The Effects And Signal Transduction Mechanisms Of Lovastatin On Cardiac Fibroblasts Proliferation In Neonatal Rat Induced By Aldosterone

Myocardial fibrosis is involved in pathological process of many cardiovascular diseases, such as dilated cardiomyopathy, left- ventricular hypertrophy, tissue repaire post myocardial infarction, which can lead to ventricular diastolic and systolic dysfunction and ultimately symptomatic heart failure. The main pathological characteristics of myocardial fibrosis were proliferation of fibroblasts, increased collagen synthesis and irregular deposition of extracellular matrix components. Cardiac fibroblasts play a pivotal role in the process of formation of myocardial fibrosis.Studies have shown that circulating (hormonal) but not haemodynamic factors are responsible for the formation of myocardial fibrosis. It is well known that the renin-angiotensin-aldosterone system (RAAS) plays an important role in myocardial fibrosis, but the current researches mainly foucs on the role of angiotensin II and its receptors. What is more, most researches about aldosterone on cardiac fibroblasts were involved with collagen production, while few studies have addressed the signal transduction pathway and proliferation of cardiac fibroblasts.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, namely statins, exerting pleiotropic actions beyond lipid-lowering effects, have been associated with reduced morbidity and mortality in patients with acute coronary artery disease. Cardiovascular protective actions of statins are independent of cholesterol lowering. Recently, numerous reports showed that statins could inhibit cell proliferation via cell cycle arrest in Go/G₁ phase in varieties of cell types including vascular smooth muscle cells, malignant cells and many tumor cells in vitro. Whether statins have the same action on cardiac fibroblasts and whether statins inhibit cardiac fibroblastsproliferation induced by aldosterone are still unclear. What is more, the molecular mechanisms for its effect are far from being elucidated.Mitogen-activated protein kinase (MAPK) is well documented to play a pivotal role in cellular growth and differentiation. In 2003, Stockand had demonstrated that aldosterone can stimulate prolifation of cardiac fibroblasts by activating Ki-RasA and MAPK 1/2 signaling, activation of Ras is a key on it. The activation of Ras may initiate downstream signals pathways cascades such as the Raf-MAPKK-MAPK pathway which MAPK can transmit extracellular signaling to cell nucleus and stimulate cell proliferation. The activation of Ras depended on mevalonate (MVA) pathway. Ras proteins are post-translationally modified by farnesyl pyrophosphate. Statins, as the rate limiting enzyme of endogenous cholesterol biosynthesis, inhibit MVA pathway and protein isoprenylation via Ras-dependent intracellular signaling pathway. So Statins and aldosterone might involved with proliferation cardiac fibroblasts through Ras-MAPK signaling and MVA pathway.In 1996, Farivar found that cardiac fibroblasts had iNOS-NO system. Recently, researches found that NO are related to cardiovascular remodeling. NO synthase induction in cardiac fibroblasts may play a pivotal role in myocardial fibrosis. But, it is unclear whether aldosterone has direct effect on iNOS-NO system activity in cardiac fibroblasts and the association with myocardial fibrosis. Statins have been found to up-regulate NOS-NO activity in endothelium cell, we speculate statins inhibite cardiac fibroblasts proliferation induced by aldosterone, which may act in part by changing of iNOS-NO system activity. So, we try to explore the relationship between the change of iNOS-NO system activity and proliferation of cardiac fibroblasts induced by statins or aldosterone via NO contents, NOS activity and iNOSmRNA and protein expression.In this study, we evaluated lovastatin effects on rat cardiac fibroblasts proliferation induced by aldosterone, activation of iNOS-NO system, and signal transduction mechanisms, in order to explore the pathophysiological and biochemical mechanisms involved in the development of cardiac