Effects Of Atorvastatin And Pravastatin On The Expression Of ET-1/ET-AR And TGF-β1/TGF-βRⅠ Systems In Cultured Neonatal Cardiac Fibroblasts Induced By Aldosterone

Cardiac fibrosis exists in lots of cardiovascular diseases, leading to ventricular dysfunction, cardiac arrhythmia, and even cardiac sudden death. Thus, the researches of cardiac fibrosis have important theoretical and practical importance. Cardiac fibroblasts, embeded between cardiac myocytes, represented 25% of the volume and 70% of the total cell number of the heart, activated after myocardial infarction, are the most source of cardiac extracellular matrix. Accumulating lines of evidence revealed that endothelin-1/endothelin subtype A receptor and transforming growth factor-β1/transforming growth factor-βreceptor types I (ET-1/ET-AR and TGF-β1/ TGF-βRⅠ) systems play a crucial role in cardiac fibrosis. Therefore, the inhibition of the overexpression of ET-1/ET-AR and TGF-β1/ TGF-βRⅠsystems is an important strategy for anti-cardiac fibrosis therapy.Aldosterone is located in cardiovascular tissues, acting in a paracrine/autocrine manner. Normal cardiac (atrial and ventricular) tissue contains abundant, specific, high-affinity receptors, mineralocorticoid receptors (MR), for aldosterone, which suggests a completely independent system of aldosterone in the cardiovascular system. Recently, aldosterone has been shown to stimulate cardiomyocytes hypertrophy and cardiac fibrosis which always accompany with conditions such as myocardial infarction and chronic heart failure. However, several in vivo studies revealed that the increase in cardiac collagen is not immediate and needs a few weeks to be observed, suggesting that the profibrotic effect of aldosterone was not direct. Accumulating lines of evidence revealed that ET-1/ET-AR and TGF-β1/ TGF-βRⅠsystems play an important role in cardiac fibrosis. Thus, the underlying mechanism of aldosterone is not well-described, further insights into its effects may have important therapeutic implications.3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to inhibit cardiac fibroblast proliferation and collagen production. However, the beneficial effects are incompletely explained by the anti-atherosclerosis. Recent studies have indicated that statins have additional benefits beyond their well-known lipid lowering effect on anti-atherosclerosis, which may involve the down-regulation of ET-1/ET-AR and TGF-β1/ TGF-βRⅠsystems. Recent studies suggest that statins exert pleiotropic effects by blocking the conversion of HMG-CoA to mevalonate. Accumulating evidence shows mevalonic acid is the precursor not only for cholesterol synthesis, but also for many isoprenoids. Studies have demonstrated that these intermediates are essential mitogenic signaling molecules act on small GTP-binding proteins, Rho and Ras. This signal transduction pathway contributes to gene expression, separated from that of statins on lipids. Studies in experimental animal models provide support for beneficial actions of statins directed toward cardiac fibroblasts. Treatment with statins reduces myocardial remodeling, fibrosis, collagen synthesis, and prolongs survival in models of myocardial injury accompanied by anti-inflammatory actions. However, the cell and molecular biological mechanisms and signal transduction passway are not clear. ET-1/ET-AR systemInvestigations showed that ET-1 was released from cardiac fibroblasts, and acted upon heart cells through the G-protein–coupled receptor subtype, ET-AR. There is increasing evidence that the potent vasoconstrictor peptide ET-1 has been implicated in the pathogenesis of heart failure, through inducing hypertrophy of cardiomyocytes, cardiac fibroblasts proliferation and collagen synthesis. However, the effects of aldosterone on the expression of ET-1/ET-AR system in cultured neonatal rat cardiac fibroblasts have not been reported. We therefore conducted the current study to test the hypothesis that aldosterone would up-regulate ET-1/ET-AR system in cultured neonatal rat cardiac fibroblasts. Recently, statins demonstrated beneficial anti-remodeling effects associated with a reduction of myocardial ET-1. Given the central role of ET-1/ET-AR system in cardiac fibrosis, we wondered whether the effects of statins are mediated by regulating the expression of ET-1/ET-AR system. We therefore conducted the present study to test the effects of