摘要
研究背景
高血压是心血管疾病最重要的危险因素,而血管重塑是导致高血压患者心血管事件发生率显著增加的独立危险因素。与高血压相伴随的血管结构和功能的改变即高血压血管重塑,主要包括管壁增厚、管壁管腔比值增大和小血管减少,以及随之产生的血管功能异常。血管重塑不仅仅是一种代偿反应,也是高血压的主要病理基础之一,是引起高血压各种严重并发症以及循环系统功能紊乱的重要原因。血管重塑可导致一系列的临床事件,如高血压、动脉粥样硬化和血管狭窄等。目前的研究表明,高血压主动脉重塑是死亡率增加的主要原因之一。因此预防和改善血管重塑也是高血压治疗的主要目标之一和评价高血压治疗效果的重要指标。血管重塑的发病机制和防治措施已成为国内外的研究热点。高血压血管重塑是一个非常复杂的病理过程,其确切的机制仍不十分明确。研究发现,血管重塑涉及多个细胞过程的变化,如细胞肥大、增殖、调亡、重塑、炎症反应、氧化应激、细胞因子的活化、细胞外基质产生与降解等。高血压主动脉重塑的中心环节是因血管平滑肌细胞肥大导致的血管中膜增厚。
蛋白激酶D(PKD)家族是一种新发现的Ca2+/钙调蛋白依赖性的丝氨酸/苏氨酸蛋白激酶,主要包括PKD1、PKD2, PKD3三个成员。PKD结构与酶的活性均和PKC家族不同。越来越多的证据表明,PKD介导的信号通路在心血管系统中发挥重要作用,尤其在调节心肌收缩、心室肥厚及重塑中的作用。最近的研究显示血管平滑肌细胞也表达PKD,且PKD的活性能被一些刺激因素激活,如血管紧张素Ⅱ,血小板源性生长因子和血栓素等。用血管紧张素Ⅱ刺激血管平滑肌细胞时,PKD通过AT1/PKCδ途经被激活。另外有研究证实PKD可通过使组蛋白去乙酰酶5(HDAC5)磷酸化介导血管紧张素Ⅱ诱导的血管平滑肌细胞肥大。然而,PKD在自发性高血压大鼠主动脉组织中的活性表达及其在主动脉重塑中的作用机制还不是很明确。
高血压患者经常伴随血脂代谢紊乱,因此他汀类药物在高血压患者中应用广泛。越来越多的证据表明,他汀类药物除了能够有效降低血脂水平外,还能发挥其降脂以外的多项心血管保护效应,如：改善血管内皮功能、稳定动脉粥样硬化斑块、抗炎抗氧化以及阻止心室肥厚、改善心室重塑等。无论患者血脂是否正常,应用他汀类药物均能降低心血管疾病的致残率和死亡率。最近的研究表明,阿托伐他汀能抑制心肌肥厚及纤维化。然而,它在血管重塑中的保护作用及其具体机制还不是很清楚。
本课题动态观察高血压大鼠主动脉组织中PKD表达和活化特点及其与主动脉重塑的关系；研究PKD在高血压主动脉重塑中的作用机制；初步探讨阿托伐他汀对高血压主动脉重塑的改善作用及其分子机制。旨在从分子角度阐明高血压血管重塑的发病机制和他汀类药物改善血管重塑的机制。为高血压血管重塑的防治提供新思路和理论依据。
目的
1.进一步明确自发性高血压大鼠主动脉重塑的发生发展过程；
2.探讨自发性高血压大鼠主动脉组织中蛋白激酶D的活性表达在血管重塑中的作用。
3.评价阿托伐他汀在改善高血压主动脉重塑发生发展中的作用及与PKD的关系。
方法
8周龄WKY大鼠40只、自发性高血压(SHR)大鼠50只。WKY大鼠随机分为四组,A组：8周龄(n=10只)；B组：16周龄(n=10只)；C组：24周龄(n=10只),G组：WKY安慰剂(n=10只)。SHR大鼠随机分为五组：D组：8周龄(n=10只)；E组：16周龄(n=10只)；F组：24周龄(n=10只)；H组：SHR安慰剂(n=10只)；Ⅰ组：SHR阿托伐他汀(ATV)50mg·kg-1·d-1 (n=10只)。以标准大鼠饲料喂养及普通饮水。另外,WKY安慰剂组、SHR安慰剂组及SHR阿托伐他汀治疗组,自16周龄开始每天以蒸馏水或阿托伐他汀分别灌胃,持续8周。于8、16、24周处死动物,留取胸主动脉组织标本备用。实验过程中,进行以下检测：(1)各组大鼠每隔2周测量体重、心率及尾动脉血压一次；(2)分别于喂养前、喂养至16周、24周抽血测定空腹血脂；(3) HE、Masson及天狼腥红染色,观察主动脉壁内膜结构、中膜厚度、弹力纤维和胶原沉积变化；(4)测量主动脉壁中层厚度(MT)、内径(LD)、胶原体积分数(VFC),并计算MT/LD比值；(5)计算羟脯氨酸含量；(6) ELISA法检测炎症因子IL-6和TNF-α; (7) Western blot法检测硝基酪氨酸、PKD、p-PKD744/748、p-PKD916蛋白的表达。
结果
1.实验动物基本情况
实验过程中共有2只大鼠死亡,SHR大鼠组16及24周龄组各一只。在普通喂养周期内死亡。SHR阿托伐他汀治疗组无死亡。共88只大鼠完成实验,其中WKY组40只,SHR组48只。
2.各组大鼠基本指标比较
喂养前,各组大鼠心率、血脂均无显著差异(P>0.05)；SHR大鼠尾动脉收缩压高于WKY大鼠,差异显著(P<0.05)。WKY大鼠的血压在整个喂养过程中无明显升高,与WKY组比较,SHR大鼠血压随周龄逐渐升高,差异有显著性意义(P<0.05)；SHR阿托伐他汀治疗组尾动脉收缩压明显降低,有显著性差异(P<0.05)
实验前各组大鼠血甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)间差异无显著性统计学意义(P>0.05)。SHR阿托伐他汀组血清TC及LDL均较WKY安慰剂、SHR安慰剂组明显降低,差异有统计学意义(P<0.05),TG和HDL-C变化不明显(P>0.05)。
