Spirolactone Protects Heart And Renal Fibrosis By Inhibiting The Endothelial-mesenchymal Transition In Isoprenaline-induced Heart Failure

Background:Chronic heart failure is one of the major issues that jeopardize healthworldwide.Cardiac remodeling is the pathophysiological basis of the occurrence and development of heart failure.Recently,the interaction of renal remodeling in heart failure has been increasingly emphasized.The direct and indirect effects of heart failure are identified as leading to acute kidney injury and dysfunction.The degree of renal function impairmentrelates to increased short-and long-term mortality.It has been linked to cardiac and renal fibrosis.Thecardiac and renal fibrosis play a key role in its development.Exploring the mechanisms of heart failure and developing effctive anti-fibrosis drugs have become the tough problems as well as hot topics in current research.Cardiac and renal fibrosisis characterized by the excess accumulation of collagen fibers,increased collagen concentration or collagen component alterationin extracellular matrix(ECM).Fibrosis a adaptive response of the organism to harmful stimulilike load,inflammation or necrosis that are common in hypertension and ischemic heart disease.In these pathological conditions,heart and kidney release a series of growth factors such as angiotensinogen,TGF-?,ET-1,TNF-?,and eventually lead to theexcess deposition of a collagen-rich extracellularmatrix[1].On the other hand,neurohormonal activation,especially circulating and local renin-angiotensin-aldosterone system,is also involved in the response of cardiac and renal fibrosis.Fibrosis results in increased tissue stiffness,promotes cardiac and renal function shifting from compensation to decompensation phase.Therefore,how to block this process is very important.Endothelial-mesenchymal transition(EndMT)is a process in which endothelial cells lose their polarityand cell-to-cell contacts and acquire mesenchymal phenotype.The important role of EndMT in theprogression of embryogenesis,wound healing and cancer have been elucidated[2].Recently,increasing evidence had suggested that EndMT contribute to the development of organ fibrosis,such as lung,kidney,mesenteric artery.Transforming growth factor ?(TGF-?)is the most important mediator in this process and can promote and induceEndMT[3,4].The discovery of EndMT in heart fibrosis by Elisabeth M Zeisberg opened a new chapter of myocardial fibrosis mechanism research[5].So what signal pathway may be involved in EndMT?Recent studies have confirmed,Notch signaling pathway and micro satellite RNA play a vital role in regulating EndMT.Activation of Notch signaling by Jagged-1 in the epithelial cells inhibited the expression of epithelial cell specific protein molecules(such as E-cadherin,?-catenin,endothelial cell nitric oxide synthetase),increased the expression of mesenchymal cell specific protein molecules,and induced the acquisition ofinesenchymal morphology and phenotype,suggesting that activation of Notch signaling pathway by Jagged-1 plays an important role in EndMT process.Aldosterone,a kind of mineralocorticoid secreted by Adrenal glomerulosa cells,regulates water-electrolytes metabolism and sodium and potassium homeostasis through mineralocorticoid receptors(MR)-dependentinteractions.Elevated aldosterone levels can upregulated the activity of matrix metalloproteinase[6],increased TGF-?1 levels[7],stimulated the synthesis of myocardial collagen,thereby resulting in cardiac fibrosis and in a dose-dependent manner[8].Therefore,aldosterone antagonist has gradually attracted people's attention.The selective aldosterone receptor antagonist-spironolact,has become one of the key anti-heart failure drugs[9].In recent years,researchers found the cardiovascular system has its own way to produce aldosterone.The renin mRNA expression was detected in local cardiovascular tissue and was not affected by circulating RAAs[10].Aldactone combines with MRs expressed in cardiomyocytes,vascular smooth muscle cells,fibroblasts and renal tubular epithelial cells to form hormone-receptor complex,it binds to hormone-response elements in the promoter of target genes to regulate gene transcription,then produce a variety of aldosterone induced protein(including luminal tubular membrane sodium channel protein,cell membrane sodium pump,mitochondrial ATPase)[11],thus affecting the cardiovascular system,urinary system and autonomic nervous system etc,participating in tissue repair,regulation of water,electrolyte and blood volume[12]With structural similarity to aldosterone,spironolactone competitively inhibit the binding of aldosterone to the mineralocorticoid receptor.As evidence mounted that spironolactone can prevent or reverse thecardiaac renal fibrosis process,spironolactonebecomes an important drug of ant-fibrosis therapy,but its pathophysiology remains unclear.We aimed to investigate the inhibitory effectsof spironolactone on cardiac and renal fibrosis and its relationships with EndMT in heart failure animal model,study the role of Notch signaling pathway in EndMT at the cellular level.Part 1:spironolactone improves cardiac remodeling in heart failure via inhibition of Endothelial-Mesenchymal TransitionWe aim to investigate the influence ofspironolactone on cardiac function,structure,the myocardial fibrosis and EndMT in isoprenaline(Iso)-induced heart failure rats.In the experiment of primary cultured human umbilical vein endothelial cells:We investigate whether aldosterone receptor-blocker spironolactone affects cell morphology and function in TGF-?