Profilin-1在高血压血管重构中的作用机制研究
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摘要
原发性高血压(Essential hypertension, EH)是目前最常见的心血管疾病之一,是全球范围内的重大公共卫生问题。目前,高血压对人类健康最大的危害是其造成的主动脉、心脏、脑等重要靶器官的结构与功能的损害。高血压患者罹患心脑血管意外的概率远高于非高血压患者,因此保护高血压患者心脑等重要靶器官的功能是目前高血压治疗的关键。现已证实血管重构(vascular remodeling, VR)与高血压导致的靶器官损害有着密切关系,研究高血压状态下血管重构的相关机制具有重要意义。
随着人们对于高血压血管重构的深入研究,发现血管重构的发生机制十分复杂,目前已公认血管活性物质、血液动力学、遗传和神经体液等多种因素均参与了血管重构,但其确切发生机制尚不明确。若能确定在血管重构中起重要作用的关键分子,则将为高血压的防治提供新靶点,蛋白质组学技术为实现这一目标提供了契机。蛋白质是生物体内生命活动的最终体现者,具有时空性和可调节性。高血压模型主动脉组织的比较蛋白质组学研究,能够提供主动脉对于在高血压状态所作反应的特定早期分子,通过进一步验证所发现的蛋白质分子的功能和其在高血压血管重构中的作用,可成为高血压的早期诊断和治疗提供重要靶点。
本研究分析了不同周龄自发性高血压大鼠(spontaneous hypertensive rat,SHR)与WYK大鼠主动脉组织中差异表达的蛋白质,其中前纤维蛋白(Profilin-1)引人瞩目。Profilin-1是一种广泛存在于除骨骼肌之外其他机体组织的,约15kd大小的肌动蛋白结合蛋白,其主要参与了肌动蛋白的聚合及解聚；并与细胞的增殖、分化和运动过程以及其信号转导密切相关。研究证实,Profilin-1在动脉粥样硬化、高血压等血管损伤中,尤其在高血压血管重构中起着重要作用。利用体内腺病毒注射技术,我们研究了体内干扰／过表达Profilin-1后SHR大鼠和WYK大鼠的病理生理变化,进一步探讨了Profilin-1在高血压血管重构中的作用机制。
第一部分
不同周龄自发性高血压大鼠主动脉组织的蛋白质组学研究研究背景
蛋白质是生物体内生命活动的最终体现者,蛋白质组学即是研究组织或细胞内全部蛋白质的组成、动态变化规律等的科学。蛋白质组学是一个比基因组学研究更加复杂的,新的研究领域。它通过分析组织、细胞内所有蛋白质的构成、表达以及修饰情况,并进一步分析他们之间的相互作用,进而从整体上阐明生命活动的本质和规律。蛋白质的表达具有时空性和可调节性,其在执行功能时并不像基因组那样是基本固定的,而是动态多样的,同时多个蛋白总是相互作用,共同参与同一生命活动中。蛋白质组学为生命科学研究和医学研究提供了新的方法和思路。
比较蛋白质组学(comparative proteomics)是通过一定的技术和方法来比较分析蛋白质组,从而鉴定出有表达差异的蛋白或者蛋白质群。近年来比较蛋白质组学被人们越来越多的用到医学研究中。比较蛋白质组学主要是比较正常和病理的个体的蛋白质组,发现在疾病中特异表达的蛋白质,而这些特异分子即可以用于疾病的早期诊断和治疗中。同位素标记的相对和绝对定量技术(isobaric tags for relative and absolute quantitation, iTRAQ)是一种新的蛋白质组学定量研究技术。iTRAQ可以同时最多对8种不同的样品来进行绝对和相对定量,不需要凝胶,而是使用基于高度敏感和精确的串联质谱方法获得相对定量结果和绝对定量信息。
高血压状态下,血管重构发生发展的原因具有多重性和复杂性。现已确定,血管的内膜、中膜和外膜等多种成分均参与了血管重构过程,另外,血流动力学、血管活性物质、神经体液因素以及炎症和氧化应激均参与其中。若能确定在血管重构中起重要作用的关键分子,则将为高血压的防治提供新靶点,蛋白质组学技术为实现这一目标提供了契机。本研究使用iTRAQ研究不同周龄SHR大鼠与WKY大鼠主动脉组织中差异表达的蛋白,为高血压防治提供了新的方向。
研究目的
1.研究SHR大鼠主动脉病变的特点,观察5周龄和17周龄SHR大鼠血压、体重及主动脉的病变情况；
2.应用iTRAQ蛋白质组学技术分别研究5周龄SHR大鼠与同周龄WKY大鼠主动脉的差异表达蛋白,17周龄SHR大鼠与同周龄WKY大鼠主动脉的差异表达蛋白；
3.比较17周龄差异表达蛋白并验证部分关键差异蛋白的表达情况,进一步确定高血压状态下血管重构发生发展的靶位蛋白及可能机制。
研究方法
选取5周龄和17周龄雄性SHR大鼠各6只,以及同周龄的雄性WKY大鼠各6只,分别分为4组：S5组,S17组,W5组,W17组。适应性饲养3天后,测量各组大鼠血压和体重情况。
1.主动脉形态学研究：用乙醚麻醉动物后,迅速分离各组大鼠主动脉组织,置入4%多聚甲酸中固定,石蜡包埋后切片,行HE染色和天狼猩红染色并测量血管壁厚度,管壁/管径比,胶原含量百分比。应用透射电镜观察主动脉组织超微结构变化。
2.蛋白质组学研究：提取各组大鼠主动脉总蛋白,后用trypsin消化蛋白得到肽段。用iTRAQ试剂标记消化后的肽段：用113标记5周龄WKY大鼠组(W5组),114标记5周龄SHR大鼠组(S5组),115标记17周龄WKY大鼠组(W17组),116标记17周龄SHR大鼠组(S17组)。将标记的肽段混合后运用强阳离子交换柱(strong cation exchange, SCX)和C18柱进行分级分离,最后用MALDI-TOF/TOF和microQ-TOF进行质谱鉴定。使用相关软件合并并处理定量及检测所得的结果。最后,使用Panther软件对差异蛋白进行分子定位,分子功能和生物过程分析。
3.部分差异蛋白功能验证：使用Western blot技术检测各组大鼠主动脉中关键差异蛋白前纤维蛋白1(Profilin-1),丝切蛋白1(Cofilin-1)和极长链酰基辅酶A脱氢酶(Acyl-Coenzyme A dehydrogenase, very long chain; ACADVL)的蛋白表达情况。使用PCR技术检测Profilin-1和Cofilin-1的mRNA表达情况。
研究结果
1.各组大鼠主动脉形态学变化
HE染色显示,W5组大鼠主动脉形态大致正常,主动脉各层分界清楚。血管内膜光滑,血管中膜弹性纤维呈同心圆排列,中膜无增厚,平滑肌排列规则,无肥大增生。W17组和S5组大鼠主动脉出现轻微血管重构情况,主动脉内膜增生不明显,部分内膜脱落；中膜弹性纤维形态不规则,部分出现断裂,平滑肌细胞无增生。S17组大鼠主动脉出现明显的血管重构表现,血管损伤较W17组和S5组均加重。发现与同龄WK.Y大鼠相比,SHR组大鼠管壁厚度和管壁／管径比均增高。天狼猩红染色显示17周龄SHR组大鼠中膜可见胶原沉积,胶原面积显著高于17周龄WKY组大鼠。5周龄SHR组大鼠和17周龄WKY组大鼠胶原沉积情况无明显差异,5周龄WKY大鼠血管中膜中无胶原沉积现象。
透射电镜下W5组血管平滑肌细胞核形态正常,细胞器分布及核染色质正常。S5组平滑肌细胞无明显异常,但平滑肌细胞与弹性膜的排列紊乱。W17组的管腔侧可见凹陷,主要是由内膜脱落造成,弹力膜不均匀,胶原成分可见。S17组可见平滑肌细胞与弹力膜的排列完全紊乱,平滑肌细胞表型改变显著,弹力膜内大量胶原成分的出现。
2. iTRAQ质谱鉴定结果
经质谱鉴定,在W5组和S5组大鼠主动脉中差异表达蛋白共92个,其中1.5倍以上差异蛋白8个；W17组和S17组大鼠主动脉中差异表达蛋白94个,其中1.5倍以上差异蛋白18个。
S5组和W5组的差异蛋白中仅有2个(ACADVL和Profilin-1)在W17组和S17组中有1.5倍以上的差异表达,其他蛋白差异表达均在1.5倍以下。W17组和S17组主动脉差异表达蛋白在S5组和W5组均有差异表达,但除却上述提到了2个蛋白外,其他蛋白在5周时表达均在1.5倍以下。
3.差异蛋白的定位分析(subcellular localization)
通过Panther软件分析差异蛋白的细胞定位,W17组和S17组的差异蛋白定位主要包括细胞内(32.9%),细胞器内(27.4%),细胞外部分(12.3%),细胞膜成分(8.2%),细胞外基质(6.8%),大分子复合物(6.8%)。
4.差异蛋白的分子功能(molecular function)及参与的生物过程分析(biological process)