atorvastatin and pravastatin on the expression of ET-1/ET-AR system in cultured neonatal cardiac fibroblasts stimulated with aldosterone from mRNA expression, protein synthesis, and secretion aspects.TGF-β1/TGF-βRⅠsystemTGF-β1 plays an important role in modulating fibroblast phenotype and gene expression, promoting extracellular matrix deposition in the infarct by up-regulating collagen and fibronectin synthesis and by decreasing matrix degradation. TGF-β1 transduces its signal from the membrane to the nucleus through distinct combinations of transmembrane type I and type II serine. Phosphorylation of the TGF-βRI activates the type I receptor kinase domain, which then propagates downstream intracellular signals. However, the effects of aldosterone on the expression of TGF-β1/TGF-βRⅠsystem in cultured neonatal rat cardiac fibroblasts have not been reported. We therefore conducted the current study to test the hypothesis that aldosterone would up-regulate TGF-β1/TGF-βRⅠsystem in cultured neonatal rat cardiac fibroblasts. Recent evidence showed that statins treatment is helpful to reduce myocardial fibrosis, which was accompanied by suppressed TGF-β1 level. Considering the importance of TGF-β1 overproduction in cardiac disease, we hypothesized that statins may alter pathological remodeling in heart failure by influencing the expression of TGF-β1. To test this hypothesis, we investigated the effects of atorvastatin and pravastatin on the expression of TGF-β1/ TGF-βRI system in cultured cardiac fibroblasts induced by aldosterone from mRNA expression, protein synthesis, and secretion aspects.Mitogen-activated protein kinasesMitogen-activated protein kinases (MAPK) locate in cardiac fibroblasts, are a family of serine/threonine protein kinases that mediate cardiac fibrosis responses to external stress signals. Upon activation of the MAPK, transcription factors present in the cytoplasm or nucleus are phosphorylated and activated, leading to expression of target genes, regulating protein sythesis and cardiac fibroblasts proliferation, resulting in cardiac fibrosis. Statins exert pleiotropic effects by blocking the conversion of HMG-CoA to mevalonate. Accumulating evidence shows mevalonic acid is the precursor for many isoprenoids, essential mitogenic signaling molecules act on small GTP-binding proteins, such as Rho and Ras. Statins can reduce cardiac fibrosis, by inhibiting the activation of Ras-MAPK. Thus, MAPK connects statins and aldosterone with cardiac fibrosis.The present study investigated the effects of aldosterone on the expression of ET-1/ET-AR and TGF-β1/TGF-βRⅠsystems in cultured neonatal rat cardiac fibroblasts with radioimmunoassay, ELISA, flow cytometry, Western blot, and RT-PCR methods. Meanwhile, the current study investigated the effects of atorvastatin and pravastatin on the expression of ET-1/ET-AR and TGF-β1/TGF-βRⅠsystems in cultured neonatal cardiac fibroblasts induced by aldosterone. Thereafter, the present study investigated the effects of aldosterone, atorvastatin and pravastatin on the expression of p44/42 MAPK and phospho-p44/42 MAPK in cultured neonatal rat cardiac fibroblasts with Western blot analysis. In summary, the present study investigated the effects of aldosterone, atorvastatin and pravastatin on the expression of ET-1/ET-AR and TGF-β1/TGF-βRⅠsystems in cultured neonatal cardiac fibroblasts from mRNA expression, protein synthesis and secretion aspects, demonstrated their cell and molecular biological mechanisms and signal transduction passway, and provided new ideas and experimental evidences for anti-myocardial fibrosis therapy. The present study included five sections as following:Section 1: Effects of aldosterone on the expression of ET-1/ET-AR system in cultured neonatal cardiac fibroblastsObjective: To investigate the effects of aldosterone on the expression of ET-1/ET-AR system in cultured neonatal rat cardiac fibroblasts.Methods: Cardiac fibroblasts were isolated by trypsin digestion method. ET concentration in conditioned medium was measured by radioimmunoassay, intracellular ET-1 level was evaluated by immunofluorescence assay, and the expression of preproendothelin-1 (ppET-1) mRNA was detected and quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) method.Results: 1. Effect of aldosterone on ET secretion: ET secretion of cardiac fibroblasts maintained low levels in the control group, whereas, aldosterone