-induced EndMT,further study the role of Notch signaling pathway in EndMT induced fibrosis and the underlying mechanism.??Animalexperimental sectionMaterial and Methods:1.Modelbuilding and group design1.1 Group design:rats were randomly divided into four groups(ten per group)for treatment:control;myocardial fibrosis(isoproerenol[Iso]);low-and high-dose spironolactone(30mg·kg-1·d-1 and 60mg·kg-1·d-1,respectively).1.2 Rat model of cardiac fibrosisFor Iso-model,Iso(5 mg·kg-1·d-1)was injected subcutaneously in male SD rats for 1 week,The same volume of saline was injected for controls.In the therapeutic groups,Iso administration was the same as in the Iso-model group,intragastric administration of spironolactone solution(30mg·kg-1·d-1 and 60mg·kg-1·d-1)for 3 weeks at the same time as Iso injection.2.1 Measurement of hemodynamicsRats were connected with a pressure-transducer input signal recorder for recording heart rate,the LV mean systolic pressure,LV end diastolic pressure,and maximum rate of change in LV pressure(+dp/dtmax,-dp/dtmax).2.2 Sample collectionAfter being humanely euthanized by bleeding.The heart was rapidly excised and rinsed in cold normal saline.Subsequently,the left and right ventricles were separated and weighed.A piece of left ventricular apical tissue(about 80mg)were cut and fixed in formalin,embedded in paraffin and sectioned.2.3 Left Ventricular Weight Index(LVWI)and Right Ventricular Weight Index(RVWI)After the left and right ventricles were separated and weighed,and the left and ight ventricular weight indices(LVWI and RVWI)were calculated as the left and right ventricular free wall mass(mg)divided by body mass(g),respectively.2.4 hematoxylin and eosin stainingHematoxylin and eosin(HE)staining was performed to eval-uate pathological and morphological changes of myocardial tissue.2.5 Masson trichrome stainingMasson's trichrome staining was performed to assess myo-cardial fibrosis.The sections were examined using light microscopy,and photographs were taken at×200 magnification.Five non-repeating visual fields were randomly selected,myocardial collagen areas were measured using Image-Pro Plus and the areas were averaged.2.6 ELISA(Enzyme-linked immunoassay:measurement of collagen ? and ? content in myocardial tissue.2.7 Immunofluorescence staining?-SMA(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence staining to investigate EndMT phenomenon.??Cell experimental section1.Primary HUVECs separation,culture and identification of HUVECs2.HUVECs Experimental designHU-VECs were randomly divided into 5 groups for treatment:blank control;vehicle control(DAPT dissoolved in DMSO);TGF-?(10 ng/ml);spironolactone+TGF-?;spironolactone+DAPT +TGF-?.HUVECs were exposed to spironolactone 24h before TGF-?.In selected experiments,cells were treated with DAPT 1h before spironolactone.3.Transwell assay were performed to observe cell migration.4.Immunofluorescence stainingvimentin(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence staining in endothelial cells to investigate EndMT phenomenon.5.CD31;vimentin and Notch protein level were examined by Western blot analysis.6.Statistical analysisData are expressed as mean ± SEM.Statistical analyses involved use of SPSS v16.0 by Student's t-test for comparing the two groups or one-way ANOVA.P,0.05 was considered statistically significant.Results:??Animalexperimental section1.Cardiac function:Iso significantly decreased LVSP,heart rate,+dp/dtmax,-dp/dtmax and increased LVEDP compared with controls(P<0.01).Spironolactone attenuated ventricular function(P<0.05).2.Organ weight index(LVWI,RVWI):LVWI,RVWI were significantly higher with Iso than control treatment(P<0.01);treatment with spironolactone decreased LVWI,RVWI as compared with Iso treatment(P<0.05).3.Histopathological observations:Heart tissues from Iso-treated rats showed collagen fibre hyperplasia,disordered myocardial structure and leukocyte infiltration compared with controls.Treatment with spironolactone alleviated these changes4.Masson's trichrome staining:Blue fibrous tissue can be seen widely in the heart tissues from of Iso-treated rats.The area of collagenwas higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone alleviated these changes.5:Types ? and ? collagen content:Content of type ?,? collagen in cardiactissue homogenate was higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone significantly decreased the content as compared with Iso treatment(P<0.01).6.Expression of CD31 and a-SMA:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).??Cell experimental section1.Transwell assayEffect ofTGF-?1 oncell migration:TGF-? significantly enhanced HUVECs' ability of proliferation,migration and adhesion as compared with the control group(P<0.01).Effect ofSPon cell migration:Pretreatment with SP for 24h inhibited cell migration stimulated by TGF-?as compared with the TGF-?group(P<0.01).Effect ofDAPTon cell migration with SP treatment:SP could inhibit HUVEC cell migration stimulated by TGF-?,and DAPT could abrogate the effect.After co-culture with the Notch inhibitor DAPT,the number of migrating cells was increased as compared with TGF-?