3.主动脉形态及组织病理观察
HE染色显示：WKY大鼠血管壁无明显增厚,平滑肌细胞体积较小,大小均一,排列整齐,中膜层弹力纤维排列有序,内膜均一,无增厚；SHR大鼠中膜明显增厚,中膜内平滑肌细胞肥大,形态不规则,排列紊乱,弹力纤维层扭曲,内皮细胞排列紊乱,内膜略有增厚,部分内皮细胞有脱落。随着周龄的增加,上述表现愈加明显。阿托伐他汀组与SHR安慰剂组相比较,内、中膜结构均明显改善,内膜较平滑均一,中膜增厚明显减弱,平滑肌细胞肥大减退,排列较规则,中层弹力纤维结构较完整有序。
Masson染色显示：WKY组弹力纤维排列整齐,胶原增生不明显；SHR组胶原纤维明显增多,弹力纤维减少、排列紊乱甚至断裂；阿托伐他汀组与SHR安慰剂组相比较,胶原增生减少,弹力层增多,排列较整齐。
天狼腥红染色显示：与WKY对照组相比,SHR组随着周龄增加,Ⅰ型胶原纤维和Ⅲ型胶原显著增加,阿托伐他汀组比SHR安慰剂组胶原纤维明显减少。
4.主动脉重塑相关指标变化
与WKY组比较,SHR组MT、MT/LD、VFC均显著增加(P<0.05)；阿托伐他汀组较SHR安慰剂组MT、MT/LD、VFC显著降低(P<0.05)。
5.羟脯氨酸含量变化
WKY大鼠在整个喂养过程中,羟脯氨酸含量无明显增加。与WKY组比较,SHR组羟脯氨酸随周龄显著增加(P<0.05)；阿托伐他汀组较SHR安慰剂组羟脯氨酸显著降低(P<0.05)。
6.炎症因子表达变化
与WKY组比较,SHR组IL-6和TNF-α均显著增加(P<0.05)；阿托伐他汀可明显降低IL-6和TNF-α(P<0.05)。
7.氧化应激
硝基酪氨酸是氧化应激的标志物,与WKY组相比,SHR组硝基酪氨酸表达明显增加(P<0.05)；阿托伐他汀可明显降低硝基酪氨酸表达(P<0.05)。
8. Westen blot检测各组主动脉组织中PKD及磷酸化PKD的表达
与同周龄WKY对照组相比,SHR8、16.24周龄组大鼠主动脉组织中p-PKD744/748、p-PKD916表达明显升高(P<0.05)；随着周龄的增加SHR组p-PKD744/748、p-PKD916表达水平逐渐升高(P<0.05)。同期WKY各组大鼠的P-PKD744/748、p-PKD916没有显著性变化。SHR阿托伐他汀组P-PKD744/748. P-PKD916较SHR安慰剂组的表达减弱,有统计学意义(P<0.05)。SHR各组总的PKD表达无明显变化。
结论
1.SHR大鼠随着喂养时间延长呈增龄性主动脉重塑,为高血压主动脉重塑发病机制的研究提供了可靠的动物模型平台。
2.SHR大鼠主动脉组织中磷酸化PKD的表达随着主动脉重塑过程逐渐升高,提示磷酸化PKD参与了自发性高血压大鼠主动脉重塑的发生发展过程。
3.阿托伐他汀可以改善自发性高血压大鼠主动脉重塑的发展,这与其抗炎抗氧化特性有关,其作用靶点可能与PKD有关。
研究背景
高血压状态下,大小动脉的结构和功能都会发生变化,从而引起血管重塑及心血管疾病的危险性增加。血管壁中膜增厚是高血压主动脉重塑最重要的特征性病理改变,其主要形成原因是管壁中层血管平滑肌细胞的肥大。高血压血管肥厚和重塑是对病理状态下血流动力学长期变化的一种代偿反应。高血压血管肥厚包含的细胞分子学过程有：VSMC肥大、增殖、迁移、凋亡、炎症反应、氧化应激和纤维化。
肾素血管紧张素系统(RAS)在高血压和心血管疾病的发生发展过程中发挥重要作用。实验证据证明,在高血压血管重塑的发生发展过程中,伴随着血管局部RAS的激活和血管紧张素Ⅱ(AngⅡ)的高水平表达。RAS的中心环节是AngⅡ识别其特异性受体,并通过一定的信号通路发挥作用。作为RAS系统的主要效应因子,AngⅡ是高血压心血管重塑的重要调节因子。AngⅡ是一种血管活性肽,通过升高血压引起血管重塑和内皮功能紊乱。然而,AngⅡ还可以通过不依赖血流动力学的作用介导血管重塑。大量的证据表明,AngⅡ通过刺激VSMC肥大而诱导病理性的血管重塑。AngⅡ还参与血管纤维化,通过TGF-β依赖性和非依赖性Smad通路引起血管纤维化。另外,AngⅡ是血管组织中ROS和促炎转录因子的主要调节因子。
ERK5是最近发现的MAPK家族的新成员,在不同类型细胞中的定位不同。多种刺激因素,如神经生长因子,表皮生长因子,血小板源性生长因子和血管紧张素Ⅱ都能激活ERK5。ERK5在将信号从细胞膜传递到细胞核的过程中激活了一个三级级联反应：MAPKKK或MEKK2/MEKK3; MAPKK或MEK5; MAPK或ERK5,受到刺激后,这些激酶持续磷酸化并活化下游的效应因子,包括肌细胞增强因子(MEF2)。ERK5在调节肥厚相关基因,尤其是MEF2依赖的c-jun基因方面有潜在作用,通过直接磷酸化MEF2使其活化,从而调节肥大反应。ERK5在AngⅡ刺激的大鼠主动脉平滑肌细胞中可激活下游的MEF2。
PKD作为肥厚调节因子,参与心脏与血管的肥厚性重塑。PKD对MAPK家族信号通路具有调节作用,如PKD通过磷酸化Ras结合蛋白RIN1而上调Ras和ERK1/2信号通路的活性；通过磷酸化c-Jun而下调JNK的活性。但是关于PKD对ERK5是否有调节作用,目前尚无相关研究。因此,本实验将进行体外AngⅡ诱导人主动脉平滑肌细胞(HASMCs)肥大实验,验证PKD/ERK5/MEF2信号通路是否参与其中,从而为高血压血管重塑的逆转提供新的治疗靶点。