17周SHR大鼠和WKY大鼠的差异蛋白主要涉及催化活性(catalytic activity)、结构分子活性(structural molecule activity)、结合功能(?)(binding)、受体活性(receptor activity)和酶调节活性(enzyme regulator activity)等分子功能。其参与的生物过程主要包括代谢过程(metabolic process)、细胞过程(cellular process)、生长过程(development process)、多种细胞器过程(multicellular organismal process)和细胞定位(location)等。
5.部分差异蛋白在主动脉组织的表达改变
为验证蛋白质组学鉴定出的部分差异蛋白在4组大鼠主动脉组织中的表达,本研究应用Western blot技术检测了关键差异蛋白：Profilin-1, Cofilin-1和ACADVL的蛋白表达情况。结果显示,骨架蛋白Profilin-1和Cofilin-1在同周龄的SHR大鼠中表达均高于WKY大鼠,且在17周龄大鼠中其差异表达更明显。ACADVL在同周龄SHR大鼠中表达较WKY大鼠均有所下降。用PCR技术检测Profilin-1和Cofilin-1的mRNA表达情况,证实两种蛋白的InRNA表达与上述Western blot检测的蛋白表达水平一致。
结论
(1) iTRAQ技术是比较蛋白质组学的新技术,灵敏性高,重复度好,可用于筛选高血压相关的差异蛋白,以寻找高血压的蛋白标志物,阐明其发病机制。
(2)17周龄WKY大鼠因血管老龄化出现血管重构现象,17周龄SHR大鼠血管重构较17周龄WKY大鼠明显加重,其血管重构是因为高血压和血管老化两方面原因。
(3)定位于胞浆的骨架蛋白和位于线粒体的能量代谢相关蛋白的差异蛋白在高血压的发生发展中起着重要作用。
(4)部分在5周龄SHR大鼠中有差异性表达的蛋白,在17周龄中并没有差异性表达,证明某些蛋白只在高血压早期发挥了作用。
第二部分
Profilin-1在自发性高血压大鼠血管重构中的作用机制研究研究背景
氧化应激是导致高血压的重要原因,高血压状态下可检测到活性氧(reactive oxygen species, ROS)产生增多。高血压状态下的血管重构是一个复杂的过程,是血管对刺激的复杂的动态反应过程,其主要过程不仅包括细胞增殖、凋亡和迁移,同样包括基质成分的合成、分解以及重新排列。氧化与抗氧化系统间的平衡紊乱参与了上述各个环节,从而导致了血管重构的发生发展。
本研究第一部分用iTRAQ蛋白质组学技术筛选出的差异蛋白中,前纤维蛋白(Profilin-1)尤为引人瞩目。Profilin-1是一种广泛存在于除骨骼肌之外其他机体组织的,约15kd大小的肌动蛋白结合蛋白,其主要参与了肌动蛋白的聚合及解聚；并与细胞的增殖、分化和运动过程以及其信号转导密切相关。现已证实Profilin-1可促进细胞外信号调节激酶1/2(ERK1/2)、Jun氨基末端激酶(JNK)及p38MAPK的磷酸化水平增加,从而在动脉粥样硬化、高血压等血管损伤过程中起到重要作用。此外,也有报道证实Profilin-1可以促进血管中的氧化应激水平,促使动脉粥样硬化的发生发展。
细胞实验和动物体内实验均证实,ROS可以促进血管重构。ROS参与了高血压血管重构的各个过程,它可以导致内皮功能失调、血管中膜增厚,平滑肌细胞迁移以及细胞外基质重构。ROS家族中的H2O2, ONOO-和HOCL等均在高血压血管重构中发挥着重要作用。其中,ONOO-可以通过氧化／硝基化作用硝化酪氨酸,对血管内皮等造成严重损伤。心血管系统中ONOO-的生成主要与NO的产生相关,在血管中诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)可以催化合成大量的NO,生成高浓度的ONOO-。 ONOO-是一个强氧化剂分子,它可以通过促进氧化损伤而造成内皮损伤,导致内皮细胞屏障功能的丧失、血小板粘附和血管调节异常,从而影响内皮功能；此外,ONOO-还可以激活与血管重构密切相关的JNK信号通路,ERK1/2信号通路。本研究采用SHR大鼠模型证实,ONOO-与高血压状态下血管重构有着密切的关系。
研究目的
1.实现SHR大鼠Profilin-1过表达腺病毒载体和Profilin-1干扰腺病毒载体的体内转染,观察Profilin-1对SHR大鼠血管重构的影响；
2.检测SHR大鼠体内炎症和氧化应激水平的变化,并探讨Profilin-1对于氧化应激的作用及其在血管重构中的作用机制；
3.在分子水平阐述Profilin-1对于高血压血管重构的作用机制。
研究方法
5周龄雄性SHR大鼠75只,同周龄雄性WKY大鼠20只,适应性饲养3天后进入实验。SHR随机分为3组：过表达组(SHR-E组)25只,干扰组(SHR-I组)25只,对照组(SHR-C组)25只。各组大鼠通过尾静脉注射分别给予3*109单位相应病毒载体两次,两次之间相隔6周。第一次注射3周后,分别处死各SHR组大鼠中每组5只大鼠。第二次注射6周后,处死其余大鼠。SHR-E组给予Profilin-1过表达腺病毒载体注射；SHR-I组给予Profilin-1干扰腺病毒载体注射；SHR-C组和WKY组给予阴性对照腺病毒载体注射。
1.实验开始前及实验中每周均测量大鼠体重各1次,并使用大鼠尾动脉间接测量法(尾套法)测量大鼠尾动脉收缩压。
2.留取转染3周的15只SHR大鼠的主动脉组织,检测血管重构的形态学变化,以及profilin-1, iNOS, p-p38的蛋白表达情况,并测定硝基酪氨酸含量。
3.第二次注射6周后处死大鼠,制作主动脉冰冻切片,于荧光显微镜下观察绿色荧光蛋白的表达,以确定转染效率；并检测主动脉组织中Profilin-1蛋白质和]mRNA表达情况,确定转染效率。
4.留取血清进行NO,IL-6检查。制作主动脉石蜡切片进行HE染色、天狼猩红饱-苦味酸染色观察主动脉形态。进行免疫组织化学染色检测相关蛋白在主动脉中的定位表达情况。并留取组织冻存,进行Western Blot和Real-time PCR检测。
研究结果
1.血压和体重情况
12周实验期间,各组大鼠体重之间差异不大。各SHR组大鼠的血压随着实验进行逐渐升高,而WKY大鼠血压变化不大,但各组SHR大鼠血压之间的差异没有统计学意义(P>0.05)。
2.腺病毒在大鼠体内转染效率
制作各组大鼠主动脉冰冻切片,并在荧光显微镜下观察大鼠主动脉壁绿色荧光蛋白(green fluorescent protein, GFP)的表达情况。接受腺病毒载体注射大鼠,其主动脉内皮细胞及平滑肌细胞内均有GFP的表达；而生理盐水组主动脉内未见GFP的表达,证明体内转染有效。
检测各组大鼠主动脉Profilin-1蛋白及mRNA表达情况,结果显示,与SHR-C组相比,Profilin-1蛋白及mRNA表达在SHR-E组均明显升高(P<0.05),而在SHR-I组则降低(P<0.05),进一步证明profilin-1过表达／干扰腺病毒体内转染有效。
3. Profilin-1转染3周对大鼠主动脉的影响
第一次腺病毒注射3周后,从SHR-E、SHR-C、SHR-I三组大鼠中,每组处死5只大鼠,观察profilin-1过表达／干扰腺病毒转染对于大鼠主动脉的影响。三组大鼠主动脉形态学研究发现,三组大鼠主动脉形态、血管内径、血管壁厚度以及血管壁／管腔比值没有显著性差异。血管形态大致正常。
Western blot检测三组大鼠主动脉profilin-1, iNOS, p-p38的蛋白表达情况,结果显示,三种蛋白在各组大鼠之间表达趋势一致,即SHR-E组大鼠三种蛋白表达均高于SHR-C组,而SHR-I组大鼠中三种蛋白表达降低。iNOS免疫组化染色所显示趋势同Western blot结果一致。
通过进行硝基酪氨酸(Nitrotyrosine)染色来评价血管内过亚硝酸根(Peroxynitrite, ONOO-)含量,SHR-E组的染色面积和H评分较SHR-C组明显增加,但SHR-I组则表现出显著的下降(P<0.05)。
4. Profilin-1转染12周对大鼠主动脉形态学影响
WKY组为正常主动脉形态。各SHR组大鼠主动脉均出现明显的血管重构改变,血管损害程度、管壁厚度都较3周时更为严重。SHR-C组大鼠主动脉出现明显的血管损害：内膜局部有隆起,部分内皮细胞脱落；中膜弹性纤维部分断裂,中膜增厚明显,平滑肌细胞增生肥大。SHR-E组大鼠主动脉损害较SHR-C组更为严重,SHR-I组大鼠内皮细胞的损害较SHR-C组轻。
WKY组大鼠中膜未见胶原沉积,而SHR-C组大鼠中膜可见部分胶原沉积,SHR-E组大鼠血管胶原沉积明显,但在SHR-I组大鼠血管中,没有明显胶原沉积。