+ SP(P<0.01).2.Immunofluorescence staining results:TGF-?decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01);compared with TGF-?,CD31 protein level was increasedand a-SMA level was decreased in SP+TGF-?group(P<0.01);compared with SP+TGF-? group,CD31 protein level was decreasedand a-SMA level was increased in TGF-?+SP+DAPT group(P<0.01).3.Werstern-blot for CD31?vimentin?Notch-1TGF-?decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01);compared with TGF-?,CD31 protein level was increasedand a-SMA level was decreased in SP+TGF-? group(P<0.01);compared with SP+TGF-? group,CD31 protein level was decreasedand a-SMA level was increased in TGF-?+SP+DAPT group(P<0.01).Notch inhibitor DAPT significantly decreased Notch1 level as compared with the control(P<0.01);compared with TGF-?,Notchl protein level was increased in SP+TGF-? group(P<0.01);compared with SP+TGF-? group,Notchl protein level was decreased in TGF-?+SP+DAPT group(P<0.01).Conclusion:Spironolactone inhibits endothelial mesenchymal transition to improve cardiac remodeling in heart failure via Notch pathway.Part 2:spironolactone improves renal remodeling in heart failure via inhibition of Endothelial-Mesenchymal TransitionFrom previous observations,we aimed to investigate whether spirolactone affects renal function in isoprenaline(Iso)-induced heart failure in rats,affects EndMT and the expression of TGF-? as well as the relation between these changes and renal fibrosis.1.Material and Methods:model building and group design1.1 Group design:rats were randomly divided into four groups(ten per group)for treatment:control;myocardial fibrosis(isoproerenol[Iso]);low-and high-dose spironolactone(30mg·kg-1·d-1 and 60mg·kg-1·d-1,respectively).1.2 Rat model of cardiac fibrosis For Iso-model,Iso(5 mg·kg-1·d-1)was injected subcutaneously in male SD rats for 1 week,The same volume of saline was injected for controls.In the therapeutic groups,Iso administration was the same as in the Iso-model group,intragastric administration of spironolactone solution(30mg·kg-1·d-1 and 60mg·kg-1·d-1)for 3 weeks at the same time as Iso injection.2.1 Measurement of hemodynamicsRats were connected with a pressure-transducer input signal recorder for recording heart rate,the LV mean systolic pressure,LV end diastolic pressure,and maximum rate of change in LV pressure(+dp/dtmax,-dp/dtmax).2.2 Sample collectionAfter being humanely euthanized by bleeding.The kidney was rapidly excised and rinsed in cold normal saline.A piece of kidney tissue(about 80mg)were cut and fixed in formalin,embedded in paraffin and sectioned.The remaining tissue were frozen in liquid nitrogen and then preserved in-80 C refrigerator for measurement.2.3 Left renal Weight Index(LVWI)and Right renal Weight Index(RVWI)After the left and right kidney were separated and weighed,and the left and right renal weight indices(LVWI and RVWI)were calculated as the left and right renal mass(mg)divided by body mass(g),respectively.2.4 hematoxylin and eosin staining Hematoxylin and eosin(HE)staining was performed to eval-uate pathological and morphological changes of kidney.2.5 Masson trichrome stainingMasson's trichrome staining was performed to assess renal fibrosis.The sections were examined using light microscopy,and photographs were taken at x200 magnification.Five non-repeating visual fields were randomly selected,renal collagen areas were measured using Image-Pro Plus and the areas were averaged.2.6 ELISA(Enzyme-linked immunoassay):measureent of collagen ? and ? content in kidney2.7 Immunofluorescence staininga-SMA(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence stainingin in endothelial cells to investigate EndMT phenomenon.2.8.CD31,?-SMA and TGF-?1 level were examined by Western blot analysis.3.Statistical analysisData are expressed as mean ± SEM.Statistical analyses involved use of SPSS v16.0 by Student's t-test for comparing the two groups or one-way ANOVA.P,0.05 was considered statistically significant.Results:1.renal weight index(KWI):KWI were significantly higher with Iso than control treatment(P<0.01);treatment with spironolactone decreased KWI as compared with Iso treatment(P<0.05).2.Histopathological observations:Kidney tissues from Iso-treated rats showed collagen fibre hyperplasiaand leukocyte infiltration compared with controls.Treatment with spironolactone alleviated these changes.3.Masson's trichrome staining:Blue fibrous tissue can be seen widely in the kidney tissues from of Iso-treated rats.The area of collagenwas higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone alleviated these changes(P<0.01).4.Types I and III collagen content:Content of type ?,? collagen in kidney tissue homogenate was higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone significantly decreased the content as compared with Iso treatment(P<0.01).5.Immunofluorescence staining results:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).6.Expression of CD31??-SMAin kidney:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).7.Expression ofTGF-?1in kidney:Iso increased TGF-?1 level as compared with the control(P<0.01),spironolactone treatment decreasedTGF-?1 level as compared with Iso treatment(P<0.01).Conclusion:Spironolactone inhibits endothelial mesenchymal transition to improve renal remodeling in heart failure.