研究目的
1.验证AngⅡ诱导人主动脉血管平滑肌细胞(HASMCs)肥大的作用；
2.明确AT1/PKCδ/PKD/ERK5/MEF2信号转导通路在Angll诱导HASMCs肥大中的作用机制；
研究方法
1.人主动脉血管平滑肌细胞的体外培养
HASMCs购自ScienCell (San Diego, CA)公司,在MEM培养基,37℃和5%CO2中培养,取代数在4-8,处于生长对数期的细胞进行实验。
4细胞分组处理
1)无刺激因素孵育细胞；
2) AngⅡ刺激细胞,筛选最佳刺激浓度和时间；
3) AngⅡ刺激+DMSO孵育细胞；
4) AngⅡ刺激+AT1拮抗剂Losartan孵育细胞；
5) AngⅡ刺激+AT2拮抗剂PD123319孵育细胞；
6) AngⅡ刺激+PKC抑制剂Go6983孵育细胞；
7) AngⅡ刺激+Control siRNA孵育细胞；
8) AngⅡ刺激+PKCδsiRNA孵育细胞；
9) AngⅡ刺激+PKD siRNA孵育细胞；
10) AngⅡ刺激+ERK5 siRNA孵育细胞。
3.对各组HASMCs进行如下检测：
1)电镜观察细胞形态学改变；
2)3H-亮氨酸摄入率测定,评估细胞肥大的程度；
3) Western blot测定PKC, p-PKCα/β,ζ,ε,δ, PKD、p-PKD744/748、P-PKD916、ERK1/2、p-ERK 1/2、ERK5、p-ERK5、MEF2C、p-MEF2C的表达,用特异性的抑制剂或si RNA阻断通路的各环节,观察下游分子的表达变化；
4)免疫荧光测定各分子的表达及ERK5在胞浆胞核之间的转位。
研究结果
1.AngⅡ刺激HASMCs的形态学变化
AngⅡ刺激组：加AngⅡ(以无血清培养液溶解)使终浓度为100nM；正常对照组(control):培养液中加等量无血清培养液。处理一定时间后在显微镜下观察细胞形态学变化。镜下观察可见在加用AngⅡ刺激后,HASMCs较正常对照组表现明显肥大。
2. AngⅡ刺激的HASMCs 3H—亮氨酸掺入率的测定
一定浓度的AngⅡ(100nM)刺激不同时间(0min、5min、15min、30min、60min),15分钟后HASMCs 3H-亮氨酸掺入较空白组明显增高(P<0.01)。当时间恒定时(15min),HASMCs 3H-亮氨酸掺入随着AngⅡ浓度(1nM、10nM、100nM、1000 nM)的增加呈剂量依赖性增加,后三组明显高于对照组,差异非常显著(P<0.01)。
3. Western blot测定AT1/PKCδ/PKD/ERK5/MEF2C信号通路
1) AngⅡ刺激HASMCs后,PKC不同亚型的激活
AngⅡ100nM不同时间(0、5、15、30、60min)刺激平滑肌细胞,观察不同亚型PKC磷酸化蛋白的表达,结果发现磷酸化PKCδ在5分钟时开始表达,15到30min达到表达高峰,以后则呈逐渐下降趋势,60min时表达恢复基线水平。PKCa/p和PKCε表达随时间无明显变化,PKCζ则在30min时开始表达,60min时表达有所增加。
2) AngⅡ对HASMs中ERK1/2和ERK5的激活
AngⅡ100nM不同时间(0、5、15、30、60min)刺激平滑肌细胞,观察ERK1/2、ERK5总蛋白及磷酸化蛋白的表达,结果发现磷酸化ERK5在5分钟时开始表达,15到30min达到表达高峰,以后则呈逐渐下降趋势,60min时表达恢复基线水平。磷酸化ERK1/2表达较早,5min表达达高峰,较0、15、30、60min有显著性差异(p<0.01)。ERK1/2、ERK5总蛋白表达在各时间段没有变化。
3)AngⅡ通过AT1激活ERK5
AngⅡ受体有两个亚型AT1、AT2。应用ATl特异性拮抗剂Losartan(0.3、1.0、3.0μmol/L)及AT2特异性拮抗剂PD123319(5、10、20μmol/L)预处理平滑肌细胞30min,然后AngⅡ100nM刺激15min,结果显示Losartan剂量依赖性阻断p-ERK5的表达,而PD123319对p-ERK5的表达没有影响。
4)AngⅡ激活ERK5依赖于PKCδ通路
应用PKC的抑制剂Go6983不同浓度(0.3、1、3μmol/L)预处理平滑肌细胞30min,AngⅡ100nM刺激15min,结果显示Go6983呈剂量依赖性抑制磷酸化ERK5的表达,由此证明PKC参与AngⅡ激活ERK5的过程。
用PKC8 siRNA预处理细胞后,再加入AngⅡ刺激,ERK5的磷酸化明显受到抑制,说明PKCδ是ERK5的上游分子。
5)AngⅡ介导的PKD激活呈AT1/PKCδ依赖性
AngⅡ诱导的PKD活化呈时间和浓度依赖性。AT1受体拮抗剂Losartan 3mmol/L阻断p-PKD的表达,而AT2受体拮抗剂PD123319 10mmol/L对p-PKD的表达没有影响；PKC抑制剂Go6983呈剂量依赖性抑制磷酸化PKD的表达；PKCδsiRNA明显抑制PKD的活化。由此证明AT1/PKCδ参与AngⅡ激活PKD的过程。
6)PKD参与AngⅡ介导的ERK5的激活
应用PKD siRNA转染HASMCs,观察下游分子ERK5的表达。结果显示：PKD siRNA抑制p-ERK5的表达,与对照组有显著性差异(P<0.01)。