fibrosis,provide theoretical baseis of preventing and treating cardiac myofibrosis. Theexperiments contained five parts as below:Part One: Primary culture and identify of cardiac fibroblastsObjective: Cardiac fibroblasts were isolated by trypsin digestion method from neonatal Sprague Dawley rats, identified by invert microscope, transmission electron microscopy, immunocytochemistry. Passage 2 and 3 cells were used in all experiments.Results: 1. Observed with invert microscope, primary cardiac fibroblasts appeared fibroblast morphology, were thin and triangular or fusiform with a light cytoplasm, often had tow or three nucleus which were big and oval. No spontaneous beat cound be observed in such cells. Trypan blue staining viable cells were >97%.2. Observed with transmission electron microscopy, there were plenty of rough endoplasmic reticulum and mitochondrion. Nucleus were big and showed round, oval or anomalo-shape. The nucleoli were clear. Cell surface were roofed collagen fibers, no myofilament and intercalated disk.3. Immunocytochemistry showed that cell stained positive for vimentin and fibronectin, negative for alpha smooth muscle actin (a -SMA). The purity of cardiac fibroblasts was 98%.Conclusion: It was succeed to primary culture neonatal rats cardiac fibroblasts by trypsin digestion method, the purity was 98% identified by invert microscope, transmission electron microscopy, immunocytochemistry. Part 2: Effects of Lovastatin on the proliferation and c-fos mRNA expression of rat cardiac fibroblasts induced by aldosterone in vitroObjective: To investigate the effects of Lovstatin on the proliferation and c-fos mRNA expression of rat cardiac fibroblasts induced by aldosterone.Methods: The DNA synthesis of CFs was determined by 3H-Thymidine (3H-TdR) incorporation. MTT colorimetry was adopted to evaluate cell number. Cell cycle was measured by flow cytometric analysis. MTT test and the observation of morphological characteristics were used to assay the proliferative capacity, reverse transcription polymerase chain reaction(RT-PCR) was used to estimate the c-fos mRNA expression.Results: 1.Compared with control [0.208±0.019, (763 + 240) counts-min"1], cell number increased at concentrations of (10'7-10"n)mol/L aldosterone group were 88.5 +13.4, 77.2+13.8, 55.1 + 11.4, 28.1+7.6, 9.7 + 2.8, respectively. 3H-TdR incorporation in every 5000 cells were (912 + 143, 1086+121, 1360+126, 1627+127, 1879+169) counts-min"1. Aldosterone promoted the proliferation of cardiac fibroblasts in a dose dependent manner, 10⁶mol/L spironolactone [0.226 + 0.013, (806+180) counts-min"1] reversed the effects of 10"7 mol/L ALD [0.039 + 0.011, (1879 + 169)countsmin"1] (PO.01).2. 10*5mol/L lovastatin had no effect on 3H-TdR incorporation and cell number of cardiac fibroblasts incubated in DMEM (1 % FBS), P>0.05.3. Observed with invert microscope, the shapes of cells without treated with lovastatin were regula, fusiform and polygon, intercellular space was small and anchoring growing. Cardiac fibroblasts of lovastatin group were shrunk and small, with many pseudopodia. Intercellular space became large, the ability of anchoring was reduced. The morphology of cardiac fibroblasts was changed more prominent, with the dose and time increase after lovastatin administration. There were some fragments of dead cells in 10⁴ mol/L lovastatin group treated 2 days.4. Lovstatin (10"8-10"5) mol/L decreased 3H-TdR incorporation and A490 values of cardiac fibroblasts in a concentration-dependent manner (PO.05), Moreover, 3H-TdR incorporation [( 1292±152), (1030±163)counts-min"1] and A49ovalues [(0.287±0.008), (0.231±0.007) ] of 10"6 mol/L lovastatin and 10'5 mol/L lovstatin group were both lower than those of control group [(1879±169) counts-min'1, 0.390±0.010], 10"7 mol/L lovastatin group [(1626±160) counts-min'1, 0.356±0.011] and 10'8 mol/L lovastatin group [(1783±184) counts-min'1,0.378±0.012].10*5mol/L lovstatin decreased cell number of cardiac fibroblasts in a time-dependent manner from 12 hours to 72 hours; lovastatin decreased 3H-TdR incorporation also in a time-dependent manner from 24 hours to 72hours. On the same time, compared with aldosterone group, A490 values of lovastatin group at 12h, 24h, 48h and 72h were lower than aldosterone group (P<0.05). 