| Background and objective Hypertension is an important risk factor for the onset of cardiovascular diseases, whereas left ventricular hypertrophy (LVH) is an independent risk factor for the increased incidence of cardiovascular events and important pathological basis for the transition of cardiac function from a"compensated"state towards a"decompensated"one. Thus preventing and treating LVH has become one of the hot research issues in an international setting. It has now been established that the abnormal activation of neurohumoral factors is an important mechanism for the development of LVH. Arginine vasopressin (AVP) is a neurohumoral factor critically involved in cardiovascular disease. It has been shown that AVP can stimulate the proliferation of neonatal rat CFs, but it remains unclear how the signal transduction is achieved. 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statins) is one of the major drug types that control the risk factors for hypertension, among which is hyperlipemia. However, a large pool of research has shown its other effects beyond cholesterol-lowing, including its role in the prevention of cardiac fibrosis. It was observed that statins could inhibit cardiomyocytes hypertrophy and cell proliferation and collagen synthesis of CFs and attenuate the LVH of spontaneously hypertensive rats (SHR). These observations indicate that statins can modulate the process of cardiac remodeling, but its mechanisms are less than well defined. caveolae are structures located in the cell membrane which harbor and transport endogenous cholesterol and serve as'hinges'for the gathering and transduction of signaling molecules. Caveolin, the protein marker of caveolae, has direct negative modulatory effects on the activation of a variety of the key signaling molecules. It has been shown to inhibit the onset and development of cardiac fibrosis. However, it still remains unclear so far whether the inhibitory effect of statins on cardiac fibrosis is influenced by caveolae. No report has ever examined the changes of caveolin in the process of CFs proliferation. In the present study, we examined the potential proliferation-inducing effect of AVP on adult rat CFs and the involvement of erk1/2 and the changes in the activation of erk1/2 in the presence of Sim. The role of caveolin1 (cav1) is also investigated. In addition, we further explored the mechanisms for the inhibitory effect of Sim on CFs proliferation, the effects of the changes in the abundance of cholesterol in the cell membrane on the expression of cav1 and the modulation of AVP-induced CFs proliferation by Sim. Our objectives are to elucidate further the mechanisms of cardiac remodeling during hypertension and further the understanding of the molecular mechanisms for statins in the prevention and treatment of cardiac remodeling, and to provide new theoretical evidences and prevention and treatment approaches for LVH of hypertension.Methods In this study, adult rat CFs isolated from Sprague-Dawley (SD) rats were cultured and used as experimental model. We employed [3H]-thymidine incorporation, flow cytometry, in vitro kinase assay, Western blot analysis and RT-PCR in the present study to investigate: (1) Effect of AVP on CFs proliferation, (2) Role of PKC and erk1/2 pathway in the proliferatory effect of AVP on CFs proliferation, (3) Effect of Sim on CFs proliferation, (4) Modulatory effect of Sim on PKC and erk1/2 pathway during CFs proliferation, (5) Changes of cav1 in the modulation of AVP-induced CFs proliferation by Sim, (6) Effect of exogenous cholesterol on the modulation of Sim on CFs proliferation and its relationship with cav1 expression.Results (1) Treating cells with 10-9 10-6 mol/L AVP increased [3H]-thymidine incorporation in a concentration-dependent manner. 10-7 mol/L and 10-6 mol/L AVP increased [3H]-thymidine incorporation by (72.0±4.0)% and(158.0±19.1)%, respectively(P < 0.05). (2) The effect of AVP on [3H]-thymidine incorporation was abolished by incubating adult rat CFs with V1 receptor antagonist, d(CH2)5[Tyr2(Me), Arg8]-vasopressin, but not V2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin. (3) Erk1/2 activation could be induced by AVP (0.1μmol/L). The activation peaked at 5 min, with subsequent decline to near baseline level at 2 hour after the initiation of the stimulation. (4) PD98059 abolished the mitogenic effect of AVP(P < 0.05). (5) Phorbol 12-myristate 13-acetate (PMA, 30 nmol/L), as well as AVP for 5 min activated erk1/2 phosphorylation as measured by Western blots(P<0.05). Depleting PKC by chronic PMA incubation (2.5μmol/L, 24 h) abolished the stimulating effect of AVP on erk1/2 phosphorylation(P < 0.05). (6) Calphostin C, a PKC inhibitor, markedly reduced [3H]-thymidine incorporation upon 0.1μmol/L AVP stimulation(P < 0.05). (7) PKC activation could readily be induced by AVP (0.1μM). The activation peaked at 5 min (approx. 