| Background and objective Myocardial fibrosis is not only one of the important pathological bases of left ventricular remodeling induced by essential hypertension but also the main cause of cardiac diastolic dysfunction.Moreover, myocardial fibrosis is regarded as a major determinant leading to cardiac functional transition from compensation to decompensation.Therefore,it is very important to prevent and regress myocardial fibrosis,and to improve cardiac function for target organic protection of hypertension.A significant body of literature demonstrates that cardiac mast cell-derived chymase is involved in the cardivascular pathologic remodeling,which has been found to play a crucial role in the progression of many cardiovascular diseases,such as hypertension, myocardial infarction,cardiomyopathy,congestive heart failure,restenosis after percutaneous transluminal coronary angioplasty,atherosclerosis,aneurysm,and so on.Recent studies suggest that chymase could inhibit proliferation of vascular smooth muscle cells and elicit hypertrophic response of cardiac myocytes.Cardiac fibroblasts(CFs) have long been considered as major effectors of myocardial fibrosis.The excessive proliferation of CFs and enhanced collagen synthesis are thus regarded as pathological foundation of myocardial fibrosis.However,it remains unknown whether chymase can exert effects on CFs proliferation and collagen synthesis,and the underlying mechanisms of signal transduction are also obscure.Although it has been reported that chymase can activate latent transforming growth factor-β1 (TGF-β1) recently,the relative role of chymase-mediated TGF-β1 forming system in CFs proliferation has not been well established.In the last few years,a major advance in understanding TGF-β1 post-receptor signaling is the identification of Smad proteins,which may specifically modulate the transcriptions of TGF-β1 target genes.It has been well documented that the activation of TGF-β1/Smads signaling pathway may participate in the fibrotic progression of many organs,including liver,lung,kidney,peritoneum and skin. Little has been known about whether TGF-β1/Smads signaling pathway could contribute to myocardial fibrosis caused by hypertension.Peroxisome proliferator-activated receptors(PPARs) are ligand-dependent nuclear receptors, including PPARα,PPARβ/δand PPARγ,.Over the past few years,extensive studies have been focused on the protective effects of PPARγ,and its agonist, thiazolidinediones,on cardiovascular tissue which could be compromised in metabolic syndrome.In contrast,the knowledge about the possible role of PPARαin the prevention of cardiovascular disease is rather sparse.Recent evidence demonstrates that PPARαis highly expressed in myocardial tissue and exert an inhibitory effect on myocardial hypertrophy,indicating that further insight into the PPARαpathway may have important therapeutic implications on hypertensive left ventricular hypertrophy.It has been confirmed that fibrates,a sort of lipid-lowering drugs,have pronounced therapeutic effects on essential hypertriglyceridemia.Since being identified as PPARαactivator,fenofibrate has attracted a great deal of attention with regard to its beneficial effect on cardiovascular system beyond its contribution to lipid metabolism.In vitro, fenofibrate could inhibit the hypertrophy of cultured cardiac myocytes induced by many proinflammatory mediators and cytokines.However,whether fenofibrate can inhibit CFs proliferation and collagen synthesis,and the signaling pathway involved remain unclear so far.Currently,it has been observed that PPARγagonist can down-regulate gene expression of TGF-β1 and thus arrest the progression of renal interstitium fibrosis.The underlying mechanism may be related to the blockade of TGF-β1/Smad signaling pathway. However,it remains unclear whether TGF-β1/Smads and PPARαsignaling pathway participate in the chymase-induced myocardial fibrosis modulated by fenofibrate and whether there exist a cross-talk between the two pathways.This study was therefore designed to observe the effect of mast cell-derived chymase on cell proliferation and collagen synthesis of cultured CFs at cellular and molecular level,and to discuss the mechanisms of its effect on myocardial fibrosis.Moreover,we also investigated the role of PPARαand its agonist fenofibrate on TGF-β1/Smads signaling pathway activited by chymase in order to elucidate the mechanisms of preventive and therapeutic effects of fenofibrate on hypertensive myocardial fibrosis.Accordingly,the study was attempted to provide noval theoretical evidence and new strategy for the clinical treatment on hypertensive left ventricular hypertrophy.Methods In this study,cultured CFs of neonatal SD rats were used as experimental models.Moreover,MTT assay,radionuclide incorporation method, flow cytometry technique,ELISA,RT-PCR and Western blot were applied to identify:(1) the effects of chymase on cell proliferation and collagen synthesis of cultured CFs in SD rats;(2) the effects of chymase on the mRNA and proteinic expression of TGF-β1,Smad2,Smad3 and Smad7 in CFs;(3) the effects of fenofibrate,a PPARαagonist,on CFs proliferation and collagen synthesis induced by chymase;(4) the intervention effects of fenofibrate on the mRNA and proteinic expression of PPARα,TGF-β1,and the proteinic expression of Smad2/3,p-Smad2/3 and Smad7 in CFs.Results(1) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 24 h significantly increased CFs number in a dose-dependent manner,and the A490 Value(0.263±0.033,0.348±0.031,and 0.387±0.026,respectively) were all significantly higher than that of control(0.201±0.019,P＜0.01).The A490 Value in 30 ng/ml chymase-treated CFs was markedly decreased by pretreatment with TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor(P＜0.05 or P＜0.01).Neither AT1 receptor blocker nor AT2 receptor blocker significantly altered the A490 Value of chymase-treated CFs(P＞0.05).