lysed in 50 mm Tris ⁄ HCl (pH 7.4), 1% Nonidet P-40, 0.1% SDS, 150 mm NaCl, 0.5% sodium deoxycho- late, 1 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, 1mm Na 3 VO 4 ,1mm NaF, and proteinase inhibitors. Lysates were centrifuged at 15 700 g at 4 °C for 15 min, and the supernatant was collected as total protein. The pro- tein concentration was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Protein was separated on an SDS ⁄ PAGE gel, and transferred to a Trans-Blot nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Monoclonal antibodies against p21 WAF1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and polyclonal antibody against HO-1 (Millipore, Bedford, MA, USA) were used according to the manufacturer’s instructions. Horseradish peroxidase-conjugated goat anti- (mouse IgG) and goat anti-(rabbit IgG) were used as sec- ondary antibodies (Sigma). Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposure to autoradiographic film. Signaling was quan- tified from scanned films using scion nih image software (Scion, Frederick, MD, USA). [ 3 H]Thymidine incorporation assay PASMCs were grown to 50–60% confluence in 24-well plates, and serum starved for 24 h (0.5% fetal bovine serum in DMEM) before the start of experiments. Cells were treated with 1 lm 5-HT or vehicle for 24 h, and this was followed by labeling with [ 3 H]thymidine (1 lCiÆmL )1 ) for 12 h. PPARc agonist was added 12 h before the stimulation of cells with serotonin. After labeling, cells were washed twice with ice-cold NaCl ⁄ P i and incubated in 5% trichloro- acetic acid for 30 min at 4 °C. Cell lysates were then washed with ice-cold NaCl ⁄ P i and solubilized by adding 0.5 mL of 0.5 m NaOH ⁄ 0.5% SDS. Cell-associated radio- activity was measured by liquid scintillation counting. Statistics Values are presented as mean ± standard deviation. Data were analyzed using one-way ANOVA with Tukey post hoc test. P < 0.05 was considered to represent significant differences between groups. Acknowledgements This work was supported by the Chinese National Science Foundation (30871116), the Tengfei Talent Project of Xi’an Jiaotong University and the start-up package to M. Li from the Second Affiliated Hospital of Medical College of Xi’an Jiaotong University, PR China. References 1 Nisbet RE, Sutliff RL & Hart CM (2007) The role of peroxisome proliferator-activated receptors in pulmo- nary vascular disease. PPAR Res 2007, 18797. 2 Duan SZ, Usher MG & Mortensen RM (2008) Peroxi- some proliferator-activated receptor-gamma-mediated effects in the vasculature. Circ Res 102, 283–294. 3 Touyz RM & Schiffrin EL (2006) Peroxisome prolifera- tor-activated receptors in vascular biology – molecular mechanisms and clinical implications. Vascul Pharmacol 45, 19–28. 4 Hansmann G, de Jesus Perez VA, Alastalo TP, Alvira CM, Guignabert C, Bekker JM, Schellong S, Urashima T, Wang L, Morrell NW et al. 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