5. Profilin-1转染12周对大鼠主动脉的影响
Western blot检测三组大鼠主动脉iNOS, p-p38的蛋白表达情况,结果显示,两蛋白在各组大鼠之间表达趋势一致,且同Profilin-1趋势相同,即SHR-E组大鼠两种蛋白表达均高于SHR-C组,而SHR-I组大鼠中两种蛋白表达降低。iNOS免疫组化染色所显示趋势同Western blot结果一致。
硝基酪氨酸染色结果趋势同3周时一致,染色程度、面积及H评分均较3周增加。
6. Profilin-1转染12周对大鼠NO，IL-6的变化。
利用硝酸还原酶法分别检测腺病毒转染12周后各组大鼠血清及主动脉中的NO含量,结果显示,四组大鼠血清中NO含量没有显著性差异(P>0.05)。对于大鼠主动脉局部NO的检测显示,各组大鼠主动脉中NO趋势同Profilin-1趋势相反。而各组中炎性因子IL-6的表达趋势同Profilin-1趋势相同。
结论
(1)SHR大鼠的血管重构伴随着体内氧化/抗氧化平衡紊乱及Profilin-1的过表达。
(2) Profilin-1过表达可以促进大鼠体内氧化应激水平和血管重构,干扰Profilin-1表达则可逆转上述变化。(3) Profilin-1对于大鼠血管重构的作用与p38/iNOS/ONOO-通路相关。
Essential hypertension is one of the most common cardiovascular disease in our country, it is also a worldwide public health problem. The major damage caused by hypertension is its effect on the target organs including heart, brain, kidney and vessels. Vascular remodeling is an important mechanism of hypertension-induced target organs damage.
The pathogenesis mechanism of vascular remodeling is complicated, it is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. With the development of proteomic techniques, a quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ) was introduced, which provides an opportunity to solve this problem. iTRAQ enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. It has been widely used in finding the pathogenesis, disease markers, the differentially expressed proteins in diseases. We resorted to iTRAQ technique of the aorta using spontaneous hypertensive rats (SHRs), SHRs are well-established animal model to study hypertension vascular remodeling. This experiment explore the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group, then validate for Profilin-1, a markedly differential protein's effect in vascular remodeling, thus provide candidate targets for clinical treatment of hypertension.
Part One
The proteomic analysis of aortic tissue of different week-old spontaneous hypertensive rats Background
Protein is the ultimate embodiment of vital movement, proteomics is a discipline which focus on all the proteins and their dynamic regularity in tissue or cell. Proteomics explicates the essence and regularity of vital movement on the whole through analyzing the composition, expression and modification of all the proteins and their interactions in tissue and cell. Proteomics is a new research field and it is more complex than genomics. The expression of protein has spatiality and regulatability, and when the protein functions, doesn't like the genome who is almost invariable, it is dynamic, and many proteins are involved in one kind of vital movement at the same time. Proteomics have provided new methods and ideas for the research on life science.
Comparative proteomics is a kind of approach that compares and analyzes the proteome to find the differences of expression of proteins. In recent years, the comparative proteomics is used in medical research more and more frequent. By comparing with the normal individual, we can find the specific proteins which only express in the abnormal individual and these specific proteins can be utilized in the early diagnosis and treatment of diseases. Isobaric tags for relative and absolute quantification (iTRAQ) is a new quantitative technique for proteomics analysis. It can be used for separating up to8different samples at the same time, with high repeatability and good quantitative effect.
It is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. iTRAQ provides an opportunity to solve this problem, which enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. We resorted to iTRAQ technique of the aorta using SHRs, explored the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group.
