Lipopolysaccharide Induced Activin A-follistatin Imbalance Affects Cardiac Fibrosis

Inflammation plays a pivotal role in cardiac remodeling, especially inmyocardial fibrosis. Abnormal growth of cardiac fibroblasts is criticallyinvolved in the pathophysiology of cardiac hypertrophy/remodeling. Previousstudy has demonstrated that many inflammation stimulating factors triggertransforming growth factor-β (TGF-β) induction and reactive myocardialfibrosis. Activin A (ACT A) is a member of TGF-β superfamily, and follistatin(FS) is an activin-binding protein, i.e., the antagonist of ACT A. Our previousstudies have shown that ACT A-FS imbalance occurs in rats with heart failureafter myocardial infarction, and overexpression of ACT A can lead toventricular remodeling after myocardial infarction, and resultant heart failure.Low expression of FS after myocardial infarction further exacerbated theformation of heart failure. The pathogenic change for overexpression of ACT Awas consistent with that of overexpression of angiotensin II (Ang-II).Ventricular remodeling includes cardiocyte remodeling and myocardialinterstitial collagen deposition and fibrosis. Therefore, the present study wasdesigned to investigate the effects of inflammatory factors on the expression ofACT A-FS and the secretion of cardiac fibroblasts in order that explore indepth the mechanism of myocardial fibrosis.Methods:To establish the heart failure model post myocardial infarction,diethylether-anesthetized rats were fixed on the operation table and the leftanterior descending coronary arteries were ligated. Primary rat cardiacfibroblasts(CF) were isolated and cultured using differential adherence method.To determine the effect of inflammatory factor--bacterial endotoxinlipopolysaccharide (LPS) on CF proliferation, primary rat cardiac fibroblasts were cultured and MTT assay was performed. To evaluate whether na ve ratcardiac fibroblasts could express activin A and FS proteins and the role of LPSon CF, cellular immunohistochemical staining were performed. Further, todetect expression of collagen type I and III, activinβA, FS, and nitric oxidesynthase (iNOS) induced by different dose of activin A on CF for24h,Real-Time quantitative PCR were performed.Statistical analysis:Data is reported as means±SD. Comparisons weremade between different treatment groups using ANOVA, followed by theDunnett post hoc test for differences. Data for percent changes were analyzedusing the Kruskal-Wallis H-test. A value of P <0.05was consideredsignificant.Results:1,Effects of LPS on cardiac fibroblasts proliferationHyperplasia of cardiac fibroblasts is an important factor leading tomyocardial remodeling. The results in the present study verified the effect ofinflammatory mediator LPS on cardiac fibroblast proliferation.24hours afterthe cultured primary rat cardiac fibroblasts were exposed to variousconcentrations of LPS, the proliferation of cardiac fibroblasts was significantlyincreased compared with the medium control (P <0.01), and the action was indose dependence, indicating that LPS may contribute to myocardialremodeling.2,Expressions of ACT A and FS in cardiac fibroblastsTo elucidate the mechanism underlying of LPS-induced cardiac fibroblastsproliferation, we analyzed the expression of activin A and FS in cardiacfibroblasts. First, we utilized immunocytochemistry staining and found that thecultured primary cardiac fibroblasts expressed ACT A and FS mature proteins.We further analyzed the changes in the expression levels of activin A andFS in LPS-treated cultured primary cardiac fibroblasts. We observed that24 hours after LPS stimulated the primary cultured cardiac fibroblasts, the levelsof ACT A and FS proteins in cardiac fibroblasts were significantly increasedand decreased respectively (P <0.01) when compared with the mediumcontrol. The resultant ACT/FS ratio was increased. The levels of ACT A andFS mRNA were significantly increased (P <0.01) and unchanged (P>0.05)compared with the medium control. The FS protein expression did not matchwith the level of gene expression, suggesting that the lowered protein levelsmay be due to its combination with ACT A. These results suggested that LPScan stimulate Activin A secretion and expression in cardiac fibroblasts, buthave no effect on FS expression. Thereby, LPS leads to ACT A-FS imbalancein cardiac fibroblasts and may potentially promote LPS-stimulated proliferationof cardiac fibroblasts.3,ACT A promotes the proliferation of cardiac fibroblasts24hours after various concentrations of ACT A stimulated cardiacfibroblasts, the proliferation of cardiac fibroblasts was significantly increasedby dose dependence compared with the medium control (P <0.01). Theseresults suggest that ACT A may be involved in the pathological process ofcardiac remodeling, and the action of LPS to promote cardiac fibroblastproliferation may be realized by stimulating the overexpression of ACT A incardiac fibroblasts.4,ACT A stimulates the synthesis of collagen in cardiac fibroblastsTwo main components of ECM are types I and III collagens, which playan pivotal role in maintenance of structure and function of the heart.24hoursafter various concentrations of ACT A stimulated cardiac fibroblasts, the levelsof types I and III collagen mRNA were significantly increased with dosedependence compared with the medium control (P <0.01). These resultssuggest that ACT A may stimulate cardiac fibroblasts to express type I and IIIcollagens, which lead to cellular interstitial remodeling, and thus contribute to myocardial remodeling.5,Levels of NO and eNOS expression in cardiac fibroblasts stimulated byACT AOur previous studies have shown that ACT A is not only involved inmyocardial fibrosis, but also has a role to promote the secretion ofinflammatory mediator NO. The present study showed that ACT A significantlystimulated cardiac fibroblasts to secret high levels of NO and overexpressiNOS mRNA compared with the medium control (P <0.01). These resultssuggest that ACT A may act on cardiac fibroblasts in the way ofautocrine/paracrine, stimulate the synthesis and secretion of NO, and thus beinvolved in the pathogenesis of myocardial remodeling.Conclusions:These results suggest that inflammatory mediator LPS canpromote ACT A-FS imbalance in cardiac fibroblasts, mainly overexpression ofACTA. Overexpression of ACT A promotes the proliferation and the secretionof collagens in cardiac fibroblasts through autocrine/paracrine of NO, and isinvolved in the pathological process of myocardial fibrosis.In conclusion, as heart failure occurs, myocardial interstitial remodelinghas a significant role in the progression of heart failure. ACT A-FS system isno longer a sexual regulatory hormone as previously believed. In thedevelopment of heart failure, the primary performance of ACT A-FS imbalanceis the increase in ACT A levels. Overexpression of ACT A may be a primaryfactor leading to myocardial fibrosis and eventually inducing heart failure.Lowered expression of FS (ACT A antagonist) further exacerbates theformation of heart failure. Inflammatory factor is a critical factor inducing ACTA-FS imbalance. Therefore, it is of significance to in-depth study thecomprehensive prevention and treatment of heart failure, includinganti-inflammatory or antagonistic cytokines, immune regulation and exerts of apleiotropic role of statins. Regulation of ACT A/FS ratio is expected to become another therapeutic target to improve heart failure.