3H-TdR incorporation of lovastatin group at 24h, 48h and 72h were lower than aldosterone group (/><0.05> PO.01, respectively), but changes of 3H-TdR incorporation in 12h group was not significant (P>0.05).5. The inhibitory effect of 10"5 mol/L lovastatin in cell number and 3H-TdR incorporation of cardiac fibroblasts induced by aldosterone were reversed by 10"3mol/L MVA group [(1883+126) counts.min1, 0.392 + 0.013] and 5 umol/LFPP group [(1867+118) counts.min"1, 0.385+ 0.01 l]o6. Lovastatin groups had increased percentage of cells in Go/Gi phase and reduced percentage of cells in S phase, decreased PI values and decreased DNA contents in a dose-dependent manner (P<0.05). Lovastatin had no effects on G2/M phase ( P>0.05). Cells treated with lovastatin for 2d, there was no apoptosis peak measured by flow cytometric analysis.MVA(10"\ 10"\ 10"3mol/L) group and FPP(1, 5, lOumol/L) group reduced percentage of cells in Go/G] phase and increased percentage of cells in S phase, PI values, DNA contents in a dose-dependent manner (PO.05).7. Aldosterone group (10"'°-10'7) mol/L increased c-fos mRNA expression in cardiac fibroblasts, with optical density (OD) score were 0.466 + 0.015, 0.604 + 0.031, 0.858 + 0.026, 1.016 + 0.028 in corresponding concentrations, which were higher than that in control group (0.035 + 0.018, PO.05). The up-regulation of c-fos mRNA expression in 10*7 mol/L aldosterone was almost completely reversed by 10"6mol/L spironolactone.The OD score in lovastatin group were 0.364 + 0.023, 0.573 + 0.046, 0.831+0.036, respectively. C-fos mRNA expression was down-regulated in lovastatin group compared with aldosterone control group (1.016 + 0.028) . Lovastatin inhibited c-fos mRNA expression in a dose dependent manner.The OD score in MVA(10'5mol/L-10"3mol/L) group and FPP(5 u mol/L)group were 0.515 ± 0.041, 0.750 ± 0.029, 1.016 ± 0.039 and 1.015 i 0.040, respectively. The down-regulation effect of c-fos mRNA of lovstatin was reversed by 10"3mol/L MVA and 5 u mol/L FPP.Conclusion: These results indicated that aldosterone could promote the proliferation of cardiac fibroblasts mediated by mineralocorticoid receptor (MR) in a dose dependent manner. Lovastatin can significantly inhibit cardiac fibroblasts proliferation and DNA contents induced by aldosterone, induce G|/S transition arrest, down-regulate c-fos mRNA expression. All the above-mentioned effects can be reversed by MVA and FPP, which are related to inhibition of MVA pathway and c-fos mRNA express by lovastatin. Part 3: The regulation mechanisms about the effects of lovastatin on NO synthesis in neonatal rat cardiac fibroblasts induced by aldosteroneObjective: To investigate the changes of NO synthase and regulation mechanisms in neonatal rat cardiac fibroblasts treated with lovastatin and/or aldosterone.Methods: Cell proliferation was assessed by MTT colorimetric assay, NO contents and iNOS activity were detected by Nitric acid reductase method and spectrophotometry respectively. Additionally, RT-PCR was used to estimate the iNOS mRNA expression; the protein expression of iNOS was detected by Western blotting.Results: 1. NO contents and iNOS activity decreased in a dose-dependent manner with different concentrations of aldosterone (P<0.05). In concentrations of 10'll-10"7mol/L, NO contents and iNOS activity in cardiac fibroblasts were (25.68 + 1.61, 25.41 + 1.75, 23.15 + 1.61, 20.51 + 1.48, and 16.62+1.27, respectively) umol/L and (4.79 + 0.38, 4.36 + 0.62, 3.97 + 0.59, 3.64 + 0.82, and 3.08 + 0.75, respectively) U/mL, after aldosterone was given for 24 hours. The down-regulation of NO contents and iNOS activity in 10"7mol/L aldosterone was completely reversed by 10"6mol/L spironolactone[(29.76 + 2.16)umol/L, (5.53 + 0.83) U/mL].Aldosterone decreased iNOS mRNA expression of cardiac fibroblasts in a dose-dependent manner. The OD score in aldosterone group were 0.569 + 0.023 (10"10mol/L), 0.476+0.006 (10"9mol/L), 0.385 + 0.012 (10'8mol/L), and 0.297 + 0.008 (10'7mol/L), with 