7)PKCδ/PKD/ERK5参与了AngⅡ介导的MEF2C的激活
AngⅡ100nM不同时间(0、5、15、30、60min)刺激HASMCs,观察MEF2C的激活。结果显示：磷酸化MEF2C在5分钟时开始表达,15min达到表达高峰(p<0.01),30min表达恢复基线水平。PKC抑制剂、PKCδsiRNA、PKD siRNA、ERK5 siRNA显著减少磷酸化MEF2C的表达(p<0.01)。
8) PKCδ/PKD/ERK5参与了Angll介导的HASMCs肥大
AngⅡ100nM刺激15分钟,HASMCs 3H-亮氨酸摄入率明显增加(p<0.01)；PKC抑制剂、PKCδsiRNA、PKD siRNA、ERK5 siRNA显著降低3H-亮氨酸摄入率(p<0.01)。
4.免疫荧光
1)免疫荧光测定各分子的表达用免疫荧光细胞化学染色观察AngⅡ100nM刺激不同时间后HASMCs PKD、ERK5和MEF2C的表达。结果显示：PKD胞质表达,MEF2C胞核表达,而ERK5随着刺激时间不同在胞浆和胞核之间转位。
2)ERK5胞浆胞核之间的转位
静息状态下,ERK5位于细胞质中,AngⅡ100nM刺激5分钟后,ERK5逐渐向细胞核转位,15分钟时基本全部位于胞核内,随后ERK5又重新向细胞质转移,在1小时时基本恢复基线水平。
3) AT1/PKCδ/PKD信号通路参与了Angll介导的ERK5转位
AngⅡ100nM刺激15分钟后,ERK5的细胞核表达水平达最高。分别应用AT1受体拮抗剂,PKC抑制剂、PKCδsiRNA和PKD siRNA干扰细胞,阻断ERK5的磷酸化,ERK5的转位明显被抑制,提示ERK5的转位依赖于其磷酸化情况。
结论
1.验证了AngⅡ具有诱导HASMCs肥大的作用；
2.首次证明AT1/PKCδ/PKD/ERK5/MEF2C信号通路参与AngⅡ诱导的HASMCs肥大的过程。
Background
Hypertension is the most important risk factor for cardiovascular diseases. Vascular remodeling (VR) in hypertensive patients is an independent risk factor for cardiovascular events. Changes of the structure and function companied with hypertension is defined as VR in hypertension, which consist of thickness of the wall, increase of medium/lumen ratio, decrease of arterioles and consequent dysfunction of the vessel. VR is not only an adaptive response, but also the main foundation of hypertension pathology, which contributes to serious vascular complications and dysfunction of the circulation. It's a prime contributor to the pathogenesis of a number of clinical disorders, including hypertension, atherosclerosis and restenosis. Current evidence shows that pathological remodeling of aorta in hypertension is one major mechanism responsible for morbidity and mortality. Thus the prevention and reversal of VR is the major objective for treatment of hypertension and as an important indicator of treatment evaluation. The study on the pathogenesis and control of VR has become a worldwide hot topic. VR is a complex course, and the pathogenesis mechanism still remains unclear. It is well-known that cell hypertrophy, hyperplasia, apoptosis, remodeling, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in VR. The main feature of aortic remodeling is thickness of medium, due to hypertrophy of vascular smooth muscle cells (VSMC).
