Therapeutic impact of follistatin-like 1 on myocardial ischemic injury in preclinical animal models
Therapeutic impact of follistatin-like 1 on myocardial ischemic injury in preclinical animal models
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发表时间：
2012
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通讯作者：
小椋 康弘
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作者：
小椋 康弘
背景——急性冠状动脉综合征（ACS）是发达国家的主要死亡原因。卵泡抑素样1 （Follistatin-like 1, FSTL1）是一种源自心肌细胞的分泌蛋白，在心肌缺血损伤时表达上调。本研究在大型和小型临床前动物缺血再灌注（I/R）模型中研究了FSTL1对急性心脏损伤的治疗作用，并探讨了其分子机制。方法和结果-在小鼠或猪I/R模型中，人FSTL1蛋白的管理显著减小心肌梗死面积，这与缺血心脏中细胞凋亡和炎症反应的减少有关。FSTL1可增强I/ r损伤心脏中amp活化蛋白激酶（AMPK）的磷酸化。在培养的心肌细胞中，FSTL1通过激活AMPK的能力抑制缺氧/再氧化时的细胞凋亡和lps刺激的促炎基因表达。I/R导致骨形态发生蛋白4 （bone morphogenetic proteins -4, BMP-4）在心脏的表达和Smad1/5/8磷酸化的增强，FSTL1抑制缺血心肌中Smad1/5/8磷酸化的增加。用FSTL1处理心肌细胞可消除bmp -4刺激的凋亡、Smad1/5/8磷酸化和促炎基因表达的增加。在培养的巨噬细胞中，FSTL1通过激活AMPK来降低lps刺激的促炎基因的表达，并消除bmp -4依赖性的促炎介质的诱导。结论-我们的数据表明，FSTL1可以通过调节AMPK-和bmp -4依赖的机制，通过抑制细胞凋亡和炎症反应来预防心肌I/R损伤，这表明FSTL1可能是ACS的一种新的治疗靶点。dd C C C与I/R后的收缩功能障碍、细胞凋亡和炎症反应有关。冠状动脉内注射FSTL1蛋白可减轻I/R后猪心肌梗死，改善心功能，这与抑制缺血心脏细胞凋亡和炎症有关。这些数据与我们之前的研究结果一致，即腺病毒介导的小鼠Fstl1递送可有效减轻小鼠心肌I/R损伤16。我们的体外实验表明，FSTL1促进心肌细胞在H/R反应中的存活，FSTL1减弱激动剂刺激的心肌细胞和巨噬细胞中促炎介质的表达。因此，FSTL1可以通过至少两种机制保护心脏免受缺血性损伤，包括减少心肌细胞死亡和抑制心肌细胞的炎症反应。由于减少心脏细胞死亡和炎症的治疗方法被认为是治疗急性心脏损伤的合乎逻辑的策略，因此给予FSTL1可能是急性冠状动脉综合征的有用辅助治疗。通过表达为一个不活跃的生长因子或一个不活跃的生长因子或车辆30分钟的生长因子来识别一个不活跃的生长因子或车辆。Western blotting分析ACC （p-ACC）和AMPK （p-AMPK）的磷酸化状态。C和D， FSTL1在I/ r损伤的小鼠(C)和猪(D)的心脏中促进AMPK信号通路。Western blot检测ACC （p-ACC）和AMPK （p-AMPK）的磷酸化水平。代表性的斑点图来自4个独立的实验。E，通过Western blot分析，AMPK抑制可消除fstl1诱导的ACC磷酸化。在MOI为10的情况下，用Ad-dn- ampk或Ad- (cid:69) -gal转导nrvm 24小时后，用FSTL1 （250 ng/ml）或载药处理nrvm 30分钟。代表性的斑点图来自3个独立的实验。F， AMPK参与FSTL1对H/ r诱导的nrvm凋亡的抑制作用。用Ad-dn- ampk或Ad- (cid:69) -gal转导nrvm，在H/R条件下，在存在或不存在FSTL1 （250 ng/ml）的情况下培养nrvm。TUNEL染色检测凋亡核（n=4）。化学化学：通过化学分析的方法对化学物质进行评价。自我评价评价的主动性与经验评价无关。B, B, B RRRolololee ofofof等离子体水平的人类FSTL1小鼠系统性注射后的人类重组FSTL1蛋白质。小鼠静脉注射重组人FSTL1蛋白（100 ng/g小鼠）后5分钟采血。Western blot检测血浆FSTL1蛋白水平（10µl）。信号强度采用重组人FSTL1蛋白（100、200、300 ng/ml）标准化，采用Image J软件定量。
: Background - Acute coronary syndrome (ACS) is a leading cause of death in developed countries. Follistatin-like 1 (FSTL1) is a myocyte-derived secreted protein, which is upregulated in the heart in response to ischemic insult. Here, we investigated the therapeutic impact of FSTL1 on acute cardiac injury in small and large preclinical animal models of ischemia/reperfusion (I/R) and dissected its molecular mechanism. Methods and Results - Administration of human FSTL1 protein significantly attenuated myocardial infarct size in a mouse or pig model of I/R, which was associated with reduction of apoptosis and inflammatory responses in the ischemic heart. Administration of FSTL1 enhanced the phosphorylation of AMP-activated protein kinase (AMPK) in the I/R-injured heart. In cultured cardiac myocytes, FSTL1 suppressed apoptosis in response to hypoxia/reoxygenation and LPS-stimulated expression of pro-inflammatory genes through its ability to activate AMPK. I/R led to enhancement of bone morphogenetic protein-4 (BMP-4) expression and Smad1/5/8 phosphorylation in the heart, and FSTL1 suppressed the increased phosphorylation of Smad1/5/8 in ischemic myocardium. Treatment of cardiac myocytes with FSTL1 abolished the BMP-4-stimulated increase in apoptosis, Smad1/5/8 phosphorylation and pro-inflammatory gene expression. In cultured macrophages, FSTL1 