| Cardiac hypertrophy and fibrosis develop in response to various cardiovascular diseases.Our laboratory has had a longstanding interest in the areas of angiotensin ?(Ang ?)-induced fibrosis and cardiac dysfunction.We have previously reported that pharmacological inhibition of the angiotension ? 1 receptor(AT1R)significantly reduces cardiac fibrosis and improves cardiac function by AT1 R blockers(ARBs)and angiotensin converting enzyme inhibitors(ACEi).Although ARBs and ACEi have been well documented as treatment options for patients with hypertension and cardiovascular diseases,limitations associated with the use of these drugs have also been indentified.Hence,adjunctive therapies to avoid these unfavorable actions and promote cardiac recovery through modulating the Ang ? system still remain an active area of investigation.Edaravone is a free radical scavenger and was used to therapy acute ischemic stroke.Many studies have demonstrated that Edaravone exerts beneficial effects on the cardiac hypertrophy or ischemic/reperfusioin injury.However,it is unknown whether edaravone had a direct effect on the pathogenesis of Ang ?-initiated cardiac injury.Aldosterone(ALDO)is a steroid hormone and is formed through the induction of Ang ?.It is known that ALDO biosynthesis occurs in the mitochondria of the zona glomerulosa in the adrenal through aldosterone synthase(AS).Moreover,steroidogenic acute regulatory protein(St AR)facilitated the step that cholesterol transfers from outer mitochondrial membrane to the inner mitochondrial membrane upon ALDO synthesis.The underlying mechanisms of St AR/AS upon myocardial fibrosis remain unclear.Therefore,in the present study,we tested the hypotheses that the edaravone inhibits Ang ? induced myocardial fibrosis and the Ang ?/AT1R/St AR/AS/ALDO signaling pathways participate in Ang ?/ADLO-induced myocardial fibrosis.We selected two rat models of transverse aortic constriction and subcutaneous Ang ? infusion to investigate the signaling mechenisms of cardiac fibrosis.The experimental methods including Western-blot,immunohistochemical staining,measurement of hemodynamics and echocardiography were used to detect the cardiac fibrosis and function.A complete understanding of the signaling events regulating Ang ?-induced ALDO biosynthesis in myocardial fibrosis may allow the identification of novel targets for clinical therapeutic interventions.Part 1: Edaravone improves cardiac hypertrophy and dysfunction by regulating AT1R?AT2R/ACE2 signal in transverse aortic constriction ratsObjective: To demonstrate whether regulation of AT1R?AT2R/ACE2 expression after treatment with Edaravone is related with improvement of cardiac hypertrophy and dysfunction in transverse aortic constriction rats.Methods:1.Experiment group: Male Sprague Dawley rats were produced transverse aortic constriction(TAC)surgery.The experimental period is lasting 8 weeks.Rats were randomly divided into 4 groups(n=6/each group).(1)Sham: rats underwent the same surgical procedure without banding the aorta;(2)TAC: rats were subjected to TAC for 8 weeks.(3)Edara: TAC plus edaravone,rats received an intraperitoneal injection of edaravone at a dose of 10mg/kg/day after TAC for 8 weeks.(4)Telmi: TAC plus telmisartan,rats were administered telmisartan via gastric gavage at a dose of 10mg/kg/day after TAC for 8 weeks.2.At the end of experiment,the chest was opened and the heart was rapidly removed.The heart body weight index(HW/BW)was calculated as heart weight divided by body weight(mg/g tissue).MSA in series was quantitatively analyzed after 6?m of tissue slides were stained with hematoxylin and eosin.3.The hearts were homogenized in phosphate buffer.The level of malonaldehye(MDA)and superoxide dismutase(SOD)activity were measured with MDA and SOD detection kit.4.Freshly frozen tissue samples were homogenized and protein concentration was measured.AT1 R and AT2 R protein level were analyzed by Western-blot assay.5.Localization and protein expression levels of ACE2 were evaluated using immunohistochemical staining in paraffin-embedded tissue section.6.At the end of experiment,the polyethylene catheter was cannulated into left ventricular through the right carotid artery and the cardiac performance was measured.7.A two-dimensional(2D)guided M-mode ultrasound system(Vivid 7)was used to assess the left ventricular systolic and diastolic function.Results:1.Chronic elevation of afterload in the left ventricle caused significant increase in HW/BW ratio and MSA size in series.Treatment with