The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases. PKD family consists of 3 isoforms PKD1, PKD2 and PKD3. It's structural and enzymatic properties distinguish it from PKC isoforms. Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. In recent years, evidence has suggested that vascular smooth muscle cells also express PKD, and PKD activity in them has been shown to be increased by physiologically important stimuli, such as angiotensinⅡ, platelet-derived growth factor, and thrombin. Functionally, PKD activity appears to regulate hypertrophy in VSMCs. Studies have indicated that PKD may control hypertrophy of VSMCs via the AT1/PKCδpathway by Ang II. Most recently, it has been proposed that PKD mediated HDAC5 phosphorylation may facilitate angiotensinⅡ-induced hypertrophy in VSMCs. However, the activation of PKD and it's possible mechanisms in hypertensive aortic remodeling remain unclear.
As hypertension is often accompanied by dyslipidemia, the treatment frequently involves statins. More and more evidences established that statins not only effectively reduced serum cholesterol level, but also exerted pleiotropic beneficial effects on cardiovascular disease, including improvement of endothelial function, stabilization of atherosclerotic plaques, antioxidant properties, inhibition of inflammatory responses and prevention of cardiac hypertrophy or remodeling. Statins reduce cardiovascular morbidity and mortality in patients with high and moderate hypercholesterolemia or even normal cholesterol levels. Recently, atorvastatin have been shown to inhibit cardiac hypertrophy and fibrosis. However, the precise mechanism of vascular protective effects of atorvastatin remains to be fully clarified. This study was therefore designed to observe the expression and activation of PKD; to analyze its relationship with hypertensive aortic remodeling; to study the effects and the mechanism of atorvastatin in the prevention and treatment of aortic remodeling. The purpose of the study is to elucidate the cellular and molecular mechanisms of vascular remodeling associated with hypertension and the interventional effects of atorvastatin on it, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive vascular remodeling.
Objectives
1. To study the development of aortic remodeling in SHR.
2. To investigate the involvement of PKD in aortic remodeling in SHR
3. To evaluate the effects and the mechanisms of atorvastatin in the prevention and treatment of aortic remodeling.
Methods
8-week-old male SHRs were obtained from Genetic Models Inc. (Indianapolis, IN). Age-matched normotensive male WKY rats were purchased from animal center of Shandong University as the control. Rats were divided into the following groups:A Group:WKY-8W; B Group:WKY-16W; C Group:WKY-24W; G Group: WKY-placebo; D Group:SHR-8W; E Group:SHR-16W; F Group:SHR-24W; H Group:SHR-placebo; I Group:SHR-atorvastatin. Each group has 10 rats for 16 weeks treatment. WKY placebo, SHR placebo and SHR atorvastatin received distilled water or atorvastatin at 50 mg/kg/day by intragastric administration from 16 weeks to 24 weeks. Animals were killed when they were 8 weeks,16 weeks and 24 weeks old by decapitation. The aortas were immediately harvested and weighed. The following parameters were measured during the study:(1) All the rats have their body weight, heart rate and tail blood pressure measured once per 2 week; (2) Blood was collected at 8 weeks,16 weeks and 24 weeks respectively. Plasma lipids were determined using routine method; (3) Changes of vascular morphology and histology were measured by special H-E, tricrome Masson and Sirius red staining methods. Thickness of the media, collagen fibers and elastic fibers were used for the detection of remodeling. (4)Media thickness (MT), luminal diameter (LD), ratio of media to lumen (M/L) and volume fraction of collagen (VFC) were measured. (5) Hydroxyproline, an indicator to reflect collagen amount in mammalian tissues was measured. (6) Measure IL-6 and TNF-a by ELISA. (7) Nitrotyrosine is considered to be an indirect marker of oxidative stress, Western blot for the expression of nitrotyrosine. (8) Western blot for the expression of PKD、p-PKD744/748、p-PKD916.