(2) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 24 h significantly increased DNA synthesis in a dose-dependent manner,and the 3H-TdR incorporation (319±29 cpm/well,372±43 cpm/well,and 401±47 cpm/well,respectively) were all remarkably higher than that of control(252±35 cpm/well,P＜0.01).The 3H-TdR incorporation in 10μmol/L chymase inhibitor-pretreated group was notablely lower than that in 30 ng/ml chymase group(P＜0.01).(3) Cell cycle analysis revealed that treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly decreased the percentages of cells in G0/G1 phase of the cell cycle(P＜0.05 or P＜0.01),and increased the percentages of cells in S phase and the proliferation index(PI) when compared with that of control(P＜0.05 or P＜0.01).Whereas The percentages of cells in G2/M phase were not significantly changed by chymase(P＞0.05).Pretreatment with 10μmol/L chymase inhibitor markedly increased G0/G1 phase percentage(P＜0.01),and decreased S phase percentage and PI when compared with that of 30 ng/ml chymase group (P＜0.01).But pretreatment with 10μmol/L chymase inhibitor had no effect on G2/M phase percentage when compared with chymase group or control group (P＞0.05).(4) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased total collagen synthesis in a dose-dependent manner,and the 3H-Proline incorporation(520±75cpm/well,684±62 cpm/well and 769±58 cpm/well,respectively) were all remarkably higher than that of control(435±60 cpm/well,P＜0.05 or P＜0.01).The 3H-proline incorporation in TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor-pretreated group was significantly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT1 receptor blocker nor AT2 receptor blocker significantly changed the 3H-proline incorporation of chymase-treated cells(P＞0.05).(5) Treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h markedly increased the mRNA levels of collagen Type-â… and -â…¢in a dose-dependent manner when compared with control(P＜0.01),whereas chymase at 7.5 ng/ml had no significant effect on the mRNA expression of collagen Type-â… and -â…¢(P＞0.05).(6) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased the protein content of collagen Type-â… and -â…¢in cultured supematant of CFs when compared with control (P＜0.01),whereas chymase at 7.5 ng/ml had no marked effect on the protein content of collagen Type-â… and -â…¢(P＞0.05).(7) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 3 h,the mRNA levels of TGF-β1 were 0.698±0.051,1.096±0.078,and 1.242±0.065,respectively,which were all notably higher than that of control(0.299±0.035,P＜0.01).The mRNA expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was remarkably lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01). Neither AT1 receptor blocker nor AT2 receptor blocker significantly modified the mRNA expression of TGF-β1 induced by chymase(P＞0.05).(8) Administration with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 6 h,the protein levels of TGF-β1 were 0.968±0.069,1.782±0.058 and 2.656±0.085,respectively,which were all significantly higher than that of control(0.333±0.023,P＜0.05 or P＜0.01).The protein expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was markedly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT1 receptor blocker nor AT2 receptor blocker significantly altered the protein expression of TGF-β1 mediated by chymase(P＞0.05).(9) Treatment with chymase at different concentrations for 6 h had no significant effects on the mRNA expression of Smad2 and Smad3 (P＞0.05).(10) Administration with chymase at 30 ng/mL for 3 h,6 h,12 h and 24 h significantly increased the p-Smad2/3 when compared with control(P＜0.05 or P＜0.01),while the protein expressionof Smad2/3 was not notably altered (P＞0.05).The protein expression of Smad7 in 3 h group was higher than that in control group(P＜0.05).However,treatment with chymase for 6 h,12 h,and 24 h markedly decreased the protein expression of Smad7 when compared with control(P＜0.05 or P＜0.01).(11) Pretreatment with fenofibrate at different concentrations decreased the number of CFs in a concentration-dependent manner,and the A49o Value in 50μmol/L group and 100μmol/L group was significantly lower than that in chymase group(P＜0.05 or P＜0.01).The A490 Value was no significant difference between the chymase group and the co-pretreated with fenofibrate and its antagonist group(P＞0.05).100μmol/L Fenofbrate alone had no significant effect on the number of CFs(P＞0.05).(12) Pretreatment with fenofibrate at different concentrations decreased the DNA synthesis of CFs in a concentration-dependent manner,and the 3H-TdR incorporation in 50μmol/L group and 100μmol/Lgroup was markedly lower than that in chymase group(P＜0.01).The 3H-TdR incorporation was not changed by fenofibrate and its antagonist co-pretreatment when compared with that of chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on 3H-TdR incorporation into CFs(P＞0.05).(13) Pretreatment with fenofibrate at different concentrations increased the percentages of cells in G0/G1 phase,and decreased those in S phase and the PI,which in 50μmol/L group and 100μmol/L group had signifcant difference when compared with that in chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on PI,the percentages of cells in G0/G1 phase or S phase (P＞0.05).