dose-dependently stimulated ET secretion (19.9%, 45.9%, and 84.7%; P<0.05, P<0.01, P<0.01, respectively). Pre-incubation with spironolactone, an antagonist for MR, abolished aldosterone-induced ET secretion. However, as comparison with controls, incubation with spironolactone alone had no influence on cardiac fibroblast ET secretion.2. Effect of aldosterone on ET-1 synthesis: The fluorescent intensity of ET-1 in cardiac fibroblasts was significantly increased in a concentration-dependent fashion (42.2%, 100.8%, and 201.9%; P<0.05, P<0.01, P<0.01, respectively). The aldosterone-induced increase in ET-1 expression was inhibited by co-incubation with spironolactone in the medium. In contrast, ET-1 expression was no different between the spironolactone alone treatment group and control group.3. Effect of aldosterone on ppET-1 and ET-AR mRNA expression:3.1 Aldosterone significantly increased ppET-1 and ET-AR mRNA expression as early as 2 h, and reach maximal level at 4 h, then decreased. Thus, 4 h was selected as incubation time for various concentrations of aldosterone.3.2 Aldosterone dose-dependently increased ppET-1 mRNA expression, about 31.9% by 10-9 mol/L and 125.2% by 10-7 mol/L (P<0.05, P<0.01, respectively). Meanwhile, ET-AR mRNA level was induced by aldosterone in the similar manner, about 34.3% by 10-9 mol/L and 163.9% by 10-7 mol/L (P<0.05, P<0.01, respectively).3.3 To investigate the effect of MR on aldosterone-activated ppET-1 and ET-AR mRNA expression, the cardiac fibroblasts was pre-incubated with spironolactone. The results showed that pretreatment of spironolactone strongly inhibited aldosterone-induced the increase in ppET-1 and ET-AR mRNA levels. In addition, incubation with spironolactone alone had no significant effect on ppET-1 and ET-AR mRNA expression.Concusion: Our results directly demonstrate that aldosterone activates the expression of ET-1/ET-AR system and that this effect is blocked by spironolactone, an inhibitor of the MR, in neonatal rat cardiac fibroblasts. Thus, the ET-1/ET-AR pathway exists within the cardiac fibrosis induced by aldosterone.Section 2:Effects of aldosterone on the expression of TGF-β1/ TGF-βRⅠsystem in cultured neonatal cardiac fibroblastsObjective: To investigate the effects of aldosterone on the expression of TGF-β1/ TGF-βRⅠsystem in cultured neonatal rat cardiac fibroblasts.Methods: Cardiac fibroblasts were isolated by trypsin digestion method. TGF-β1 concentration in conditioned medium was measured by ELISA, intracellular TGF-β1 level was evaluated by Western blot, and the expression of TGF-β1/ TGF-βRⅠmRNA were detected and quantified using RT-PCR method.Results: 1. Effect of aldosterone on TGF-β1 secretion: TGF-β1 secretion of cardiac fibroblasts maintained low levels in the control group, whereas, aldosterone dose-dependently stimulated TGF-β1 secretion (P=0.038, P<0.01, P<0.01, respectively). Pre-incubation with spironolactone, an antagonist for MR, abolished aldosterone-induced TGF-β1 secretion. However, as comparison with controls, incubation with spironolactone alone had no influence on cardiac fibroblast TGF-β1 secretion.2. Effect of aldosterone on TGF-β1 synthesis: TGF-β1 in cardiac fibroblasts was significantly increased in a concentration-dependent fashion (P =0.071, P<0.01, P<0.01, respectively). The aldosterone-induced increase in TGF-β1 expression was inhibited by co-incubation with spironolactone in the medium. In contrast, TGF-β1 expression was no different between the spironolactone alone treatment group and control group.3. Effect of aldosterone on TGF-β1 and TGF-βRⅠmRNA expression: 3.1 Aldosterone significantly increased TGF-β1 mRNA expression as early as 4 hour, and reach maximal level at 8 hour. Aldosterone also transiently increased the expression of TGF-βRⅠmRNA in the similar fashion. And then, 8 hour was selected as incubation time for various concentrations of aldosterone.3.2 Aldosterone dose-dependently increased TGF-β1 mRNA expression (P<0.01, P<0.01, P<0.01, respectively). Meanwhile, TGF-βRⅠmRNA level was induced by aldosterone in the similar manner (P=0.189, P<0.01, P<0.01, respectively).3.3 To investigate the effect of MR on aldosterone-activated TGF-β1 and TGF-βRⅠmRNA expression, the cardiac fibroblasts was pre-incubated with spironolactone. The results showed that pretreatment of spironolactone strongly inhibited aldosterone-induced the increase in TGF-β1 and