Results
1. The experimental animals
Two rats of SHR group(E and F) died in the entire experiment. A total of 88 rats finished the study,40 rats in WKY group,48 rats in SHR group.
2. Comparisons of HR, SBP and Lipids level between WKY and SHR groups
There were no significant differences in terms of heart rat and lipids at the beginning of the experiment. SBPs in SHRs at 8.16 and 24 weeks are higher than those in WKY rats (P<0.05). In SHR group, SBP increased during the course, while the one in WKY group remained unchanged. After treatment with atorvastatin, SBP decreased significantly (P<0.05). The serum lipid levels of SHRs and those of WKY rats were not significantly different at the beginning of the experiment. Atorvastatin at 50 mg/kg/day significantly reduced the serum cholesterol and LDL-cholesterol of SHRs (P<0.05). However, no significant changes were found in TG and HDL-cholesterol levels with atorvastatin treatment.
3. Changes of vascular morphology and histology
H-E staining:In SHR group, aortic media were thickening; VSMCs in media were hypertrophy and disordered; elastic fibers were decreased and unregular; More smoothing aortic tunica intima, thinner vascular wall, less media VSMCs hypertrophy, more regular array of media VSMCs and elastic fibers were observed in atorvastatin group.
Masson staining:Aortic elastic fibers of WKY rats arranged in order and no hyperplasia of collagen; Aortic wall of untreated SHR was thickening; collagen fibers in media were hyperplastic; elastic fibers were decreased, disordered, and even ruptured; Aortic elastic fibers in Atorvastatin group were fairly ordered, collagen fibers were not hyperplastic significantly compared with SHR group.
Sirius red staining:Under polarization microscope, type I collagen is in colour red or orange, and type III collagen is in colour green. Much more collagen I and III existing in the vascular wall of untreated SHR group compared with WKY rats, which decreased significantly in the atorvastatin group.
4. Aortic remodeling parameters
Arterial MT of untreated SHR were significantly higher than WKY rats, and atorvastatin treated animals had lower MT compared with untreated animals in the model group. Luminal diameter (LD) in each group was no significant difference. The ratio of MT to LD in untreated SHR was significantly higher than WKY rats, whereas animals in the atorvastatin group had lower ratio of MT to LD compared with untreated SHR. Volume fraction of collagen (VFC) was significantly higher in untreated SHR than it in WKY rats, and it decreased in the atorvastatin group.
5. Effect of atorvastatin on hydroxyproline
Compared to WKY group, the groups of SHRs in 16W and 24W showed a significant rise in hydroxyproline content in aorta tissue (P<0.05), which were reduced by atorvastatin treatment.
6. Effect of atorvastatin on inflammatory cytokines
Compared to WKY group, IL-6 and TNF-a in SHRs increased significantly(P<0.05), which were reduced by atorvastatin treatment.
7. Effect of atorvastatin on oxidative stress in SHRs
Nitrotyrosine is considered to be an indirect marker of oxidative stress. In SHR group, nitrotyrosine increased during the course (P<0.05). while these in WKY group remained unchanged. These changes were attenuated by atorvastatin (P<0.05).
8. Expression of PKD, p-PKD744/748 and p-PKD916 by Western-blot
Compared to WKY group, the groups of SHRs in 16W and 24W showed a significant rise in p-PKD protein in aorta tissue (P<0.05). In SHR group, expression of p-PKD increased during the course (P<0.05). while these in WKY group remained unchanged. These changes were attenuated by atorvastatin (P<0.05).
Conclusions
1. The age-related aortic remodeling occured with the development of hypertension in SHRs, which offers reliable animal model for the researches on the mechanisms of hypertensive aortic remodeling.
2. The over-expression of p-PKD has been confirmed in the aorta tissue of SHR, which suggested that activation of PKD was involved in the development and process of aortic remodeling in SHR.
3. Atorvastatin could partially reverse the hypertension-induced aortic remodeling through its anti-inflammatory and anti-oxidant effects. Another mechanism for atorvastatins to reverse remodeling is down-regulation of PKD activation.
Background
In hypertension small and large arteries undergo structural, mechanical and functional changes that contribute to vascular remodeling and increased cardiovascular risk. Increase of vascular media thickness due to hypertrophy of vascular smooth muscle cell is the most important pathological change in hypertensive aortic remodeling. It is well established that vascular hypertrophy and remodeling in hypertension is an adaptive process in response to chronic changes in hemodynamic conditions during development and vascular pathologies. The cellular events underlying vascular hypertrophy during hypertension involve VSMC hypertrophy, hyperplasia, migration, apoptosis, inflammation, oxidative stress and fibrosis.
