摘要
背景
心房重构是指心房由于损伤或负荷增加等因素所导致的心房大小、形状、功能和组织结构等一系列变化,是在病理情况下,心房修复、代偿及继发的病理生理反应过程。一般来说,心房重构包括心房的结构重构和心房电重构两个方面,在这两个方面中,结构重构又是心房电生理重构发生和维持的重要因素。大量研究显示,心房相关性疾病的发生和发展过程中,往往存在心房重构。近年来的相关研究进一步显示,改善心房重构,拮抗心房肌肥大,阻滞心房纤维化的发生,可以有效的阻止心房疾病的发生和发展。由此可见,开展针对心房重构影响的研究是寻找防治心房性疾病治疗途径的重要方法。
之前的研究显示,肾素—血管紧张素系统(renin-angiotensin system, RAS)作为心血管系统中主要的调节系统,在心血管疾病发生发展过程中发挥着重要作用,是心房重构过程重要参与因素。血管紧张素转化酶—2系统(angiotensin converting enzmy-2, ACE2)是近几年来发现的RAS系统的新的组成成分。ACE2是ACE的同系物,二者具有相似的分子结构,在体内ACE2的主要生理作用是水解血管紧张素Ⅰ和Ⅱ产生血管紧张素1-7(angiotensin1-7, Angl-7)和血管紧张素1-9(angiotensin1-9, Ang1-9)。研究显示,ACE2系统是心血管系统中不可或缺的重要调节因子,在心血管疾病发生发展过程发挥着有重要的保护作用。随着进一步深入研究的开展,发现ACE2对心血管的保护调节作用相当一部分是通过其代谢产物Ang1-7和Angl-9来完成的。
目前针对Angl-7已有大量的研究,对Angl-7在心血管疾病中的作用有了较为深入的了解。研究显示,Ang1-7具有重要的心血管保护作用,在心血管系统中它能够发挥扩张血管,抗增殖,抗纤维化和阻滞心脏重构的作用。研究发现,Ang1-7具有抗心室重构和纤维化的作用,可明显减少室性心律失常的发生；具有降低心房纤维化程度的作用,可以增强心房对房颤(atrial fibrillation AF)的易损性。与Ang1-7相比,目前有关Angl-9的研究相对较少,对Angl-9的作用还缺乏认识,甚至有学者认为Angl-9在体内是不具有生物学活性的中间代谢产物。
最近的研究发现,在依那普利改善心梗后心肌重构过程中,ACE2活性改变的同时存在Angl-9血清浓度的改变,提示ACE2在缺血性心肌重构过程可能是通过改变Angl-9浓度来发挥作用的。另外一项体外研究也发现Angl-9具有兴奋血管紧张素—2受体(angiotensin receptor2, AT2),抑制心肌细胞肥大的作用。这些研究结果提示,Angl-9可能与Angl-7一样,在体内是有活性的生物分子,具有心血管保护作用。另外,我们的前期研究发现,房颤患者在存在心房重构的情况下伴随有血清Ang1-9浓度的降低,提示Angl-9可能参与了病理情况心房重构的过程,其可能与Ang1-7一样,对心房重构及纤维化方面具有良性阻滞作用,可拮抗病理情况下心房重构的发生。因此,我们在综合分析国内外相关文献的基础上,提出了在Ang1-9能够影响病理情况下心房重构的实验假说,并拟通过体内和体外实验予以验证。
目的
1.明确Ang1-9在体内对心房重构的影响。
2.探讨Ang1-9在体外对心房肌细胞HL-1和心房成纤维细胞的影响
3.探讨Angl-9影响心房重构的可能机制。
方法
1.动物实验：
实验分组：通过皮下植入胶囊渗透泵持续给予大鼠药物刺激4周,具体刺激方案如下：①对照组：给予生理盐水;②AngⅡ组：给予AngⅡ (100ng/kg per min);③AngⅡ+Ang1-9组：给予AngⅡ (100ng/kg per min)+Angl-9(450ng/kg per min);④AngⅡ+Angl-9+A779组：给予AngⅡ (100ng/kg per min)+Angl-9(450ng/kg per min)+A779(100ng/kg per min)干预。在干预前后分别检测大鼠的体重、血压、心率和相关的心脏超声指标。所有实验鼠经药物干预4周后,取左心房组织标本进行组织病理学和分子生物学检查。
2.细胞培养：
HL-1细胞是由美国路易斯安那州立大学的Claycomb博士赠与。大鼠心房成纤维细胞参照以往的研究,从Wistar大鼠心房组织中提取。为了观察Ang1-9在体外对心房肌细胞和心房成纤维细胞的影响,将HL-1细胞和心房成纤维细胞各自分为空白对照组、Ang Ⅱ组、AngⅡ+Angl-9组、AngⅡ+Angl-9+A779组、AngII+Angl-9+PD123319组,分别给予生理盐水、AngⅡ100nM、Ang Ⅱ100nM+1μM Angl-9、AngⅡ100nM+1μ M Ang1-9+500nM A779、AngⅡ100nM+1μ M Ang1-9+500nM PD123319刺激。