TGF-βRⅠmRNA levels. In addition, incubation with spironolactone alone had no significant effect on TGF-β1 and TGF-βRⅠmRNA expression.Conclusion: Our results directly demonstrate that aldosterone activates the expression of TGF-β1/TGF-βRⅠsystem and the response to aldosterone appears to require MR, in neonatal rat cardiac fibroblasts.Aldosterone-mediated stimulation of TGF-β1/TGF-βRⅠsystem could aggravate cardiac function under conditions such as occur with heart failure. Thus, aldosterone may in pathological states exert profibrotic effects through this pathway, and the change in TGF-β1 and TGF-βRⅠobserved could be connected to the salutatory effects of spironolactone.Section 3:Effects of atorvastatin and pravastatin on the expression of ET-1/ET-AR system in cultured neonatal cardiac fibroblasts induced by aldosteroneObjective: To investigate the effects of atorvastatin and pravastatin on the expression of ET-1/ET-AR system in cultured neonatal rat cardiac fibroblasts induced by aldosterone.Methods: Cardiac fibroblasts were isolated by trypsin digestion method. ET concentration in conditioned medium was measured by radioimmunoassay, intracellular ET-1 level was evaluated by immunofluorescence assay, and the expression of ppET-1 and ET-AR mRNA was detected and quantified using RT-PCR method.Results: 1. Effects of statins on aldosterone-induced ET secretion: Aldosterone (10-7 mol/L) significantly stimulated ET secretion, which was inhibited by atorvastatin (10-6, 10-5, 10-4 mol/L) in a concentration-dependent manner (16.7%, 30.3%, and 40.2%; P<0.01, respectively). Similarly, pravastatin (10-5, 10-4, 10-3 mol/L) inhibited the effect (11.4%, 27.1%, and 38.1%; P<0.01, respectively). When added with mevalonate, the ET level reduced by atorvastatin (10-4 mol/L) and pravastatin (10-3 mol/L) recovered to the level similar to the group treated with aldosterone.2. Effect of statins on aldosterone-induced ET-1 synthesis: ET-1 was significantly up-regulated by aldosterone (10-7 mol/L). The increased level of ET-1 was dose-dependently reduced when the cells were pretreated with atorvastatin (36.2%, 49.9%, and 63.9%; P<0.01, respectively) or pravastatin (21.7%, 44.7%, and 57.2%; P<0.05, P<0.01, P<0.01, respectively). Similarly, our data again showed a recovery of ET-1 expression in the presence of mevalonate.3. Effect of statins on aldosterone-induced ppET-1 and ET-AR mRNA expression: Aldosterone stimulated ppET-1 mRNA expression, but was inhibited by atorvastatin (about 22.9% by 10-6 mol/L and 52.3% by 10-4 mol/L (P<0.05, P<0.01, respectively) or pravastatin (about 15.3% by 10-5 mol/L and 51.2% by 10-3 mol/L (P<0.05, P<0.01, respectively) in a concentration-dependent fashion. Similarly, atorvastatin (10-6, 10-5, 10-4 mol/L) or pravastatin (10-5, 10-4, 10-3 mol/L) resulted in the decrease of aldosterone-induced ET-AR mRNA expression (about 24.3% by 10-6 mol/L and 61.2% by 10-4 mol/L in atorvastatin, P<0.05, P<0.01, respectively; about 13.5% by 10-6 mol/L and 57.9% by 10-4 mol/L in pravastatin, P=0.08, P<0.01, respectively). To investigate the pathway of atorvastatin and pravastatin inhibited aldosterone-activated ppET-1 and ET-AR mRNA expression, the cardiac fibroblasts was pre-incubated with mevalonate. The results showed that mevalonate effectively restored aldosterone–induced the increase in ppET-1 and ET-AR mRNA expressions.Conclusion: We demonstrate that aldosterone activates the expression of ET-1/ET-AR system and that this effect is blocked by pravastatin and atorvastatin through the mevalonate pathway in neonatal rat cardiac fibroblasts. Given that pathological ET-1/ET-AR system was an important contributor to the adverse cardiac remodeling, the current study supported the hypothesis that statins regulated cardiac structure via a mechanism that involves the ET-1/ET-AR system rather than their lipid lowering properties.Section 4:Effects of atorvastatin and pravastatin on the expression of TGF-β1/ TGF-βRⅠsystem in cultured neonatal cardiac fibroblasts induced by aldosteroneObjective: To investigate the effects of atorvastatin and pravastatin on the expression of TGF-β1/ TGF-βRⅠsystem in cultured neonatal rat cardiac fibroblasts induced by aldosterone.Methods: Cardiac fibroblasts were isolated by trypsin digestion method. TGF-β1 concentration in conditioned medium was measured by ELISA, intracellular TGF-β1 level was evaluated by Western blot, and the expression