edaravone or telmisartan comparatively reduced the HW/BW ratio and MSA size.2.Constriction of ascending artery caused a significant upregulation in expression of AT1 R and downregulation of AT2 R.Along with these changes,ACE2 expression and SOD activity decreased and MOD content increased after TAC 8 weeks.After treatment with edaravone and telmisartan,protein level of the AT1 R was reduced and AT2 R was upregulated,as evidenced by the reduced ratio of AT1 over AT2 receptor(0.57±0.2 vs 3.16±0.39,p<0.05),the reduced expression of ACE2 in the intracardiac vessels and myocardium was reversed.Simultaneously,the reduced MDA content and increased SOD activity were detected in Edara and Telmi group.3.The cardiac systolic function decreased after TAC 8weeks.Relative to TAC group,cardiac systolic function was preserved,as shown by increased left ventricular systolic pressure(LVSP,204±51 vs 110±19mm Hg,p<0.05)and ejection fraction(EF,82%±3% vs 60%±5%,p<0.05)after intraperitoneal injection of edaravone or oral administration of telmisartan.Conclusion: Data suggest that transverse aortic constriction induces cardiac hypertrophy and dysfunction through mechamisms independed of blood pressure.Edaravone treatment ameliorates cardiac hypertrophy and improves left ventricular function,suppresses production of reactive oxygen species(ROS)through inhibiting AT1R-mediated signaling pathways.Part 2: Edaravone improves the cardiac fibrosis after transverse aortic constriction through reducing the number of accumulated macrophages,expression of TGF-?1/SMADs and proliferation of myofibroblasts.Objective: The present study aimed to investigate whether edaravone improves cardiac fibrosis after TAC through reducing the number of accumulated macrophages,expression of TGF-?1/SMADs and proliferation of myofibroblasts.Methods:1.The rats were randomly divided into 4 groups,Sham,TAC,TAC plus edaravone and TAC plus telmisartan as described in Study part 1.2.The expression and localization of macrophage,?-SMA myofibroblasts and Collagen I in myocardium and perivascular region were analyzed using immunohistochemical staining method.3.The protein expression level of TGF-?1,Smad2,3,4,7 and Collagen ?I were detected by Western-blot.4.Myocardial fibrosis in 6?m paraffin-embedded tissue sections was determined using Masson's trichrome method.Results:1.TAC caused a significant increase of the number of macrophages in the intravascular region and interstitial myocardium.Administration of edaravone or telmisartan during TAC comparatively reduced the number of accumulated macrophages,expression of TGF-?1,and proliferation of myofibroblasts at week 8.2.Administration of edaravone or telmisartan comparatively reduced protein level of Smad2/3 and enhanced the level of Smad7 compared with TAC group.In accordance with the change of SMADs,the deposition of Collagen I not only occurred in the matrix of vascular endothelium,but also was excessively expressed in the myocardium.The production of fibrotic tissue as identified by Masson's trichrome staining was significantly augemented by TAC.Treatment with edaravone or telmisartan comparatively reduced the collagen deposition and fibrosis.Conclusion:These results indicated that transverse aortic constriction can induce myocardial fibrosis through mechamisms independed of blood pressure.Edaravone and telmisartan inhibits the collagen deposition and cardiac fibrosis by suppressing the AT1 R.The underlying mechanisms may be associated with an inhibition of TGF-?1/SMADs signaling pathways and decrease of accumulate macrophages and proliferation of myofibroblasts.Part 3: The role of locally produced aldosterone in the heart in the regulation of angiotensin ?-induced cardiac hypertrophy and fibrosisObjective: To demonstrate whether locally enhanced expression of aldosterone in the heart is associated with Ang ?-induced cardiac hypertrophy and fibrosis.Methods:1.Studies were performed on male Sprague-Dawley rats weighing 200 ± 10 g.Animals were randomized into one of the following treatment groups,n=6/ group.The procedures of rat tissue removement,tissue preservation and embedding tissues in paraffin blocks are same as described by part 1.(1)Sham: rats were infused with a saline pump.(2)Ang ?: rats were subjected to Ang ? infusion at a rate of 500ng/kg/min for 4 weeks.(3)Telmi: Ang? plus telmisartan,rats were administrated with telmisartan via gastric gavage at a dose of 10mg/kg/day during Ang ? infusion for 4 weeks.