3. Masson染色实验：
观察Ang Ⅱ和Angl-9干预后,实验鼠心房组织纤维化程度的变化。
4. qRT-PCR实验：
观察干预前后,心房组织和细胞中ANP、BNP、MMP-1和MMP-9mRNA含量的变化。
5. western blot实验：
观察干预前后,心房组织和细胞中胶原蛋白Ⅰ和TGF-β1蛋白含量的变化。
6.统计学分析
实验数据应用SPSS16.0软件包进行统计分析。主要统计指标均进行正态性检验,实验资料所得结果均以平均值±标准差表示(x±s)表示。对照组、不同药物干预组,多组间均数比较采用One-Way ANOVA方差分析。检验水准α=0.05,P<0.05为差异有统计学意义。
结果
1.Ang1-9降低Ang Ⅱ诱导的大鼠心脏体重比的增高
本实验中共包括80只Wistar雄性大鼠,经4周干预刺激后,对照组、Ang Ⅱ、AngⅡ+Ang1-9组和Ang Ⅱ+Ang1-9+A779组大鼠在体重、左室重量、左室射血分数(left ventricular ejection fraction, LVEF)、左心房内径及循环中Angl-7浓度方面无明显差异,但与对照组相比,AngⅡ、AngⅡ+Angl-9组和AngⅡ+Ang1-9+A779组大鼠动脉收缩压和心率明显增高。与Ang Ⅱ组相比,AngⅡ+Angl-9组和AngⅡ+Angl-9+A779组大鼠在收缩压和心率方面无明显差异。进一步的分析发现,在能够反映心室重构程度的指标—左心室／体重比方面,AngⅡ+Angl-9组和AngⅡ+Angl-9+A779组大鼠明显低于Ang Ⅱ组(P<0.05)。4周末测量4组大鼠循环中Angl-7浓度,结果显示4组间无明显的统计学差异。
2.Angl-9在体内可以抑制Ang Ⅱ导致心房肥大指标的增高
持续灌注Ang Ⅱ可以明显增加大鼠心房组织中ANP和BNP mRNA的表达量(ANP,P=0.007vs.对照组；BNP,P=0.015vs.对照组),联合应用Ang1-9后,大鼠心房组织中ANP和BNP mRNA水平与AngⅡ组相比明显降低(P<0.05)。AngⅡ+Angl-9+A779组和AngⅡ+Angl-9组相比,大鼠心房组织中ANP和BNP mRNA水平无统计学差异。结果提示,Angl-9在体内可以抑制Ang Ⅱ心房肥大作用；A779不影响Angl-9对AngⅡ的拮抗作用。
3.Angl-9在体内可以拮抗AngⅡ致心房纤维化作用
Masson染色结果显示,与对照组相比,应用Ang Ⅱ4周后,大鼠心房间质纤维化程度明显增高；与Ang Ⅱ组相比,AngⅡ+Angl-9组大鼠心房纤维化程度明显降低；与AngⅡ+Angl-9组相比,AngⅡ+Angl-9+A779组大鼠心房纤维化程度无明显差异。
Western blot结果显示,与对照组相比,Ang Ⅱ干预大鼠的心房组织中纤维化相关因子—TGF-β1和胶原蛋白Ⅰ的蛋白含量明显的增高；与Ang Ⅱ组相比,同时应用Angl-9可以明显的降低TGF-β1和胶原蛋白Ⅰ的蛋白表达；与Ang Ⅱ+Angl-9组相比,AngⅡ+Ang1-9+A779组大鼠心房组织中TGF-β1和胶原蛋白Ⅰ的蛋白含量无明显变化。以上结果提示,Ang1-9在体内可以拮抗Ang Ⅱ致心房纤维化的作用,而这种拮抗作用不被A779所阻滞。
4.Ang1-9在体外阻滞Ang Ⅱ诱导心房肌细胞HL-1肥大作用
与对照组相比,AngⅡ使HL-1细胞ANP mRNA表达增高了2.9倍(P=0.009)；与Ang Ⅱ组相比,同时应用Angl-9可以阻滞Ang Ⅱ使HL-1细胞ANP mRNA表达的增多；同时结果显示Ang1-9可以阻滞AngⅡ诱导的HL-1细胞面积的增加；与Ang1-9同时应用AT2R拮抗剂——PD123319可以拮抗Angl-9的阻滞作用,使HL-1细胞中ANPmRNA的含量和HL-1细胞面积增高；与Angl-9同时应用Mas受体拮抗剂—A779不影响Ang1-9的阻滞作用。
5.Angl-9在体外拮抗Ang Ⅱ在大鼠心房成纤维细胞中的促纤维化作用
与对照组相比,Ang Ⅱ刺激组的成纤维细胞中TGF-β1和胶原蛋白Ⅰ蛋白表达量明显增加；与Ang Ⅱ组相比,同时应用Angl-9可以阻滞Ang Ⅱ使心房成纤维细胞中TGF-β1和胶原蛋白Ⅰ蛋白表达量增多的作用；与Angl-9同时应用PD123319可以拮抗Ang1-9对AngⅡ阻滞作用,使心房成纤维细胞中TGF-β1和胶原蛋白工蛋白含量增加；与Ang1-9同时应用A779对Ang1-9的阻滞作用没有影响。