of TGF-β1/ TGF-βRⅠmRNA were detected and quantified using RT-PCR method.Results: 1. Effects of statins on aldosterone-induced TGF-β1 secretion: Aldosterone (10-7 mol/L) significantly stimulated ET-1 secretion, which was inhibited by atorvastatin (10-6, 10-5, 10-4 mol/L) in a concentration-dependent manner (P=0.113, P<0.01, P<0.01, respectively). Similarly, pravastatin (10-5, 10-4, 10-3 mol/L) inhibited the effect (P=0.214, P<0.01, P<0.01, respectively). When added with mevalonate, the ET-1 level reduced by atorvastatin (10-4 mol/L) and pravastatin (10-3 mol/L) recovered to the level similar to the group treated with aldosterone.2. Effect of statins on aldosterone-induced TGF-β1 synthesis: TGF-β1 was significantly up-regulated by aldosterone (10-7 mol/L). The increased level of TGF-β1 was dose-dependently reduced when the cells were pretreated with atorvastatin (P=0.058, P<0.01, P<0.01, respectively) or pravastatin (P=0.15, P<0.01, P<0.01, respectively). Similarly, our data again showed a recovery of TGF-β1 expression in the presence of mevalonate.3. Effect of statins on aldosterone-induced TGF-β1 and TGF-βRⅠmRNA expression: Aldosterone stimulated TGF-β1 mRNA expression, but was inhibited by atorvastatin (P<0.01, respectively) or pravastatin (P<0.01, respectively) in a concentration-dependent fashion. Similarly, atorvastatin (10-6, 10-5, 10-4 mol/L) or pravastatin (10-5, 10-4, 10-3 mol/L) resulted in the decrease of aldosterone-induced TGF-βRⅠmRNA expression (P<0.01, respectively). To investigate the pathway of atorvastatin and pravastatin inhibited aldosterone-activated TGF-β1 and TGF-βRⅠmRNA expression, the cardiac fibroblasts was pre-incubated with mevalonate. The results showed that mevalonate effectively restored aldosterone–induced the increase in TGF-β1 and TGF-βRⅠmRNA expressions.Conclusion: These results showed that atorvastatin and pravastatin inhibit the expression of TGF-β1/TGF-βRⅠsystem in aldosterone-induced cardiac fibroblasts by a MAV mechanism. Because pathological elevated TGF-β1/TGF-βRⅠsystem is thought to play an important role in cardiac remodeling, these data suggested a new possible mechanism by which statins attenuated cardiac fibrosis progression.Section 5:Effects of p44/42 MAPK on the secretion of ET and TGF-β1 in cultured neonatal cardiac fibroblastsObjective: To investigate the effects of p44/42 MAPK on the secretion of ET and TGF-β1 in cultured neonatal cardiac fibroblasts.Methods: Cardiac fibroblasts were isolated by trypsin digestion method. ET and TGF-β1 concentration in conditioned medium was measured by radioimmunoassay and ELISA, respectively. Intracellular p44/42 MAPK level and phospho-p44/42 MAPK were evaluated by Western blot.Results: 1. Effect of PD 98059 on ET secretion: ET secretion of cardiac fibroblasts maintained low levels in the control group, whereas, aldosterone stimulated ET secretion (P<0.01). Pre-incubation with PD 98059, an antagonist for p44/42 MAPK, abolished aldosterone-induced ET secretion(P<0.01).2. Effect of PD 98059 on TGF-β1 secretion: TGF-β1 secretion of cardiac fibroblasts maintained low levels in the control group, whereas, aldosterone stimulated TGF-β1 secretion (P<0.01). Pre-incubation with PD 98059, an antagonist for MAPK, abolished aldosterone-induced TGF-β1 secretion(P<0.01).3. Effect of aldosterone on the expression of p44/42 MAPK in cultured cardiac fibroblasts: The expression of phospho-p44/42 MAPK in cardiac fibroblasts was significantly increased by aldosterone (P<0.01). The aldosterone-induced increase in phospho-p44/42 MAPK expression was inhibited by co-incubation with spironolactone in the medium(P<0.01). However, the expression of total p44/42 MAPK was not affected by aldosterone(P>0.05).4. Effect of atorvastatin and pravastatin on the expression of p44/42 MAPK in cultured cardiac fibroblasts induced by aldosterone: Phospho-p44/42 MAPK was significantly up-regluated by aldosteron(eP<0.01). The increased level of phospho-p44/42 MAPK was reduced when the cells were pretreated with atorvastatin or pravastatin(P<0.01). Similarly, our data again showed a recovery of phospho-p44/42 MAPK expression in the presence of mevalonate(P<0.01).Concusion: Our results demonstrate that the p44/42 MAPK pathway may exist within the cardiac fibrosis induced by aldosterone. Meanwhile, these data suggested a new possible mechanism by which statins attenuated cardiac fibrosis progression.