(4)Spiron: Ang? plus spironolactone,rats received an oral treatment of spironolactone at a dose of 100mg/kg/day during Ang ? infusion for 4 weeks.(5)T+S: rats were administrated with both telmisartan and spironolactone via gastric gavage during Ang ? infusion for 4 weeks.(6)ADX: rats were subjected to adrenalectomy and were provided with 0.9% saline in place of drinking water during Ang ? infusion for 4 weeks.2.Non-invasive blood pressure(NIBP)was measured in conscious condition using a NIBP system with a pulse transducer.3.MSA was quantitiatively analyzed after 6?m of tissue slides were stained with Masson's trichrome method.HW/BW was calculated as part 1 reported.4.The expression and localization of macrophages and ?-SMA myofibroblasts in myocardium and perivascular region were analyzed by immunohistochemical staining.5.The protein level of TGF-?1,total and phosphorylated forms of Smad2,3,Collagen I and ?I were detected by Western-blot.6.Myocardial fibrosis in 6?m paraffin-embedded tissue sections was determined using Masson's trichrome method.Results:1.Administration of spironolactone,telmisartan or adrenalectomy during Ang ? infusion reduced the increase of MSA and HW/BW.However,there still exist significant cardiac hypertrophy after adrenalectomy and demonstrated the locally enhanced expression of aldosterone in heart was more responsible for cardiac hypertrophy.2.The accumulation of macrophages,proliferation of fibroblasts and protein expression level of TGF-?1/Smad2?3 were suppressed in spironolactone,telmisartan or adrenalectomy groups.Consistent with the the above change,increased synthesis of Collagen I,?I and the production of fibrotic tissue in the perivascular area and interstitial myocardium were significantly inhibited by oral treatment with spironolactone or telmisartan.The myocardial fibrosis still significantly present even after adrenalectomy indicating the locally produced ALDO in heart was more important in Ang ?-mediated cardiac fibrosis.Conclusion:Aldosterone plays an important role in Ang ?-induced myocardiac fibrosis.Telmisartan that regulates the upstream AT1/St AR/AS signaling pathway inducing ALDO production,spironolactone that blocks the binding of ALDO with mineralocorticoid receptor(MCR),and adrenalectomy all decrease the cardiac fibrosis.The underlying mechanism may include that decreased number of accumulated macrophages and proliferation of fibroblasts,downregulation of TGF-?1/Smad2?3 signaling pathway.The ALDO locally produced in heart is more responsible for Ang ?-induced cardiac fibrosis.Part 4: The role of steroidogenic acute regulatory protein/aldosterone synthase in the regulation of Ang ? induced aldosterone production in heartObjective: To illustrate whether steroidogenic acute regulatory protein/aldosterone synthase involved in the regulation of enhanced expression of aldosterone in Ang ?-induced myocardial fibrosis.Methods:1.The animal model and groups were same as described in part 3.2.The protein expression level of AT1 R,StAR,AS,ERK1/2 and p38 were measured using Western-blot method.The aim is to demonstrate the upstream signaling molecular and regulative mechanism of Ang ? induced ALDO production.3.The expression and localization of TNF-? in myocardium was detected by immunohistochemical staining.The protein expression of Akt and MCP-1 were analyzed by western-blot method.The purpose is to investigate the signaling pathway of accumulation of macrophages induced by ALDO.Results:1.The protein expression of St AR and AS upregulated in Ang ? induced myocardial fibrosis,implying that St AR/AS signaling pathway involved into Ang ? infusion-induced ALDO secretion.The process can be blocked by oral administration of telmisartan,indicating that Ang ?-regulated ALDO production depends on activated AT1 R.2.Treatment with telmisartan downregulated the expression of ERK1/2 and p38 protein,in consistant with inhibitation of St AR protein.3.Oral administration of sipronolactone decreased the number of accumulated macrophages through suppressing the TNF-?/Akt/MCP-1 signaling pathway.Conclusion: Data suggest that the protein St AR and AS contribute to the enhancement of ALDO secretion in Ang ? induced cardiac fibrosis.This process depended on activating of AT1 R.Simultaneously,MAPKs signaling pathway participated in the regulation of St AR expression.Treatment with MCR blocker spironolactone inhibited the upregulation of TNF-?/Akt/MCP-1 signaling pathway and decreased the number of accumulated macrophages and myocardial fibrosis. |
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