进一步的研究显示,Ang1-9可以拮抗Ang Ⅱ在心房成纤维细胞中影响MMP-1和MMP-9mRNA的表达的作用；PD123319可以消除Ang1-9的对Ang Ⅱ拮抗作用,而A779不影响Ang1-9的作用。以上结果提示,Angl-9可以拮抗Ang Ⅱ在大鼠心房成纤维细胞中的促纤维化作用。
6.Angl-9抑制Ang Ⅱ在HL-1细胞和大鼠心房成纤维细胞中ERK信号通路的激活
为进一步明确Angl-9的作用机制,本研究中我们又进一步检测了HL-1细胞和大鼠心房成纤维细胞中ERK信号通路相关蛋白的表达情况,实验结果显示,与对照组相比,AngⅡ可以激活MARK-ERK信号通路,使HL-1细胞和心房成纤维细胞中P-ERK蛋白量增高(P=0.00,in HL-1cells；P=0.00,in atrial fibroblasts)；与Ang Ⅱ相比,应用Angl-9后抑制Ang Ⅱ对MARK-ERK信号通路的激活作用,降低P-ERK蛋白水平(P=0.00, in HL-1cells； P=0.00, in atrial fibroblasts)；PD123319拮抗Angl-9在HL-1细胞和心房成纤维细胞抑制MARK-ERK信号通路激活的作用(P=0.016, in HL-1cells； P=0.010, in atrial fibroblasts)；A779对Angl-9的作用没有影响。以上结果提示,Angl-9阻滞Ang Ⅱ促心房肥大和纤维化作用是(至少部分是)通过激活AT2R受体后降低MARK-ERK信号通路的活化来完成的。
结论
1.Angl-9自身是ACE2系统中具有活性的生物学因子,在体内和体外均具有拮抗心房肌肥大和心房纤维化,改善心房重构的作用,其作用的发挥不依赖于Angl-7的产生。
2.Angl-9阻滞Ang Ⅱ促心房肥大和纤维化作用是通过激活AT2R受体,影响MARK-ERK信号通路来完成的。
背景
心房颤动(房颤)是临床上最常见的持续性心律失常,与无房颤患者相比,房颤患者生命质量明显下降,血栓形成和栓塞性疾病的发生率明显增加,患者的致病率、死亡率明显上升。房颤本身具有自我维持的趋势,随着其发作时间的延长,患者转复和维持窦性心律的能力均受到了损害,给治疗带来了困难。现阶段虽然临床上针对房颤治疗有多种手段,包括抗心律失常药物治疗、外科手术治疗及介入射频消融治疗等,但是这些治疗方法对房颤治疗效果还不能让人满意,造成这种情况的原因是对房颤发生的始动因素和维持因素尚不能完全明确。因此,开展针对房颤发生和维持机制的研究,进一步明确房颤的发生机制,对制定有效的房颤治疗策略具有重要意义。
随着研究的深入,发现房颤发生和维持的电生理异常是在心房结构重构的基础上发生的,即心房结构重构是房颤发生、维持的基础原因之一。相对于心房电重构中的电生理功能改变而言,心房结构的重构是一种组织结构上的改变,表现为心房肌细胞的凋亡、坏死,心房的扩大和纤维化的发生。其中心房纤维化是房颤发生时心房重构的重要的病理基础之一。研究显示,在慢性房颤患者和房颤动物模型中,均存在显著的心房间质的纤维化,间质纤维化干扰了兴奋／冲动的传导,产生局部传导异常,诱发房颤。目前认为,心房组织的纤维化可以引起心房内电生理传导的不连续,同时增加传导的空间离散程度,增加了心房内可容纳的折返子波数,从而促进了房颤的发生和维持。由此可见,心房纤维化是房颤时心房重构的重要病理特征和结构基础,也是房颤治疗过程中主要治疗靶点之一。目前已有大量的关于房颤时心房纤维化发生机制及其治疗方法的研究,但是时至今日,有关房颤时心房重构和纤维化发生具体机制尚不明确,临床上对房颤时心房纤维化的治疗效果并不理想,仍需开展进一步的研究。
小窝蛋白是细胞膜小窝结构的基本组成单位。小窝是一种分布于细胞质膜表面呈烧瓶样内陷的囊泡状结构,直径约50-100nm,存在于多种高度分化的细胞表面。小窝主要由脂类和蛋白质组成。小窝蛋白家族有三种类型：小窝蛋白-1(caveolin-1, Cav-1)、Cav-2和Cav-3。其中,Cav-1和Cav-2在大多数细胞均可表达,尤其在高度分化的细胞表达丰富,如成纤维细胞、Ⅰ型肺泡细胞、脂肪细胞、上皮细胞,心房肌细胞等。其中Cav-1,作为小窝的主要标志性蛋白,在这个过程中扮演着重要的角色。研究显示Cav-1可引起机体器官和细胞的功能的异常,可导致多种器官纤维化的发生。但是,目前对Cav-1与对房颤心房重构及易损性的研究目前国内外尚未见相关报道。