0.608 + 0.032 in control group. The down-regulation effect of iNOS mRNA expression of 10⁷ mol/L aldosteronewas reversed by lO^mol/L spironolactone (0.606+0.053).Aldosterone decreased iNOS protein expression of cardiac fibroblasts in a dose-dependent manner, compared with control group (100.0). The OD score in aldosterone group were 84.6+7.2 (10'10mol/L), 68.3 + 5.7 (10'9mol/L), 49.5+2.9 (10-8mol/L), and 33.4 + 4.1 (10*7mol/L), all significantly lower than that in control group. The down-regulation effect of iNOS protein expression of 10 ⁷ mol/L aldosterone (33.4 + 4.1) was reversed by 10"6mol/L spironolactone (97.4 + 2.8) .2. Compared with 10⁷mol/L aldosterone group, in concentrations of (10⁸-10"5) mol/L, NO contents and iNOS activity were 19.96+1.92, 25.46+1.74, 28.91 + 2.13, and 35.56+1.92) umol/L and (3.99 + 0.63, 4.62 + 0.62, 5.41+0.97, and 6.62 +0.8 l)U/mL, respectively, 24 hours after lovastatin was given. NO contents and iNOS activity increased in a dose-dependent manner with different concentrations of lovastatin (PO.05).Lovastatin increased iNOS mRNA expression of cardiac fibroblasts in a dose-dependent manner, the OD score in lovastatin group were 0.569 + 0.023 (10-8mol/L), 0.476 + 0.006 (10-7mol/L), 0.385 + 0.012 (lO^mol/L), and 0.297 + 0.008 (10"5mol/L), with 0.297 + 0.008 in aldosterone control group.Lovastatin increased iNOS protein expression of cardiac fibroblasts in a dose-dependent manner. Compared with control group (100.00), 10'7mol/L aldosterone control group was 33.37 + 4.14, the OD score in lovastatin group were 52.73+ 4.04(10-8mol/L), 80.87+ 3.50(10"7mol/L), 121.13 + 6.90(10"6mol/L), 176.47 + 6.50 (10'5mol/L)3. Compared with 10⁵ mol/L lovastatin and 10⁷mol/L aldosterone group, NO contents and iNOS activity in 10'3mol/L MVA group and 5 umol/L FPP group were (16.65+1.44, 17.45+ 1.56) umol/L and (3.35 + 0.74, 3.17 + 0.64) U/mL after MVA and FPP were given for 24 hours. The OD score of iNOS mRNA expression in 10'3mol/L MVA group and 5 umol/L FPP group were 0.309 ± 0.014, 0.313 ± 0.017. Compared with 10⁷mol/L aldosterone control group(100), the OD score of iNOS protein expression in 10'3mol/L MVA group and 5 umol/L FPP group were 110.23 ±6.13, 108.77 ± 4.43.4. With aldosterone concentrations of 10"10-10'7mol/L for 24 hours, there were significant positive correlations between NO contents and iNOS activity, iNOS activity and iNOS protein expressions, iNOS protein expressions and iNOS mRNA expressions (r=0.837, 0.815, 0.956, respectively, PO.01). There were significant negative correlations between NO contents and A490 values ( r =-0.857, p<0.01).Cultured with 10⁵ mol/L lovastatin and 10^<sup>7mol/L aldosterone for 24 hours, there were significant positive correlation between NO contents and iNOS activity, iNOS activity and iNOS protein expressions, iNOS protein expressions and iNOS mRNA expressions (r=0.826^ 0.896, 0.986, respectively, P<0.01) in cardiac fibroblasts. There were significant negative correlation between NO contents and A490 values (r=-0.908, p<0.01).Conclusion: Aldosterone down-regulated iNOS-NO system activity in neonatal rat CFs via MR in a concentration-dependent manner, which could be related to the proliferation of cardiac fibroblasts induced by aldosterone. Lovastatin up-regulated iNOS-NO system activity via MYA pathway in a concentration-dependent manner, which could be related to the inhibition of lovastatin in cardiac fibroblasts proliferation induced by aldosterone. Lovastatin and aldosterone might affect cardiac fibroblasts proliferation via regulating iNOS-NO system activity.Part 4: Ras-MAPK signal transduction mechanisms in the inhibitive effects of lovastatin on cardiac fibroblasts proliferation in neonatal rat induced by aldosteroneObjective: To provide experimental evidence for Ras-MAPK signal regulating cardiac fibroblasts proliferation induced by lovastatin and aldosterone.Methods: The DNA synthesis of cardiac fibroblasts was determined byH-Thymidine (3H-TdR) incorporation, Western blotting analysis detectsphosphorylated p42/44 MAPK protein expression. The expressions ofphosphorylated p42/44 MAPK in cardiac fibroblasts