diminished LPS-stimulated expression of proinflammatory genes via activation of AMPK, and also abolished BMP-4-dependent induction of pro-inflammatory mediators. Conclusions - Our data indicate that FSTL1 can prevent myocardial I/R injury by inhibiting apoptosis and inflammatory response through modulation of AMPK- and BMP-4-dependent mechanisms, suggesting that FSTL1 could represent a novel therapeutic target for ACS. d d C C C dd ooonnn systolic dysfunction, apoptosis, and inflammatory responses following I/R. Intracoronary injection of FSTL1 protein attenuated myocardial infarction and improved cardiac function in pigs after I/R, which was associated with suppression of apoptosis and inflammation in the ischemic heart. These data are consistent with our previous findings that adenovirus-mediated delivery of mouse Fstl1 is effective at attenuating myocardial I/R injury in mice 16 . Our in vitro experiments showed that FSTL1 promotes the survival of cardiac myocytes in response to H/R and that FSTL1 attenuated agonist-stimulated expression of pro-inflammatory mediators in cardiac cells and macrophages. Thus, FSTL1 administration can protect the heart from ischemic damage through at least two mechanisms involving reduction of cardiomyocyte death and suppression of inflammatory responses in myocardial cells. Because therapeutic approaches to minimize cell death and inflammation in the heart are believed to be logical strategies to treat acute cardiac injury, administration of FSTL1 may be a useful adjunctive therapy for acute coronary syndrome. IdIdentitifificacatitionon oof fofollllisisttatin-n-liikeke 11 bby y exeprpresessisionn as an n acacactititvavavatototor r r ofoo ggroroowtwtwhhh didiifffffferererenenentititaaatitiiononn fffacactototor or vehicle 30 minutes. The phosphorylation status of ACC (p-ACC) and AMPK (p-AMPK) was analyzed by Western blotting. C and D , FSTL1 promotes AMPK signaling pathways in I/R-injured hearts of mice (C) and pigs (D). The phosphorylation of ACC (p-ACC) and AMPK (p-AMPK) was assessed by Western blot analysis. Representative blots are shown from 4 independent experiments. E , AMPK inhibition abolishes FSTL1-induced ACC phosphorylation as assessed by Western blot analysis. After transduction with Ad-dn-AMPK or Ad- (cid:69) -gal at a MOI of 10 for 24 hours, NRVMs were treated with FSTL1 (250 ng/ml) or vehicle for 30 minutes. Representative blots are shown from 3 independent experiments. F , AMPK is involved in the suppressive action of FSTL1 on H/R-induced apoptosis of NRVMs. NRVMs were transduced with Ad-dn-AMPK or Ad- (cid:69) -gal and cultured in the presence or absence of FSTL1 (250 ng/ml) under conditions of H/R. Apoptotic nuclei were identified by TUNEL staining (n=4). sschchhemememicicic hhheaeaeartrt aaassssssesesssesesd bybyby anananalalalysysysisisi. ReReReprprpresesseenentatatatititiveveve ndependent t exexexpepeperiririmemementntntss. B,B,B RRRolololee ofofof Plasma levels of human FSTL1 in mice after systemic injection of human recombinant FSTL1 protein. Blood was collected from mice at 5 minutes after intravenous injection of recombinant human FSTL1 protein (100 ng/g mouse). FSTL1 protein levels in plasma (10 µ l) was determined by Western blot analysis. The signal intensities were standardized by recombinant human FSTL1 protein (100, 200, 300 ng/ml) and quantified by using Image J software.