Objectives
1. To research the characteristics of aortic pathological changes of SHR, to observe the blood pressure, weight and aortic pathological changes of five-week-old and seventeen-week-old SHR.
2. To explore the differentially expressed proteins of aorta in five-week-old and seventeen-week-old SHR and the same week-old WKY rats by iTRAQ.
3. To find out the aortic differentially expressed proteins of five-week-old and seventeen-week-old rats, further confirm the target proteins and potential mechanism of vascular remodeling under hypertension.
Methods
Male SHRs (n=6,5weeks old; n=6,17weeks old), male WKY (n=6,5weeks old; n=6,17weeks old) assigned to4groups respectively:S5group, S17group, W5group, W17group. After3days adaptive feeding, blood pressure and weight were measured.
1. Aortic morphology:After anesthetized, aortic tissue of rats were extracted, and fixed in10%formalin, then embedded in paraffin for HE staining, the internal circumference and wall thickness of the thoracic aorta were determined using Image-Pro Plus. Aortic fibrosis was shown on histological pictures, where red and yellow fibers represent collagen I and green fibers show collagen Ⅲ. Picrosirius red staining was observed by polarized light and measured by Image-Pro Plus to obtain the percentage of collagen per media area. Or fix the aortic tissue in glutaraldehyde for ultrastructure observation using electron microscope.
2. Proteomic analysis:The proteins-enriched fractions were digested by tripsin. The resulting complex peptide mixtures were labeled with iTRAQ reagents (113for the W5group,114for the S5group,115for the W17group and118for the S17group respectively), All the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges. After being labeled, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF to evaluate the effectiveness of reagents labeling. Panther software was applied to analyze protein functions.
3. Western blot and PCR:4differentially expressed proteins were validated using western blot and PCR.
Results
1. Aortic Morphology
Aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of5-week SHRs and17-week WKY, and17-week SHRs showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes. Compared with17-week WKYs,17-week SHRs showed increased collagen deposition in the media.
Under electron microscopy, normal ultrastructure was observed in the aortic tissue of W5, and the rupture of vascular elastic membrane and the increased collagen fibers were observed in the aorta of W17and S5rats. Moreover, ultrastructure changed more obviously in S17rats.
2. Mass Spectrometry Identify the Differentially Expressed Proteins
We identified92differential proteins between W5group and S5group, among which the expression of8proteins was significantly changed;94differential proteins between W17group and S17group, among which the expression of18proteins was significantly changed.
3. Subcellular localization analysis vascular remodeling associated proteins
We performed the localization analysis of the identified proteins by Panther software. Cellular component of differentially expressed proteins among W17group and S17group mainly included cell part, extracellular region, organelle.
4. Protein function analysis vascular remodeling associated proteins
Molecular function of differentially expressed proteins among W17group and S17group mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.
5. Differentially expressed proteins in aorta
Protein and mRNA levels of Profilin-1and Cofilin-1were higher in SHRs than in same aged WKYs, protein level of AC AD VL was lower in SHRs.
Conclusions
1. As a new technique of quantitative proteomics, iTRAQ is more accurate and reproducible, enables comparative analysis for many groups. We obtained reliable differentially expressed proteins from different aged SHRs and WKYs, and find potential new drug targets for hypertension and its complications.
2. Molecular function of differential proteins we identified mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.
Part Two
Profilin-1promotes the development of hypertension-induced artery remodeling
Background
Vascular hypertrophy and remodeling in hypertension is an adaptive process in response to chronic changes in hemodynamic conditions during the development of vascular diseases. The ensuing changes in the size and/or composition of a remodeling blood vessel allow blood vessels to adapt and heal but they also underlie the pathogenesis of major cardiovascular diseases.
Profilin-1is a ubiquitous, small (12-15kD) actin-binding protein that plays an important role in the regulation of actin polymerization and cytoskeleton remodeling by activating hypertrophic signaling cascades, such as mitogen activated protein kinase (MAPK) signaling, to thus contribute to vascular hypertrophy and hypertension. Numerous studies have provided strong evidence for the important role of profilin-1in vascular inflammation and vascular remodeling, and have linked profilin-1to inducible nitric oxide synthase (iNOS) and peroxynitrite production. Also, peroxynitrite has been linked to vascular remodeling because it activates MAPKs (c-Jun N-terminal kinase [JNK], extracellular signal-regulated kinase1/2[ERK1/2]). However, the relationship between profilin-1, iNOS and the associated remodeling in SHRs remains to be fully clarified. Here, we studied the effects of profilin-1on hypertension-induced vascular remodeling and its potential relationship with iNOS. We used SHRs as a classical, genetic hypertensive animal model. Overexpression of profilin-1significantly promoted arterial structural remodeling in SHRs through a p38-iNOS-peroxynitrite-pathway. Profilin-1may be a potential target for genetic therapy of hypertension-induced arterial structural remodeling.
Objective
1. To investigate the effect of Profilin-1on vascular remodeling by transfection of pAd-profilin-1-IRES-EGFP and pAd-miR-profilin-1into SHRs;
2. To study the effect of Profilin-1on vascular inflammation and oxidative stress, and the relationship between oxidative stress and vascular remodeling.
3. To explore the mechanism of Profilin-1on vascular remodeling in molecular, histological and functional levels.
Methods
Five-month-old male SHRs (n=75) and Wistar-Kyoto rats (WKYs)(n=20) were obtained from Slac Laboratory Animal Co. SHRs were divided into3groups (n=25each) for treatment:profilin-1overexpression adenovirus vector (SHR-E), profilin-1miRNA adenovirus vector (SHR-I) or negative control adenovirus (SHR-C) after3days adaptation. WKYs (n=20) were treated with negative control adenovirus at the same time. The adenoviruses were injected twice into the tail vein at3x109infectious units per rat with an interval of6weeks between injections. At3weeks after the first administration,15SHRs from each of the three SHR groups (n=5each) were killed: At6weeks after the second administration of adenovirus, the remaining SHRs (n=60) and WKYs were killed.
1. Systolic blood pressure (SBP) and body weight were measured in conscious animals before the start of treatment and weekly during treatment. Systolic blood pressure (SBP) was measured by means of a tail-cuff sphygmomanometer.
2. Six days after the second injection of adenovirus, five rats in each group were killed and thoracic aortas were removed to determine transfection efficiency. Thoracic aorta specimens were sectioned at5μm for enhanced green fluorescent protein (EGFP) observation. Tissues underwent sky blue staining to shield the autofluorescence of the vascular tissues and DAPI staining was used to locate the nuclei. EGFP expression in the vascular tissues was observed by fluorescence microscopy.
3. Changes of vascular morphology and histology were measured by HE staining, Picrosirius-red-staining.
4. Nitrotyrosine is considered to be an indirect marker of oxidative stress, measured by immunohistochemical analysis.
5. Measure IL-6and NO by ELISA.
6. Western blot for the expression of p-p38, Profilin-1and iNOS, RT-PCR for mRNA expression of Profilin-1and iNOS.
Results
1. Transfection Efficiency of Adenovirus Vector
The high transfection efficiency was confirmed by observing EGFP expression under fluorescence microscopy and detecting the mRNA and protein expression of Profilin-1. EGFP expression was observed in more than90%cells for each transduction group. Profilin-1expression was higher in HUVECs and aortic tissues after profilin-1overexpression by adenovirus vector transfection, but was reduced by profilin-l miRNA vector treatment.