The renin-angiotensin system (RAS)-plays a key role in the development and pathophysiology of hypertension and cardiovascular disease (CVD). Evidences show that activation of local RAS and levels of angiotensinⅡ(AngⅡ) are elevated in the development of vascular remodeling in hypertension.The central part of RAS is recognizing of different receptors by AngⅡand then carrying out its functions through different signal pathways. AngⅡ, the main effector of the renin-angiotensin system (RAS), is one of the major mediators of vascular remodeling in hypertension. Ang II, a potent vasoactive peptide, induces vascular remodeling and endothelial dysfunction in association with increases in levels of BP. However, AngⅡis able to induce vascular remodeling independently of its haemodynamic effects. Mounting evidence shows that AngⅡactivation contributes to pathological vascular remodeling, largely by stimulating VSMCs hypertrophy. Ang II also is crucially involved in fibrosis, Ang II induced vascular fibrosis via both TGF-β-dependent and-independent Smad signaling pathways. Furthermore, Ang II is a strong modulator of ROS production and proinflammatory transcription factors in the vasculature.
ERK5 has recently been identified as a new member of the MAPK family, the subcellular localization of ERK5 is cell type specific. Various stimuli, including nerve growth factor, epidermal growth factor, platelet-derived growth factor, and angiotensin II (Ang II), activate ERK5. The central part of ERK5 cascades involved in signal transduction from the membrane to the nucleus is structured by three sequentially activated kinases:a MAPKKK family, or MEKK2/MEKK3; a MAPKK family, or MEK.5; and a MAP kinase, or ERK5. On stimulation, these kinases are consecutively phosphorylated and thus activated, leading to the activation of downstream effectors including MEF2. ERK5 has a potential role in the transcriptional regulation of hypertrophic genes, in particular MEF2-dependent genes such as c-jun gene through direct phosphorylation and activation of MEF2C, and govern the hypertrophic growth. ERK5 was recently found to interact with MEF2C on Ang II stimulation in rat aortic SMCs.
As a hypertrophic modulator, PKD was involved in cardiac and vascular hypertrophy. PKD regulates the signal pathway of the MAPK family. For example, PKD upregulates Ras activity and ERK1/2 signaling by phosphorylating the Ras-binding protein RIN1, and downregulates JNK activity, possibly by phosphorylating c-Jun. But, to our knowledge, little is known about the effect of PKD on ERK5. Therefore the aim of this study is to investigate the role of PKD/ERK5/MEF2C signaling pathway in Ang II induced hypertrophy of VSMC, which may provide a new target for reversal of hypertensive vascular remodeling.
Objectives
1. To observe the hypertrophy of HASMCs stimulated by Ang II.
2. To explore the AT1/PKCδ/PKD/ERK5/MEF2C pathway in Ang II-induced hypertrophy of HASMCs.
Methods
1. Cell culture
The human aortic smooth muscle cells, which was purchased from ScienCell (San Diego, CA), was cultured in MEM growth medium at 37℃and 5% CO2. Cells in generation 7-15 were used in the treatment.
2. Cells were divided into several groups:
1) Control Group:no stmulation factor;
2) AngⅡstmulation Group:different dosges of AngⅡwith different times;
3) AngⅡstmulation +DMSO Group;
4) AngⅡstmulation+AT1 antagonist Losartan Group;
5) AngⅡstmulation+AT2 antagonist PD123319 Group;
6) AngⅡstmulation+PKC inhibitor Go6983 Group;
7) AngⅡstmulation+Control siRNA Group;
8) AngⅡstmulation+PKCδsiRNA Group;
9) AngⅡstmulation+PKD siRNA Group;
10) AngⅡstmulation+ERK5 siRNA Group.
3. Detecting
1) Observing changes of HASMCs form by electron microscope;
2) 3H-leu incorporation rate, evaluated the level of cell hypertrophy;
3) Western blot dectected expression of p-PKCα/β,ζ,ε,δ,PKD、p-PKD744/748、
p-PKD916、ERK1/2、p-ERK1/2、ERK5、p-ERK5、MEF2C、p-MEF2C;
4) Detecting the expression of PKD、ERK5 and MEF2C and EKR5 translocation between cytoplasm and nucelus by immunofluorescence.
Results
1. Morphological changes of HASMCs stimulated by AngⅡAngⅡgroup:the final concentration is 100nM; Control group:HASMCs were incubated in serum-free medium. Morphological changes were observed by electron microscope after stimulation by AngⅡ. Hypertrophy phenomenon of HASMCs was significantly greater after stimulation with AngⅡ.
2. Measurement of [3H]-Leu incorporation HASMCs which were incubated in 24 orifice plates were made quiescent by incubation in serum-free DMEM medium for 24 h. Cells were stimulated by different concentrations of AngⅡ(0 nM, 1nM, 10nM,100 nM and 1000nM). [3H]-Leu incorporation were determined using a scintillation counter. [3H]-Leu incorporation was concentration dependent, beginning at 10 nmol/l AngⅡand with maximum effect at 1000 nmol/l AngⅡ.100nM AngⅡrapidly increased [3H]-Leu incorporation with peak incorporation at 15 and 30 min.
3. Western blot for AT1/PKCδ/PKD/ERK5/MEF2C signal-transduction pathway
1) Activation of PKCs by AngⅡinHASMCs HASMCs were stimulated by AngⅡat 100nM for different times (0,5,15,30, 60min), AngⅡ(100 nmol/L) induced phosphorylation of PKCδafter 5 min, with peak phosphorylation between 15 and 30 min (P<0.01), which returned to base line after 1 hr. However, the phosphorylation of PKCaα/βand PKCεremain the same, PKCζphosphorylation was later than that of PKCδ.