3-fold induction, P<0.05) and subsequently declined to near baseline level at 30 min after the initiation of AVP stimulation. (8) Ca2+ chelating agent BAPTA had no significant effect on DNA synthesis stimulated by AVP (P>0.05). (9) The protein level of p27Kip1 was markedly attenuated upon AVP treatment to (21.7±1.6)% of control level, while expression of cyclins D1, A and E increased to (5.7±0.5)-fold,(5.4±0.5)-fold and (6.0±0.7)-fold of control(P < 0.05). Inhibiting erk1/2 activation by PD98059 (30μmol/L) abolished the effect of AVP on protein expression of p27Kip1 as well as cyclins D1, E and A. (10) 0.1μmol/L AVP induced cell cycle progression from G0/G1 into S phase, accompanied by increased proliferation index (PI) (P<0.05). The effects of AVP on G0/G1-S phase progression and PI were inhibited by PD98059. (11) When pretreating cells with simvastatin (10-8 10-5 mol/L) for 24 h before 10-7 mol/L AVP stimulation for another 24 h, [3H]-thymidine incorporation in adult rat CFs were (172.0±9.8)%, (151.3±7.7)%, (128.3±7.6)%, and (121.7±11.5)%, respectively, of control, all being significantly lower (P<0.05) than AVP single-drug treated cells, except in the 10-8 mol/L simvastatin treated group. (12) When pretreating cells with simvastatin (10-8 10-5 mol/L) for 24 h before 10-7 mol/L AVP stimulation for another 24 h, cells in S stage and PI tended to decrease, with those of 10-7 mol/L and 10-6 mol/L Sim groups being significantly lower (both P<0.05) than AVP single-drug treated cells. And PI was significantly lower (P<0.05) in 10-7 10-5 mol/L Sim cells than in AVP single-drug treated cells. (13) When pretreating cells with simvastatin (10-8 10-5 mol/L) for 24 h before 10-7 mol/L AVP stimulation for 5 min, erk1/2 activation reached (2.5±0.2)-fold, (1.9±0.1)-fold, (1.5±0.1)-fold, and(1.3±0.1)-fold, respectively, over control level, being significantly lower (P<0.05) in 10-7 10-5 mol/L Sim cells than in AVP single-drug treated cells. (14) When pretreating cells with simvastatin (10-8 10-5 mol/L) for 24 h before 10-7 mol/L AVP stimulation for 5 min, PKC activation reached (24.8±2.4)%, (21.5±2.6)%, (17.3±1.8)% and(15.0±1.3)%, respectively, of control level, being significantly lower (P<0.05) in 10-6 mol/L and 10-5 mol/L Sim cells than in AVP single-drug treated cells. (15) MVA pretreatment reversed the inhibitory effect of 10-6mol/L simvastatin on 10-7mol/L AVP induced increase in [3H]-thymidine incorporation and PKC and erk1/2 activation. (16) Geranylgeranyl pyrophosphate (GGPP) reversed the inhibitory effect of 10-6mol/L simvastatin on 10-7mol/L AVP induced increase in [3H]-thymidine incorporation and erk1/2 activation, whereas farnesyl pyrophosphate (FPP) had no significant effect. (17) 10-7 mol/L simvastatin significantly inhibited 10-7 mol/L AVP-induced increase in MBS-P expression(P<0.05). (18) Pretreating cells with 10μmol/L GGTI or 5μmol/L Y27632 for 24 h before 10-7 mol/L AVP stimulation for 5 min significantly inhibited the AVP-induced erk1/2 activation (P<0.05). (19) Cav1 antisense oligonucleotides significantly increased [3H]-thymidine incorporation, PKC and erk1/2 activation and the expression of cyclins D1, A and E in CFs, and decreased the expression of p27kip1 (all P < 0.05). (20) The cav1 expression in CFs were (86.7±4.6)%, (79.0±8.6)%, (59.7±3.7)% and (46.0±3.1)% of control when treating cells with 10-9 10-6 mol/L AVP, with the expression levels in 10-7 mol/L and 10-6 mol/L AVP groups being significantly lower than that of control (P < 0.05). (21) When pretreating cells with simvastatin (10-8 10-5 mol/L) for 24 h before 10-7 mol/L AVP stimulation for 24 h, cav1 expression were (88.7±2.7)%, (70.3±2.6)%,(60.7±2.2)% and (56.3±1.9)% of AVP single-drug treated cells, respectively, being significantly lower (P<0.05) in 10-7 10-5 mol/L Sim cells than in AVP single-drug treated cells. The addition of 10-4 mol/L MVA to 10-7 mol/L AVP significantly increased cav1 expression (P < 0.05). (22) 10μg/ml, 20μg/ml and 30μg/ml cholesterol significantly increased cav1 expression in cells treated with 10-6mol/L simvastatin and 10-7mol/L AVP (all P < 0.05). (23) When cells were incubated with 10% FBS, the addition of 20μg/ml cholesterol showed no apparent effect on cav1 protein expression (P > 0.05). However, when cells were incubated with 2% Methyl-β-cyclodextrin (MβCD), a maneuver to deplete cells of cholesterol, the restoration of cholesterol by MβCD-cholesterol complex (containing 20μg/ml cholesterol) caused a (2.7±0.2)-fold increase from the cholesterol-depleted condition (P < 0.05). (24) Exogenous cholesterol increased the cholesterol content in cells treated with 2% MβCD and 10-7 mol/L AVP combined with 10-6 mol/L simvastatin. Exogenous cholesterol also inhibited the increase in [3H]-thymidine incorporation upon 2% MβCD treatment, whereas it had little effects on cellular cholesterol and the increase in [3H]-thymidine incorporation when cells were treated with 10% FBS. (25) Erk1/2 activation was significantly attenuated when 20μg/ml cholesterol was added simultaneously with 10-6 mol/L simvastatin to 10-7 mol/L AVP treated cells when compared with AVP and simvastatin treated cells (P < 0.05).Conclusions (1) AVP acts as a growth-factor for adult rat CFs. (2) The mitogenic effect of AVP is mediated via type 1 receptor and PKC–erk1/2 pathway. (3) AVP modulates the expression of cell cycle regulatory proteins p27Kip1 and cyclins D1, A and E, which lie down-stream of erk1/2 activation, and induces cell cycle progression of adult rat CFs. (4) Simvastatin inhibits AVP induced CFs proliferation, PKC and erk1/2 pathway activation, an effect that can be reversed by MVA. (5) Cav1 restoration by cholesterol enhances the inhibitory effect of simvastatin on AVP-induced CFs proliferation.In summary, AVP can stimulate the proliferation of adult rat CFs via type 1 receptor and PKC–erk1/2 pathway, an effect can be inhibited by simvasatin. The inhibitory effect of simvastatin on AVP-induced CFs proliferation can be enhanced by exogenous cholesterol via the induction of cav1 exression.
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