2. Effect of Profilin-1on Body Weight and SBP of SHRs
Body weight did not differ among the four groups during the12weeks of treatment. SBP for the three SHR groups increased with age, no significant difference between SHR-C and the other SHR-groups (p>0.05).
3. Effect of profilin-1on3-week-treatment SHRs
At3weeks after the administration of the adenovirus, we found that the lumen size (internal circumference), wall thickness and wall to lumen ratio (wall thickness: internal diameter) did not differ among the three SHRs groups. Protein level of iNOS and phosphorylation of p38were higher in SHR-E than in SHR-C tissues, but were decreased by the knockdown of profilin-1. We also found an increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E (0.530±0.028) and a decrease in SHR-I (0.072±0.016), as compared with SHR-C.
4. Profilin-1Promotes Vascular Hypertrophy and Fibrosis
This increased Profilin-1expression was accompanied by vascular remodeling in SHRs, as evidenced by a36.8%increase in wall thickness, a12%increase in wall-lumen ratio (the wall thickness:internal diameter) and increased vascular fibrosis. Compared with SHR-C, SHR-E showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes.
5. Effect of Profilin-1on Remodeling in SHR via the p38iNOS and Peroxynitrite-dependent Mechanism
Total p38protein expression did not differ among the four treatment groups, but phosphorylation of p38was higher in SHR-E than in SHR-C. In contrast, phosphorylation of p38was decreased by the knockdown of profilin-1. Thoracic aorta expression of iNOS showed a similar trend to that of p38phosphorylation.
Our results showed a significant increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E tissues but a significant decrease in SHR-I tissues, as compared with SHR-C. IL-6level was significantly higher in SHR-E as compared with SHR-I and SHR-C.
Next, we investigated the production of NO in aortic tissues and found a lower level of NO in SHR-E as compared with SHR-I (1.45±0.15vs.2.34±0.16μmol/g protein nitrites, p<0.05) and as compared with SHR-C (1.88±0.14μmol/g protein nitrites). We also tested the NO levels in the serum of rats and found no significant difference among the three SHR groups, indicating that changes in NO levels are a local phenomenon rather than a systemic reaction.
Conclusions
1. Vascular remodeling of SHRs was companied with oxidative stress reactions and over expression of Profilin-1.
2. Over expression of Profilin-1could facilitate the vascular remodeling and oxidative stress reactions, and knockdown of Profilin-1reversed those changes.
3. Prohypertrophic effects of profilin-1are mediated, at least in part, by suppression of the p38-iNOS-peroxynitrite pathway.
随着人们对于高血压血管重构的深入研究,发现血管重构的发生机制十分复杂,目前已公认血管活性物质、血液动力学、遗传和神经体液等多种因素均参与了血管重构,但其确切发生机制尚不明确。若能确定在血管重构中起重要作用的关键分子,则将为高血压的防治提供新靶点,蛋白质组学技术为实现这一目标提供了契机。蛋白质是生物体内生命活动的最终体现者,具有时空性和可调节性。高血压模型主动脉组织的比较蛋白质组学研究,能够提供主动脉对于在高血压状态所作反应的特定早期分子,通过进一步验证所发现的蛋白质分子的功能和其在高血压血管重构中的作用,可成为高血压的早期诊断和治疗提供重要靶点。
本研究分析了不同周龄自发性高血压大鼠(spontaneous hypertensive rat,SHR)与WYK大鼠主动脉组织中差异表达的蛋白质,其中前纤维蛋白(Profilin-1)引人瞩目。Profilin-1是一种广泛存在于除骨骼肌之外其他机体组织的,约15kd大小的肌动蛋白结合蛋白,其主要参与了肌动蛋白的聚合及解聚；并与细胞的增殖、分化和运动过程以及其信号转导密切相关。研究证实,Profilin-1在动脉粥样硬化、高血压等血管损伤中,尤其在高血压血管重构中起着重要作用。利用体内腺病毒注射技术,我们研究了体内干扰／过表达Profilin-1后SHR大鼠和WYK大鼠的病理生理变化,进一步探讨了Profilin-1在高血压血管重构中的作用机制。
第一部分
不同周龄自发性高血压大鼠主动脉组织的蛋白质组学研究研究背景
蛋白质是生物体内生命活动的最终体现者,蛋白质组学即是研究组织或细胞内全部蛋白质的组成、动态变化规律等的科学。蛋白质组学是一个比基因组学研究更加复杂的,新的研究领域。它通过分析组织、细胞内所有蛋白质的构成、表达以及修饰情况,并进一步分析他们之间的相互作用,进而从整体上阐明生命活动的本质和规律。蛋白质的表达具有时空性和可调节性,其在执行功能时并不像基因组那样是基本固定的,而是动态多样的,同时多个蛋白总是相互作用,共同参与同一生命活动中。蛋白质组学为生命科学研究和医学研究提供了新的方法和思路。