以往大量研究证实,房颤心房纤维化的过程中TGF-β1发挥着至关重要的作用。如研究显示,与窦性心律组患者相比,房颤组患者心房组织TGF-β1的表达明显上调。在使小鼠过度表达TGF-β1可以促进心房纤维的发生,同时增强心房内电生理传导的异向性,增加心房房颤的易损性。由此可见,若降低TGF-β1水平或阻滞TGF-β1作用的相关信号通路,便有可能阻滞甚至逆转心房纤维化的发生。以往的研究发现Cav-1可以抑制TGF-β1相关的信号通路,是体内TGF-β1的拮抗因子。Cav-1拮抗TGF-β1的作用,可能使其具有阻滞房颤心房纤维化的能力。最近的研究显示,在Cav-1沉默后小鼠中存在小鼠心房结构和功能的异常。因此,结合以上的研究我们推测Cav-1可能对心房重构方面具有良性阻滞作用,可拮抗病理情况下心房重构的发生,增强心房易损性,抑制房颤等房性心律失常发生和维持。国内目前尚未见相关文献报道。
以往的研究证实,Cav-1羧基尾巴上的脚手架结构域,即Cav-1羧基82-101残基是其与其它分子相互作用的主要结构。以该段结构为模板衍生的多肽—CSD肽具有与Cav-1蛋白相同的功能,该多肽能完全穿透细胞膜,在细胞内发挥作用,是研究Cav-1功能的常用工具,已被广泛地用在各种有关Cav-1的组织学或细胞学研究的功能。因此,在本研究中我们拟在明确房颤的心房组织中Cav-1变化的基础上,在体外通过应用Cav-1衍生肽(the peptide derived from Cav-1,CSD肽)和siRNA技术研究Cav-1在房颤心房纤维化过程中作用,明确其相关机制,并观察CSD肽在体外对心房纤维化的影响,为更好地防治房颤心房重构提供新的理论依据和实验数据。
目的
1.明确房颤心房纤维化过程中Cav-1的变化情况。
2.探讨Cav-1影响房颤心房纤维化的相关机制。
3.明确CSD肽对心房纤维化影响。
实验方法
1.入选对象：
本研究连续收录了2012年1月11日到2013年6月30日就诊于山东大学齐鲁医院的102名诊断为风湿性心脏病行心脏瓣膜置换手术的患者,其中54名患者诊断具有房颤病史(房颤组),48名患者无房颤病史(窦性心律组)。
2.细胞实验：
人心房成纤维细胞(human atrial fibroblats, HAFs):由人心房组织标本提取。实验中仅第2-4代细胞被用于研究。细胞被种植于6孔板中,当细胞融合度达到90%时,将细胞培养基中的血清移除,饥饿细胞24小时。24小时后,直接给予干预刺激,或者进行siRNA转染后再进行刺激。实验组细胞先用CSD肽或Scr肽(5μM)孵育细胞30分钟,然后给予100ng/ml TGF-β1刺激,对照组细胞给予PBS刺激,培养24小时或48小时,收获细胞用于qRT-PCR或Western blot实验分析。
3. Masson染色：
观察入选对象心房组织纤维化程度。
4. qRT-PCR实验：
观察入选对象心房组织Cav-1mRNA含量的不同,及不同干预对HAFs中Cav-1、MMP-2、MMP9、胶原蛋白Ⅰ和ⅢmRNA的含量的影响。
5. western blot实验：
观察入选对象心房组织中Cav-1、TGF-β1、胶原蛋白Ⅰ和Ⅲ蛋白含量不同及干预对HAFs由Cav-1、Smad信号通路相关蛋白、胶原蛋白Ⅰ和Ⅲ蛋白含量的影响。
6.统计学分析
实验数据应用SPSS16.0软件包进行统计分析。主要统计指标均进行正态性检验,实验资料所得结果均以平均值±标准差(x±s)表示。窦性心律与房颤组两组间均数比较采用t检验；对照组、不同干预组,多组间均数比较采用One-Way ANOVA方差分析。检验水准α=0.05,P<0.05为差异有统计学意义。
结果
1.房颤患者心房组织在Cav-1低表达的同时伴有TGF-β1水平的增高和纤维化的增强。
Western blot和qRT-PCR结果显示,与窦性心律组相比,房颤组患者心房组织中Cav-1蛋白表达量降低了约30%(P<0.001)。同时,与窦性心律组相比,房颤组患者心房组织中TGF-β1的表达量增加了约2.5倍(P<0.001)。患者心房组织中TGF-β1和Cav-1的表达情况呈负相关关系(r=0.75,P=0.012)。Masson染色发现,在房颤患者心房组织纤维化程度明显高于窦性心律组。房颤组心房组织中胶原蛋白Ⅰ和Ⅲ的含量明显高于窦性心律组。
2. TGF-β1可使HAFs中Cav-1的表达降低。
以100ng/ml浓度刺激HAFs48h后,发现HAFs中Cav-1蛋白的表达在48h后与对照组相比降低了约42%(P=0.004)。