2) Activation of ERK1/2 and ERK5 by AngⅡinHASMCs HASMCs were stimulated by AngⅡat 100nM for different times (0,5,15,30, 60min), AngⅡ(100 nmol/L) induced phosphorylation of ERK5 after 5 min, with peak phosphorylation between 15 and 30 min (P<0.01), which returned to base line after 1 hr. However, the phosphorylation of ERK1/2 were earlier than that of ERK5, with peak phosphorylation at 5 min (P<0.01). Total protein level of ERK5 and ERK1/2 did not change.
3) AngⅡstimulates ERK5 activation through a AT1-dependent pathway AngⅡreceptor has two subtypes:AT1 and AT2, cells were pretreated for 30 mins with losartan (0.3,1.0,3.0μmol/L, a specific antagonist for AT1, or PD123319 (5, 10,20μmol/L), an antagonist for AT2, then stimulated with AngⅡ(100 nmol/L) for 15 mins. Losartan inhibited AngⅡ-induced ERK5 activation in a dose-dependent manner, whereas PD123319 had no effect on AngⅡactivation of ERK5.
4) Activation of ERK5 by AngⅡis PKCδ-dependent Cells were pretreated with the general PKC inhibitor Go6983 (0.3,1,3μmol/L) or PKCδsiRNA before exposure to AngⅡ(100 nmol/L) for 15 mins. Go6983 blocked ERK5 phosphorylation in a dose-dependent manner, PKC8 siRNA also inhibited the activation of ERK5, which suggests that PKC8 is involved in AngⅡ-stimulated ERK5 phosphorylation in HASMCs.
5) Ang II induces AT1/PKCδ-dependent activation of PKD in HASMCs AngⅡtime-and dose-dependently induces PKD phosphorylation both at Ser744/748 and at Ser916. Losartan (3 mmol/L) but not PD123319 (10 mmol/L) abolished AngⅡ-induced PKD phosphorylation, suggesting that AngⅡinduces activation of PKD via ATI receptor. Go 6983 also dose-dependently inhibited AngⅡ-stimulated PKD activation, PKC8 siRNA blocked PKD phosphorylation, which demonstrated that PKD activation is PKCδdependent.
6) Knockdown of PKD by siRNA inhibited AngⅡ-induced activation of ERK5 in HASMCs HASMCs were transfected with PKD siRNA and then stimulated with AngⅡfor the time indicated. Treatment of HASMCs with PKD siRNA significantly reduced endogenous PKD expression, whereas control siRNA had no effect. Decreasing PKD expression by its siRNA significantly inhibited AngⅡ-induced ERK5 activation in HASMCs.
7) PKCδ/PKD/ERK5 pathway is involved in Ang II-induced activation of MEF2C
AngⅡsignificantly (P<0.05) stimulated the phosphorylation of MEF2C by 15 min of treatment and returned to basal levels by 30 min. Go 6983 markedly blocked MEF2C activation while knocking down PKCδ, PKD and ERK5 expression by siRNA significantly attenuated activation of MEF2C.
8) PKCδ/PKD/ERK5 pathway is implicated in AngⅡ-stimulated HASMCs hypertrophy [3H]-leucine incorporation were significantly greater in AngⅡ(100 nM) treated cells than in controls (P<0.05). PKCs inhibitor, PKCδ, PKD and ERK5 siRNA greatly suppressed AngⅡ-induced [3H]-leucine incorporation (P<0.05).
3. Immunofluorescence staining
1) Immunofluorescence for expression of all molecules The expression of p-PKD744/748、p-PKD916、p-ERK5、p-MEF2C was observed by immunofluorescence staining. After stimulation by AngⅡfor 15 min, P-PKD744/748 and p-PKD916 were expressed in cytoplasm while p-ERK5 and p-MEF2C were in the nucleus.
2) Translocation of ERK5
Before stimulation of AngⅡ, ERK5 was located primarily in the cytoplasm of HASMCs, ERK5 nuclear entry was seen at 5 min after AngⅡstimulation, striking translocation from the cytoplasm to the nucleus was observed by 15 min after addition of AngⅡ, after 60 min of AngⅡtreatment, ERK5 gradually shuttled back to the cytoplasma.
3) Ang II stimulates ERK5 translocation via ATl/PKCδ/PKD-dependent Pathway
The pharmacological inhibitors for AT1 and PKC significantly inhibited AngⅡ-induced ERK5 translocation. Knocking down PKCδand PKD expression by siRNA also greatly attenuated AngⅡ-induced translocation of ERK5 in HASMCs, which suggest that ERK5 phosphorylation plays an important role in ERK5 translocation.
Conclusions
1. AngⅡinduced hypertrophy of HASMCs.
2. AT1/PKC8/PKD/ERK5/MEF2C signal pathway was involved in hypertrophy of HASMCs induced by AngⅡ.
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