比较蛋白质组学(comparative proteomics)是通过一定的技术和方法来比较分析蛋白质组,从而鉴定出有表达差异的蛋白或者蛋白质群。近年来比较蛋白质组学被人们越来越多的用到医学研究中。比较蛋白质组学主要是比较正常和病理的个体的蛋白质组,发现在疾病中特异表达的蛋白质,而这些特异分子即可以用于疾病的早期诊断和治疗中。同位素标记的相对和绝对定量技术(isobaric tags for relative and absolute quantitation, iTRAQ)是一种新的蛋白质组学定量研究技术。iTRAQ可以同时最多对8种不同的样品来进行绝对和相对定量,不需要凝胶,而是使用基于高度敏感和精确的串联质谱方法获得相对定量结果和绝对定量信息。
高血压状态下,血管重构发生发展的原因具有多重性和复杂性。现已确定,血管的内膜、中膜和外膜等多种成分均参与了血管重构过程,另外,血流动力学、血管活性物质、神经体液因素以及炎症和氧化应激均参与其中。若能确定在血管重构中起重要作用的关键分子,则将为高血压的防治提供新靶点,蛋白质组学技术为实现这一目标提供了契机。本研究使用iTRAQ研究不同周龄SHR大鼠与WKY大鼠主动脉组织中差异表达的蛋白,为高血压防治提供了新的方向。
研究目的
1.研究SHR大鼠主动脉病变的特点,观察5周龄和17周龄SHR大鼠血压、体重及主动脉的病变情况；
2.应用iTRAQ蛋白质组学技术分别研究5周龄SHR大鼠与同周龄WKY大鼠主动脉的差异表达蛋白,17周龄SHR大鼠与同周龄WKY大鼠主动脉的差异表达蛋白；
3.比较17周龄差异表达蛋白并验证部分关键差异蛋白的表达情况,进一步确定高血压状态下血管重构发生发展的靶位蛋白及可能机制。
研究方法
选取5周龄和17周龄雄性SHR大鼠各6只,以及同周龄的雄性WKY大鼠各6只,分别分为4组：S5组,S17组,W5组,W17组。适应性饲养3天后,测量各组大鼠血压和体重情况。
1.主动脉形态学研究：用乙醚麻醉动物后,迅速分离各组大鼠主动脉组织,置入4%多聚甲酸中固定,石蜡包埋后切片,行HE染色和天狼猩红染色并测量血管壁厚度,管壁/管径比,胶原含量百分比。应用透射电镜观察主动脉组织超微结构变化。
2.蛋白质组学研究：提取各组大鼠主动脉总蛋白,后用trypsin消化蛋白得到肽段。用iTRAQ试剂标记消化后的肽段：用113标记5周龄WKY大鼠组(W5组),114标记5周龄SHR大鼠组(S5组),115标记17周龄WKY大鼠组(W17组),116标记17周龄SHR大鼠组(S17组)。将标记的肽段混合后运用强阳离子交换柱(strong cation exchange, SCX)和C18柱进行分级分离,最后用MALDI-TOF/TOF和microQ-TOF进行质谱鉴定。使用相关软件合并并处理定量及检测所得的结果。最后,使用Panther软件对差异蛋白进行分子定位,分子功能和生物过程分析。
3.部分差异蛋白功能验证：使用Western blot技术检测各组大鼠主动脉中关键差异蛋白前纤维蛋白1(Profilin-1),丝切蛋白1(Cofilin-1)和极长链酰基辅酶A脱氢酶(Acyl-Coenzyme A dehydrogenase, very long chain; ACADVL)的蛋白表达情况。使用PCR技术检测Profilin-1和Cofilin-1的mRNA表达情况。
研究结果
1.各组大鼠主动脉形态学变化
HE染色显示,W5组大鼠主动脉形态大致正常,主动脉各层分界清楚。血管内膜光滑,血管中膜弹性纤维呈同心圆排列,中膜无增厚,平滑肌排列规则,无肥大增生。W17组和S5组大鼠主动脉出现轻微血管重构情况,主动脉内膜增生不明显,部分内膜脱落；中膜弹性纤维形态不规则,部分出现断裂,平滑肌细胞无增生。S17组大鼠主动脉出现明显的血管重构表现,血管损伤较W17组和S5组均加重。发现与同龄WK.Y大鼠相比,SHR组大鼠管壁厚度和管壁／管径比均增高。天狼猩红染色显示17周龄SHR组大鼠中膜可见胶原沉积,胶原面积显著高于17周龄WKY组大鼠。5周龄SHR组大鼠和17周龄WKY组大鼠胶原沉积情况无明显差异,5周龄WKY大鼠血管中膜中无胶原沉积现象。
透射电镜下W5组血管平滑肌细胞核形态正常,细胞器分布及核染色质正常。S5组平滑肌细胞无明显异常,但平滑肌细胞与弹性膜的排列紊乱。W17组的管腔侧可见凹陷,主要是由内膜脱落造成,弹力膜不均匀,胶原成分可见。S17组可见平滑肌细胞与弹力膜的排列完全紊乱,平滑肌细胞表型改变显著,弹力膜内大量胶原成分的出现。
2. iTRAQ质谱鉴定结果
经质谱鉴定,在W5组和S5组大鼠主动脉中差异表达蛋白共92个,其中1.5倍以上差异蛋白8个；W17组和S17组大鼠主动脉中差异表达蛋白94个,其中1.5倍以上差异蛋白18个。
S5组和W5组的差异蛋白中仅有2个(ACADVL和Profilin-1)在W17组和S17组中有1.5倍以上的差异表达,其他蛋白差异表达均在1.5倍以下。W17组和S17组主动脉差异表达蛋白在S5组和W5组均有差异表达,但除却上述提到了2个蛋白外,其他蛋白在5周时表达均在1.5倍以下。
3.差异蛋白的定位分析(subcellular localization)
通过Panther软件分析差异蛋白的细胞定位,W17组和S17组的差异蛋白定位主要包括细胞内(32.9%),细胞器内(27.4%),细胞外部分(12.3%),细胞膜成分(8.2%),细胞外基质(6.8%),大分子复合物(6.8%)。
4.差异蛋白的分子功能(molecular function)及参与的生物过程分析(biological process)
17周SHR大鼠和WKY大鼠的差异蛋白主要涉及催化活性(catalytic activity)、结构分子活性(structural molecule activity)、结合功能(?)(binding)、受体活性(receptor activity)和酶调节活性(enzyme regulator activity)等分子功能。其参与的生物过程主要包括代谢过程(metabolic process)、细胞过程(cellular process)、生长过程(development process)、多种细胞器过程(multicellular organismal process)和细胞定位(location)等。
5.部分差异蛋白在主动脉组织的表达改变
为验证蛋白质组学鉴定出的部分差异蛋白在4组大鼠主动脉组织中的表达,本研究应用Western blot技术检测了关键差异蛋白：Profilin-1, Cofilin-1和ACADVL的蛋白表达情况。结果显示,骨架蛋白Profilin-1和Cofilin-1在同周龄的SHR大鼠中表达均高于WKY大鼠,且在17周龄大鼠中其差异表达更明显。ACADVL在同周龄SHR大鼠中表达较WKY大鼠均有所下降。用PCR技术检测Profilin-1和Cofilin-1的mRNA表达情况,证实两种蛋白的InRNA表达与上述Western blot检测的蛋白表达水平一致。
结论
(1) iTRAQ技术是比较蛋白质组学的新技术,灵敏性高,重复度好,可用于筛选高血压相关的差异蛋白,以寻找高血压的蛋白标志物,阐明其发病机制。
(2)17周龄WKY大鼠因血管老龄化出现血管重构现象,17周龄SHR大鼠血管重构较17周龄WKY大鼠明显加重,其血管重构是因为高血压和血管老化两方面原因。
(3)定位于胞浆的骨架蛋白和位于线粒体的能量代谢相关蛋白的差异蛋白在高血压的发生发展中起着重要作用。
(4)部分在5周龄SHR大鼠中有差异性表达的蛋白,在17周龄中并没有差异性表达,证明某些蛋白只在高血压早期发挥了作用。
第二部分
Profilin-1在自发性高血压大鼠血管重构中的作用机制研究研究背景
氧化应激是导致高血压的重要原因,高血压状态下可检测到活性氧(reactive oxygen species, ROS)产生增多。高血压状态下的血管重构是一个复杂的过程,是血管对刺激的复杂的动态反应过程,其主要过程不仅包括细胞增殖、凋亡和迁移,同样包括基质成分的合成、分解以及重新排列。氧化与抗氧化系统间的平衡紊乱参与了上述各个环节,从而导致了血管重构的发生发展。