(14) Pretreatment with fenofibrate at 10μmoFL,50μmol/L and 100μmol/L remarkably decreased the 3H-proline incorporation in a concentration-dependent manner,and the 3H-proline incorporation at 50μmol/L and 100μmol/L was significantly lower than that of chymase group(P＜0.01). The 3H-proline incorporation had no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05). 100μmol/L fenofibrate alone had no apparente effect on 3H-proline incorporation into CFs(P＞0.05).(15) Pretreatment with fenofibrate at different concentrations decreased the mRNA expression of collagen Type-â… and -â…¢in a concentration-dependent manner,and the mRNA levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The mRNA expression of collagen Type-â… and -â…¢had no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no marked effect on the mRNA expression of collagenâ… andâ…¢(P＞0.05).(16) Administration with chymase at different concentrations for 6 h lowered the mRNA expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.01).Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L increased mRNA expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group (P＜0.05 or P＜0.01).100μmol/L fenofibrate alone notably elevated the mRNA expression of PPARαwhen compared with control(P＜0.01).(17) Treatment with chymase at different concentrations for 12 h decreased the protein expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L up-regulated the protein expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.01).100μmol/L fenofibrate alone remarkably elevated the protein expression of PPARαwhen compared with control(P＜0.01).(18) Pretreatment with fenofibrate at different concentrations down-regulated the mRNA expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The expression level was not altered in fenofibrate and its antagonist co-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of TGF-β1(P＞0.05).(19) Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L down-regulated the protein expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no marked effect on the protein expression of TGF-β1(P＞0.05).(20) Administration with chymase at different concentrations for 12 h had no significant effect on the protein expression of Smad2/3(P＞0.05) No changes of Smad2/3 in protein expression was observed when CFs were pretreated with different concentrations of fenofibrate(P＞0.05).(21) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h up-regulated the protein expression of p-Smad2/3 when compared with control(P＜0.05 or P＜0.01).Pretreatment with fenofibrate at different concentrations down-regulated the protein expression of p-Smad2/3,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no notable effect on the protein expression of p-Smad2/3(P＞0.05).(22) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h down-regulated the mRNA expression of Smad7 when compared with control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the mRNA expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of Smad7(P＞0.05).(23) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 12 h down-regulated the protein expression of Smad7 when compared with control(P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the protein expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no remarkable effect on the protein expression of Smad7(P＞0.05).Conclusion(1) Cardiac mast cell-derived chymase can promote cell proliferation and collagen synthesis of CFs in a dose-dependent manner, indicating that cardiac chymase may play a significant role in the formation and progression of myocardial fibrosis.(2) Chymase can up-regulate TGF-β1 expression,promote Smad2/3 phosphorylation and down-regulate Smad7 expression,suggesting that chymase can activate TGF-β1/Smads signaling pathway.(3) TGF-β1 neutralizing antibody and serine/threonine kinase inhibitor exert significant inhibitory effects on cell proliferation,collagen synthesis and TGF-β1 expression caused by chymase while angiotensinâ…¡receptor blocker (ARB) valsartan and PD123319 fail to exhibit similar changes.The result further indicates that TGF-β1/Smads pathway is involved in myocardial fibrosis caused by chymase.(4) Fenofibrate,a PPARαagonist,can suppress cell proliferation and collagen synthesis of CFs induced by chymase.Thus,it may exert reverse effect on myocardial fibrosis.(5) Fenofibrate can not only activate PPARαand restrain TGF-β1 production,but also down-regulate p-Smad2/3 expression and up-regulate Smad7 expression.This may be one of the molecular and biological mechanisms involved in the inhibitory effects of fenofibrate on cell proliferation,collagen synthesis of CFs and then result in the regression of myocardial fibrosis.(6) There may exist cross-talk between PPARαand TGF-β1/Smads pathway.That is to say,activated PPARαcan exert negative modulation on TGF-β1/Smads pathway by down-regulating TGF-β1 expression, inhibiting Smad2/3 phosphorylation and up-regulating Smad7 expression.In a summary,cardiac mast cell-derived chymase can induce cell proliferation and collagen synthesis of CFs and therefore,it can promote myocardial fibrosis.Up-regulation of TGF-β1 expression,phosphorylation of Smad2/3 and down-regulation of Smad7 expression are considered to be one of intra-cellular signal transduction mechariisms.Fenofibrate,a PPARαagonist, can reverse myocardial fibrotic response and thus exert protective effects on cardiovascular system apart from its effect on lipid metabolism.It may be one of the fenofibrate-mediated potential mechanisms that suppression of TGF-β1 expression caused by activating PPARαmay result in down-regulation of Smad2/3 phosphorylation and up-regulation of Smad7 expression.Therefore, this study may provide theoretical evidence and a novel therapeutic target for left ventricular hypertrophy caused by hypertension.
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