were detected byimmunohistochemistry method.Results: 1. Aldosterone promoted the proliferation of cardiac fibroblasts in a dose dependent manner, PD98059 in concentration of 10⁵mol/L [(782 + 152)counts.min"'] reversed the effects of 10⁷ mol/L aldosterone [(1879 + 169)counts-min*1].2. Taken serum-free DMEM as control group (OD score=l), 10"7 mol/L aldosterone significantly increased the levels of the activated (phosphorylated) P42/44 MAPK protein, and the levels of peak of activity of phospho-P42/44MAPK (8.11+0.46 fold at 4 hours). By 24 houes, the relative levels of phospho-P42/44MAPK did not differ from basal levels at time 0 ( 1.22 + 0.09 fold), compared with control group.3. Aldosterone with a concentration of 10⁷ mol/L significantly increased the phosphorylated P42/44 MAPK protein levels at 4 hours (8.11 + 0.46), while PD98059 in a concentration of 10⁵mol/L significantly decreased the phosphorylated P42/44 MAPK protein levels (1.21+0.10, PO.01). The result of p44 and p42 were same.4. Taken OD score of blank control group as 1, the OD score in 10"7 mol/L aldosterone group was 8.12 + 0.54, 10⁵mol/L lovastatin group was 2.42 + 0.47. Lovastatin in a concentration of 10⁵mol/L decreased phosphorylated P42/44 MAPK protein levels induced by 10"7mol/L aldosterone (PO.01).The OD score of phosphorylated P42/44 MAPK protein stimulated by 10" 3mol/L MVA group and 5 umol/L FPP group were 7.59 ± 0.37 and 7.70 ± 0.49, higher than that in 10"5mol/L lovastatin (2.42 + 0.47, /?<0.01). Compared with 10"7 mol/L aldosterone group, there was no significant difference (P >0.05).Lovastatin, MVA and FPP had no effects on nonphosphorylated MAPK.5. After cardiac fibroblasts were rested for 24 hours and stimulated by 10" 7mol/L aldosterone, phosphorylated P42/44 MAPK appeared in cytoplasm at 15 min, starting to move to the nucleus as early as 30 min and becoming predominantly nuclear at 2 hours, disappeared from nuclear at 4 hours. Lovastatin of 10⁵mol/L could block MAPK activity and nuclear translocation induced by 10"7 mol/L aldosterone.Conclusion: Aldosterone could stimulate MAPK activity and translocation to the nucleus, which led to cell proliferation. Lovastatin can down-regulate phosphorylated MAPK protein levels induced by aldosterone, that can be reversed by MVA and FPP. Lovastatin can block MAPK activity, nuclear translocation and proliferation in cardiac fibroblasts induced by aldosterone. Part 5: Effects of Aldosterone and lovastatin on the production of ET, NO and TGF-fli from myocardial fibroblasts in neonatal ratObjective: To investigate the effects of aldosterone and lovastatin on the production of ET, NO and TGF-0, from CFs.Methods: The changes of ET, NO and TGF^Pi production from CFs were examined by radioimmunoassay, nitrate reductase-dependent assay and by ELISA, respectively.Results: 1. Cultured with aldosterone in concentrations of 10'l0-10"7mol/L for 72 hours, ET levels from cardiac fibroblasts were 145.68 + 8.62, 155.17 + 13.96, 185.17+17.36, and 213.45 + 16.82 pg/ml, TGF-0, were 212.50 + 14.80, 245.22+12.93, 286.93 + 18.30, and 316.19+ 17.48 pg/ml, respectively. Aldosterone increased ET and TGF-Pi synthesis in cardiac fibroblasts in a concentration-dependent manner. NO contents were 26.90+1.20, 22.46+ 1.57, 20.71 + 1.73, 16.73 + 1.62 umol/L, respectively, Aldosterone also inhibited NO synthesis in cardiac fibroblasts in a concentration-dependent manner.The regulation of NO contents, ET and TGF-pi secretion in cardiac fibroblasts with 10'7mol/L aldosterone could completely be reversed by 10⁶mol/L spironolactone.2. Cultured with lovastatin in concentrations of 10"8-10"5mol/L for 72 hours, the ET levels from cardiac fibroblasts were 195.65+15.48, 171.57+17.04, 145.04+14.07, and 129.86 + 8.95 pg/ml, TGF-p, were 270.88+12.38, 257.74 + 13.50, 231.52 + 9.57, and 199.38+11.35 pg/ml, respectively. Lovastatin decreased ET and TGF-Pi synthesis in cardiac fibroblasts in a concentration-dependent manner. Lovastatin increased NO contents synthesis in cardiac fibroblasts in a concentration-dependent manner with NO contents of 19.20+1.36, 21.82+1.56, 25.46+1.24, 27.91 + 1.01 umol/L, in...