3.应用siRNA降低Cav-1表达明显增强TGF-β1相关的Smad信号通路的活性
与对照组相比,siRNA-cav可明显降低HAFs中Cav-1蛋白的表达。细胞中Cav-1蛋白低表达可以导致细胞中TGF-β1相关的Smad信号通路活性的增强,细胞中TGF-β1诱导的磷酸化Smad2and Smad3含量增高,进而引起胶原蛋白表达量的增加。
4.CSD肽可以抑制TGF-β1在HAFs中胶原蛋白的产生和Smad信号通路的激活
与TGF-β1刺激组相比,CSD肽干预刺激组中胶原蛋白的含量降低了78%,Smad信号通路的活性明显降低,而其对照肽—Scr与TGF-β1刺激组相比无统计学差异。同时qRT-PCR结果显示,CSD肽可以拮抗TGF-β1导致HAFs中的MMPs表达的减少,而其对照肽Scr肽对HAFs中TGF-β1对MMPs的作用没有影响。
结论
1.Cav-1参与了房颤心房纤维化的过程。
2.Cav-1可以调节TGF-β1促心房纤维化的作用,影响TGF-β1对Smad通路的激活,拮抗TGF-β1促进胶原生成的作用,可以成为治疗房颤心房纤维化的新的靶点。
3.CSD肽可以发挥Cav-1蛋白样的作用,阻滞TGF-β1诱导的Smad通路的激活和胶原生成,拮抗TGF-β1促心房纤维化的作用,可以成为治疗心房纤维化的潜在的新型药物。
Backgroud:
The angiotensin-converting enzyme2(ACE2) system, a newly discovered and important rennin-angiotensin system (RAS) member, counterbalances the effect of the traditional RAS system and plays a protective role in heart disease pathophysiology. ACE2is the homolog of ACE which hydrolyzes angiotensin (Ang) II and Ang I to Angl-7and Ang1-9, respectively. Several reports demonstrated that the ACE2/Angl-7/Mas axis exerts protective actions in the pathophysiology of heart diseases and inhibits many detrimental cardiovascular disease phenotypes.
In contrast to Angl-7, there has been scarce research on the effect of Ang1-9on the cardiovascular system, because it was initially thought to have no biological activity and to be active only after conversion to Angl-7. However, recent studies subverted the above point and clarified that, as Ang1-7, Ang1-9is a counter-regulator of the RAS system. Ang1-9was reported to block left cardiac hypertrophy in a rat myocardial infarction model and to attenuate ventricular fibrosis in the stroke-prone spontaneously hypertensive rat, indicating that Ang1-9is an active endogenous factor with cardiovascular protective effects. To date, the role of Ang1-9on atrial remodeling remains poorly understood. The present study therefore investigated the effect of Angl-9on atrial remodeling in the Ang II-induced hypertension rat model.