本研究第一部分用iTRAQ蛋白质组学技术筛选出的差异蛋白中,前纤维蛋白(Profilin-1)尤为引人瞩目。Profilin-1是一种广泛存在于除骨骼肌之外其他机体组织的,约15kd大小的肌动蛋白结合蛋白,其主要参与了肌动蛋白的聚合及解聚；并与细胞的增殖、分化和运动过程以及其信号转导密切相关。现已证实Profilin-1可促进细胞外信号调节激酶1/2(ERK1/2)、Jun氨基末端激酶(JNK)及p38MAPK的磷酸化水平增加,从而在动脉粥样硬化、高血压等血管损伤过程中起到重要作用。此外,也有报道证实Profilin-1可以促进血管中的氧化应激水平,促使动脉粥样硬化的发生发展。
细胞实验和动物体内实验均证实,ROS可以促进血管重构。ROS参与了高血压血管重构的各个过程,它可以导致内皮功能失调、血管中膜增厚,平滑肌细胞迁移以及细胞外基质重构。ROS家族中的H2O2, ONOO-和HOCL等均在高血压血管重构中发挥着重要作用。其中,ONOO-可以通过氧化／硝基化作用硝化酪氨酸,对血管内皮等造成严重损伤。心血管系统中ONOO-的生成主要与NO的产生相关,在血管中诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)可以催化合成大量的NO,生成高浓度的ONOO-。 ONOO-是一个强氧化剂分子,它可以通过促进氧化损伤而造成内皮损伤,导致内皮细胞屏障功能的丧失、血小板粘附和血管调节异常,从而影响内皮功能；此外,ONOO-还可以激活与血管重构密切相关的JNK信号通路,ERK1/2信号通路。本研究采用SHR大鼠模型证实,ONOO-与高血压状态下血管重构有着密切的关系。
研究目的
1.实现SHR大鼠Profilin-1过表达腺病毒载体和Profilin-1干扰腺病毒载体的体内转染,观察Profilin-1对SHR大鼠血管重构的影响；
2.检测SHR大鼠体内炎症和氧化应激水平的变化,并探讨Profilin-1对于氧化应激的作用及其在血管重构中的作用机制；
3.在分子水平阐述Profilin-1对于高血压血管重构的作用机制。
研究方法
5周龄雄性SHR大鼠75只,同周龄雄性WKY大鼠20只,适应性饲养3天后进入实验。SHR随机分为3组：过表达组(SHR-E组)25只,干扰组(SHR-I组)25只,对照组(SHR-C组)25只。各组大鼠通过尾静脉注射分别给予3*109单位相应病毒载体两次,两次之间相隔6周。第一次注射3周后,分别处死各SHR组大鼠中每组5只大鼠。第二次注射6周后,处死其余大鼠。SHR-E组给予Profilin-1过表达腺病毒载体注射；SHR-I组给予Profilin-1干扰腺病毒载体注射；SHR-C组和WKY组给予阴性对照腺病毒载体注射。
1.实验开始前及实验中每周均测量大鼠体重各1次,并使用大鼠尾动脉间接测量法(尾套法)测量大鼠尾动脉收缩压。
2.留取转染3周的15只SHR大鼠的主动脉组织,检测血管重构的形态学变化,以及profilin-1, iNOS, p-p38的蛋白表达情况,并测定硝基酪氨酸含量。
3.第二次注射6周后处死大鼠,制作主动脉冰冻切片,于荧光显微镜下观察绿色荧光蛋白的表达,以确定转染效率；并检测主动脉组织中Profilin-1蛋白质和]mRNA表达情况,确定转染效率。
4.留取血清进行NO,IL-6检查。制作主动脉石蜡切片进行HE染色、天狼猩红饱-苦味酸染色观察主动脉形态。进行免疫组织化学染色检测相关蛋白在主动脉中的定位表达情况。并留取组织冻存,进行Western Blot和Real-time PCR检测。
研究结果
1.血压和体重情况
12周实验期间,各组大鼠体重之间差异不大。各SHR组大鼠的血压随着实验进行逐渐升高,而WKY大鼠血压变化不大,但各组SHR大鼠血压之间的差异没有统计学意义(P>0.05)。
2.腺病毒在大鼠体内转染效率
制作各组大鼠主动脉冰冻切片,并在荧光显微镜下观察大鼠主动脉壁绿色荧光蛋白(green fluorescent protein, GFP)的表达情况。接受腺病毒载体注射大鼠,其主动脉内皮细胞及平滑肌细胞内均有GFP的表达；而生理盐水组主动脉内未见GFP的表达,证明体内转染有效。
检测各组大鼠主动脉Profilin-1蛋白及mRNA表达情况,结果显示,与SHR-C组相比,Profilin-1蛋白及mRNA表达在SHR-E组均明显升高(P<0.05),而在SHR-I组则降低(P<0.05),进一步证明profilin-1过表达／干扰腺病毒体内转染有效。
3. Profilin-1转染3周对大鼠主动脉的影响
第一次腺病毒注射3周后,从SHR-E、SHR-C、SHR-I三组大鼠中,每组处死5只大鼠,观察profilin-1过表达／干扰腺病毒转染对于大鼠主动脉的影响。三组大鼠主动脉形态学研究发现,三组大鼠主动脉形态、血管内径、血管壁厚度以及血管壁／管腔比值没有显著性差异。血管形态大致正常。
Western blot检测三组大鼠主动脉profilin-1, iNOS, p-p38的蛋白表达情况,结果显示,三种蛋白在各组大鼠之间表达趋势一致,即SHR-E组大鼠三种蛋白表达均高于SHR-C组,而SHR-I组大鼠中三种蛋白表达降低。iNOS免疫组化染色所显示趋势同Western blot结果一致。
通过进行硝基酪氨酸(Nitrotyrosine)染色来评价血管内过亚硝酸根(Peroxynitrite, ONOO-)含量,SHR-E组的染色面积和H评分较SHR-C组明显增加,但SHR-I组则表现出显著的下降(P<0.05)。
4. Profilin-1转染12周对大鼠主动脉形态学影响
WKY组为正常主动脉形态。各SHR组大鼠主动脉均出现明显的血管重构改变,血管损害程度、管壁厚度都较3周时更为严重。SHR-C组大鼠主动脉出现明显的血管损害：内膜局部有隆起,部分内皮细胞脱落；中膜弹性纤维部分断裂,中膜增厚明显,平滑肌细胞增生肥大。SHR-E组大鼠主动脉损害较SHR-C组更为严重,SHR-I组大鼠内皮细胞的损害较SHR-C组轻。
WKY组大鼠中膜未见胶原沉积,而SHR-C组大鼠中膜可见部分胶原沉积,SHR-E组大鼠血管胶原沉积明显,但在SHR-I组大鼠血管中,没有明显胶原沉积。
5. Profilin-1转染12周对大鼠主动脉的影响
Western blot检测三组大鼠主动脉iNOS, p-p38的蛋白表达情况,结果显示,两蛋白在各组大鼠之间表达趋势一致,且同Profilin-1趋势相同,即SHR-E组大鼠两种蛋白表达均高于SHR-C组,而SHR-I组大鼠中两种蛋白表达降低。iNOS免疫组化染色所显示趋势同Western blot结果一致。
硝基酪氨酸染色结果趋势同3周时一致,染色程度、面积及H评分均较3周增加。
6. Profilin-1转染12周对大鼠NO，IL-6的变化。
利用硝酸还原酶法分别检测腺病毒转染12周后各组大鼠血清及主动脉中的NO含量,结果显示,四组大鼠血清中NO含量没有显著性差异(P>0.05)。对于大鼠主动脉局部NO的检测显示,各组大鼠主动脉中NO趋势同Profilin-1趋势相反。而各组中炎性因子IL-6的表达趋势同Profilin-1趋势相同。
结论
(1)SHR大鼠的血管重构伴随着体内氧化/抗氧化平衡紊乱及Profilin-1的过表达。
(2) Profilin-1过表达可以促进大鼠体内氧化应激水平和血管重构,干扰Profilin-1表达则可逆转上述变化。(3) Profilin-1对于大鼠血管重构的作用与p38/iNOS/ONOO-通路相关。
Essential hypertension is one of the most common cardiovascular disease in our country, it is also a worldwide public health problem. The major damage caused by hypertension is its effect on the target organs including heart, brain, kidney and vessels. Vascular remodeling is an important mechanism of hypertension-induced target organs damage.