Aims:
1. To study the effect of angiotensin-(1-9) on the atrial remodeling in vivo.
2. To study the effect of angiotensin-(1-9) on the atrial caridomycytes--HL-1cells and atrial fibroblasts in vitro.
3. To study the mechanism of angiotensin-(1-9) affecting the atrial remodeling.
Materials and Methods:
1. Animal Experimental Protocols
Eighty male Wistar rats (213.3±7.6g) were housed in cages with ad libitum access to rat chow and water. Rats were randomly assigned to sham, Ang Ⅱ, Angl-9and A779groups. There were no differences in body weight and systolic blood pressure among the four groups before treatment onset. Hypertension and cardiac remodeling were induced by chronic subcutaneous infusion of Ang Ⅱ (100ng/kg per min) delivered for4weeks by osmotic minipump (model2ML4, Alzet, Cupertino, CA). In the sham, Angl-9and A779groups, osmotic minipumps contained0.9%saline, Ang Ⅱ (100ng/kg per min)+Angl-9(450ng/kg per min), or a combination of Ang Ⅱ (100ng/kg per min), Ang1-9(450ng/kg per min) and A779(100ng/kg per min), respectively. Each treatment group included6rats, and no rodents died during the experimental treatment. Indirect blood pressure was recorded before and after4weeks treatment by tail-cuff plesthymography. Echocardiography was performed after4weeks treatment under anesthesia. Images were obtained from two-dimensional, M-mode, pulsed-wave Doppler and tissue Doppler imaging. After echocardiography, hearts were removed by blunt dissection; the atria were separated from the ventricles and discarded, and the heart ventricles including both the LV and right ventricle (RV) as well as the interventricular septum were weighed. A section of left atrial tissue from each rat was immediately frozen in liquid nitrogen for RNA and protein isolation and an adjacent cross section was fixed in10%neutralized formalin for histopathology analysis.
2. Cell culture
HL-1atrial myocytes were kindly donated by Dr. Claycomb. Rat atrial fibroblasts were derived from8week-old Wistar rats, and cells of passages2-4were used. For fibroblasts and HL-1cells, serum was withdrawn24h before preincubation with1μM Angl-9for90min, after which100nM Ang II was added. The effects of A779[a mas antagonist for Angl-7] and PD123319[an angiotensin type2(AT2) receptor antagonist for Ang1-9] were evaluated by preincubating the cells with A779(500nM) or PD123319(500nM) for30min before Angl-9was added. The cultures were incubated for24h for qRT-PCR analysis or for48h for western blot assays.
Results:
1. Angl-9attenuates Ang Ⅱ-induced atrial remodeling in vivo
Chronic infusion of Ang Ⅱ caused a significant increase in systolic blood pressure and heart beat in rats compared with saline infused animals, which was unchanged by co-infusion of Ang1-9and A779. And after4weeks treatment, body weight increased in all four groups compared with baseline weight; the latter increase was mumerically lower in the Ang Ⅱ group, however, differences were not significantly different among treatments (P=0.197). Additionally, ventricular weights were similar among all four groups (P=0.663). The calculated heart weight-to-body weight ratio was significantly higher in the Ang Ⅱ than in the sham group, while the ratio was lower in the Angl-9and A779groups compared to the Ang Ⅱ group. Left atrial diameter in rats in the Ang Ⅱ, Ang1-9, and A779groups was not different from that in saline-infused animals.
Infusion of Ang Ⅱ markedly increased ANP and BNP relative atrial gene expression compared with atria of animals infused with saline (P=0.007and0.015, respectively). Concomitant treatment with Angl-9significantly attenuated the increase in both ANP and BNP mRNA levels compared with infusion of Ang II alone (P=0.007and0.029, respectively). Co-treatment with A779had no significant effect on relative ANP or BNP mRNA expression in vivo compared with that in rats infused with Ang1-9+AngⅡ.
Masson-trichrome-stained left-atrium cross sections showed chronic infusion of Ang II caused a marked increase in atrial interstitial fibrosis compared with saline-infused animals. The increase in interstitial fibrosis with Ang Ⅱ infusion was significantly attenuated by the co-administration of Ang1-9. Consistent with the result of masson-trichrome stained atrial tissue, the amount of fibrotic markers (TGF-β1and collagen I) in atrial tissue of Ang II-treated rats was markedly greater than that of rats in the sham group. The amount of fibrotic markers was lower in Angl-9-treated rats compared to Ang Ⅱ-treated rats, and A779did not affect the suppressive effect on atrial fibrosis of Angl-9in vivo.
2. Angl-9attenuates effects of Ang Ⅱ on HL-1cells and atrial fibroblats
Ang II elevated ANP expression by2.9-fold compared to the control group in HL-1cells (P=0.009). This effect was significantly prevented by Angl-9. A779incubation had no effect on the Ang1-9-dependent suppression of ANP expression while co-administration of PD123319with Angl-9eliminated the suppressive effect.
Isolated primary cultures of neonatal atrial fibroblasts were used to confirm direct effects of Angl-9on atrial fibroblasts. Co-administration of Angl-9(1μM) inhibited Ang Ⅱ-induced increases in TGF-β1and collagen I production.However, the addition of PD123319to Ang1-9-stimulated fibroblasts reversed the anti-fibrotic effect of Angl-9while A779did not, which indicates that Angl-9mediates antiproliferative effects on atrial fibroblasts via the AT2R. Meanwhile, Angl-9inhibited the Ang Ⅱ-induced activation of ERK signal pathway.
To evaluate collagen degradation, we assessed the effects of Angl-9on expression of two principal matrix metalloproteinase collagenases (MMP-1and MMP-9), which degrade the extracellular matrix. The results showed that Angl-9attenuated the ability of Ang Ⅱ to downregulate MMP-1and MMP-9. PD123319abolished the effect of Angl-9on upregulation of MMPs expression, whereas A779did not.
Conclusion
1. Angiotensin-(1-9) is an endogenous active factor.
2. Angiotensin-(1-9) could reversed angiotensin Ⅱ-induced adverse atrial structural remodeling in vivo and in vitro.
3.Angiotensin-(1-9) plays its protective roles via AT2R and MARK-ERK signal pathway.
Backgroud:
Caveolae are50-to100-nm omega-shaped invaginations of the cytoplasmic membrane, which were first reported as early as the middle of the last century. Over the past decade, the study on caveolae has blossomed into a rapidly expanding field, and caveolae have been identified as an important member participating in the transcytosis of macromolecules, cholesterol transport and signal transduction in various types of cells. Caveolin-1is the first member of the caveolae gene family consisting of three structurally related proteins:caveolin-1(Cav-1), caveolin-2(Cav-2), and caveolin-3(Cav-3). Cav-1is the principal structural component of caveolae organelles cells.In the cardiovascular system, Cav-1and Cav-2are co-expressed in a variety of cells and tissue types but are most abundantly present in fibroblasts and endothelial cells, whereas Cav-3is strictly expressed in cardiomyocytes.
Atrial fibrillation(AF), the most common cardiac arrhythmia, is frequently accompanied by atrial interstitial fibrosis. A plethora of studies in animal models of atrial fibrillation (AF) and clinical AF have verified that AF is associated with progressive atrial structural and electrical remodeling. During the development of cardiac fibrosis, the transforming growth factor-β1(TGF-β1) is considered to be the key profibrotic cytokine. Cav-1-knockout animals displayed enhanced TGF-β1signaling activities, as reflected by more widespread collagen deposition accompanied by reduced expression of matrix metalloproteinases MMP-8and MMP-13mRNAs in the heart. This would imply that Cav-1can cause subordinate alterations in cardiac structure and function by regulating cardiac fibrosis.
In view of all these findings, we were prompted to propose a hypothesis that Cav-1might produce an anti-AF effect by participating in the atrial structural remodeling process through its anti-fibrotic action. The carboxyl tail of Cav-1, or the Cav-1scaffolding domain (CSD; residues82-101in Cav-1), is the primary structure that interacts with other molecules. The peptides derived from CSD (the CSD peptide) are able to elicit the same cellular functions as Cav-1, which is fully cell permeable and has been widely used as a mimic of the full-length Cav-1in studies of variety cellular functions associated with Cav-1, indicating the peptide is a superior gain-of-function tool for studying the function of Cav-1. Therefore, basing on results of our research on changes in the atrial tissue of AF, an in vitro study was conducted to examine our hypothesis whether Cav-1could reverse the pathological atrial structural remodeling in patients with AF by using a gain-of-function approach with the CSD peptide and a loss-of-function approach with siRNA.
Aims:
1. To study changes of Cav-1in atrial fibrosis during atrial fibrillation
2. To study the mechanism of changes of Cav-1in patients with AF.
3. To study the effect of CSD petides on atrial fibriosis.
Materials and Methods:
1. Patients
The subjects included102patients,54females and48males, with rheumatic heart disease undergoing mitral/aortic valve replacement from January11,2012to June30,2013, with the age of52.58±11.0(range from29to76years old). Fifty-four were diagnosed with AF (AF group) and48with no history of AF (sinus rhythm (SR) group). After written informed consent was obtained, atrial tissue samples from the right atrial appendage were obtained from all subjects. The samples were then used for primary culturing of human atrial fibroblasts, or frozen and stored at-80℃for RNA and protein extraction for qRT-PCR and Western blot, respectively, or fixed by10%neutralized formalin for histopathology analysis.
2. Cell culture
Human atrial fibroblasts (HAFs) were derived from biopsies of the right atrial appendage. Cells of passages2-4were plated in6-well plates. Serum was withdrawn24h before incubation with100ng/ml recombinant human TGF-|31for48h. HAFs were also pretreated for30min with the CSD peptide (5μM) or the scrambled peptide (Scr peptide;5μM), and subsequently incubated for48h with or without100ng/ml TGF-β1. Cells were then harvested from each experimental group for Western blot analysis or qRT-PCR.
3. siRNA transfection
HAFs were plated in6-well plates with80%final density, and transfected with caveolin-1siRNA (sense5'-GCCGUGUCUAUUCCA UCUA-3'; antisense5'-UAGAUGGAAU AGACACGGC-3') or a non-silencing negative control siRNA (sense5'-UUCUCCGAACGUGU CACGU-3'; antisense5'-ACGUGACACGUUC GGAGAA-3') using Hiperfect transfection reagent (Introgen) following protocols provided by the manufacturer. After48h of incubation the cells were either harvested for protein extraction or treated with TGF-β1as described above.
4. Statistical analysis
Results were presented as mean±SE. Between group differences were determined using one-way ANOVA followed by Fisher's protected least significant difference (Fisher's PLSD) tests. The statistical tests were two-tailed, with a p-value of<0.05for significance. The statistical analysis was performed with SPSS16.0software (SPSS, Chicago, IL).
Results:
1. Downregulation of atrial Cav-1and increase of atrial fibrosis in AF patients
There were no significant differences between the SR and AF group in sex, age, types of valve disease, ejection fraction and degrees of cardiac function. Left atrial diameter in AF group was bigger than that in SR group (59.4±10.5vs.47.8±4.9P=0.004).
Western blot and qRT-PCR analyses showed the protein level of Cav-1was approximately30%lower in AF subjects than in the SR group (P<0.001). The Cav-1mRNA was also downregulated, even to a greater extent, in AF relative to SR (P<0.001).
The protein level of transforming growth factor beta1(TGF-β1) was significantly elevated (-2.5fold) in AF patients relative to SR subjects (P<0.001). And the statistics showed a negative correlation between TGF-beta1level and Cav-1level (r=0.75, P=0.012). Meanwhile, there was much higher percentage of fibrotic tissues, as indicated by the more widespread areas stained with Masson-trichrome, in AF than in SR tissues. Furthermore, the level of collagen I, the main component of the extracellular matrix in cardiac fibrosis, and collagen III were markedly greater in atrial tissues from AF patients than from SR subjects.
2. Downregulation of Cav-1by TGF-β1in HAFs
HAFs were incubated with TGF-β1of a fixed concentration of100ng/ml for48h, the results showed that Cav-1was reduced by42%(P=0.004).
3. Enhanced TGF-β1induced-Smad signal pathway and collagen production by knock-down caveolin-1with siRNA in HAFs
The siRNA of caveolin-1could suppressed the expression of caveolin-1. The downregulation of caveolin-1subsequently increased the amount of phosphorylated Smad2and Smad3and the expression of collagens in HAFs.
4. Inhibition of TGF-β1induced-collagen production and the activation of Smad signal pathway by the CSD peptide in HAFs
The CSD peptide inhibited the TGF-β1-induced activation of Smad2/3and pronouncedly reduced the TGF-β1-induced increase in production of collagens by78%. By comparison, the Scr peptide (5μM), as a negative control, did not alter the effects of TGF-β1.
The CSD peptide (5μM) attenuated TGF-β1induced-decrease of MMPs, whereas the Scr peptide did not significantly affect the changes of MMPs induced by TGF-β1.
Conculsion:
Our data suggested that Cav-1is an important regulator of AF and a promising therapeutic target for AF. However, this in vitro study merely lays the groundwork for future studies on the role of Cav-1in AF. Additional studies are warranted to confirm the results.
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