The pathogenesis mechanism of vascular remodeling is complicated, it is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. With the development of proteomic techniques, a quantitative proteomics-isobaric tags for relative and absolute quantification (iTRAQ) was introduced, which provides an opportunity to solve this problem. iTRAQ enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. It has been widely used in finding the pathogenesis, disease markers, the differentially expressed proteins in diseases. We resorted to iTRAQ technique of the aorta using spontaneous hypertensive rats (SHRs), SHRs are well-established animal model to study hypertension vascular remodeling. This experiment explore the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group, then validate for Profilin-1, a markedly differential protein's effect in vascular remodeling, thus provide candidate targets for clinical treatment of hypertension.
Part One
The proteomic analysis of aortic tissue of different week-old spontaneous hypertensive rats Background
Protein is the ultimate embodiment of vital movement, proteomics is a discipline which focus on all the proteins and their dynamic regularity in tissue or cell. Proteomics explicates the essence and regularity of vital movement on the whole through analyzing the composition, expression and modification of all the proteins and their interactions in tissue and cell. Proteomics is a new research field and it is more complex than genomics. The expression of protein has spatiality and regulatability, and when the protein functions, doesn't like the genome who is almost invariable, it is dynamic, and many proteins are involved in one kind of vital movement at the same time. Proteomics have provided new methods and ideas for the research on life science.
Comparative proteomics is a kind of approach that compares and analyzes the proteome to find the differences of expression of proteins. In recent years, the comparative proteomics is used in medical research more and more frequent. By comparing with the normal individual, we can find the specific proteins which only express in the abnormal individual and these specific proteins can be utilized in the early diagnosis and treatment of diseases. Isobaric tags for relative and absolute quantification (iTRAQ) is a new quantitative technique for proteomics analysis. It can be used for separating up to8different samples at the same time, with high repeatability and good quantitative effect.
It is reported that cell hypertrophy, apoptosis, inflammation, oxidative stress, cytokines and extracellular matrix changes are involved in vascular remodeling, but the exact mechanism remained unclear. iTRAQ provides an opportunity to solve this problem, which enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. We resorted to iTRAQ technique of the aorta using SHRs, explored the protective mechanisms of vascular remodeling by searching the differential protein among SHR group and WKY group.
Objectives
1. To research the characteristics of aortic pathological changes of SHR, to observe the blood pressure, weight and aortic pathological changes of five-week-old and seventeen-week-old SHR.
2. To explore the differentially expressed proteins of aorta in five-week-old and seventeen-week-old SHR and the same week-old WKY rats by iTRAQ.
3. To find out the aortic differentially expressed proteins of five-week-old and seventeen-week-old rats, further confirm the target proteins and potential mechanism of vascular remodeling under hypertension.
Methods
Male SHRs (n=6,5weeks old; n=6,17weeks old), male WKY (n=6,5weeks old; n=6,17weeks old) assigned to4groups respectively:S5group, S17group, W5group, W17group. After3days adaptive feeding, blood pressure and weight were measured.
1. Aortic morphology:After anesthetized, aortic tissue of rats were extracted, and fixed in10%formalin, then embedded in paraffin for HE staining, the internal circumference and wall thickness of the thoracic aorta were determined using Image-Pro Plus. Aortic fibrosis was shown on histological pictures, where red and yellow fibers represent collagen I and green fibers show collagen Ⅲ. Picrosirius red staining was observed by polarized light and measured by Image-Pro Plus to obtain the percentage of collagen per media area. Or fix the aortic tissue in glutaraldehyde for ultrastructure observation using electron microscope.
2. Proteomic analysis:The proteins-enriched fractions were digested by tripsin. The resulting complex peptide mixtures were labeled with iTRAQ reagents (113for the W5group,114for the S5group,115for the W17group and118for the S17group respectively), All the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges. After being labeled, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF to evaluate the effectiveness of reagents labeling. Panther software was applied to analyze protein functions.
3. Western blot and PCR:4differentially expressed proteins were validated using western blot and PCR.
Results
1. Aortic Morphology
Aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of5-week SHRs and17-week WKY, and17-week SHRs showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes. Compared with17-week WKYs,17-week SHRs showed increased collagen deposition in the media.
Under electron microscopy, normal ultrastructure was observed in the aortic tissue of W5, and the rupture of vascular elastic membrane and the increased collagen fibers were observed in the aorta of W17and S5rats. Moreover, ultrastructure changed more obviously in S17rats.
2. Mass Spectrometry Identify the Differentially Expressed Proteins
We identified92differential proteins between W5group and S5group, among which the expression of8proteins was significantly changed;94differential proteins between W17group and S17group, among which the expression of18proteins was significantly changed.
3. Subcellular localization analysis vascular remodeling associated proteins
We performed the localization analysis of the identified proteins by Panther software. Cellular component of differentially expressed proteins among W17group and S17group mainly included cell part, extracellular region, organelle.
4. Protein function analysis vascular remodeling associated proteins
Molecular function of differentially expressed proteins among W17group and S17group mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.
5. Differentially expressed proteins in aorta
Protein and mRNA levels of Profilin-1and Cofilin-1were higher in SHRs than in same aged WKYs, protein level of AC AD VL was lower in SHRs.
Conclusions
1. As a new technique of quantitative proteomics, iTRAQ is more accurate and reproducible, enables comparative analysis for many groups. We obtained reliable differentially expressed proteins from different aged SHRs and WKYs, and find potential new drug targets for hypertension and its complications.
2. Molecular function of differential proteins we identified mainly included catalytic activity, binding and structural molecular activity, they were mainly involved in metabolic processes, cellular processes, developmental processes and cellular component organization or biogenesis.
Part Two
Profilin-1promotes the development of hypertension-induced artery remodeling
Background
Vascular hypertrophy and remodeling in hypertension is an adaptive process in response to chronic changes in hemodynamic conditions during the development of vascular diseases. The ensuing changes in the size and/or composition of a remodeling blood vessel allow blood vessels to adapt and heal but they also underlie the pathogenesis of major cardiovascular diseases.
Profilin-1is a ubiquitous, small (12-15kD) actin-binding protein that plays an important role in the regulation of actin polymerization and cytoskeleton remodeling by activating hypertrophic signaling cascades, such as mitogen activated protein kinase (MAPK) signaling, to thus contribute to vascular hypertrophy and hypertension. Numerous studies have provided strong evidence for the important role of profilin-1in vascular inflammation and vascular remodeling, and have linked profilin-1to inducible nitric oxide synthase (iNOS) and peroxynitrite production. Also, peroxynitrite has been linked to vascular remodeling because it activates MAPKs (c-Jun N-terminal kinase [JNK], extracellular signal-regulated kinase1/2[ERK1/2]). However, the relationship between profilin-1, iNOS and the associated remodeling in SHRs remains to be fully clarified. Here, we studied the effects of profilin-1on hypertension-induced vascular remodeling and its potential relationship with iNOS. We used SHRs as a classical, genetic hypertensive animal model. Overexpression of profilin-1significantly promoted arterial structural remodeling in SHRs through a p38-iNOS-peroxynitrite-pathway. Profilin-1may be a potential target for genetic therapy of hypertension-induced arterial structural remodeling.
Objective
1. To investigate the effect of Profilin-1on vascular remodeling by transfection of pAd-profilin-1-IRES-EGFP and pAd-miR-profilin-1into SHRs;
2. To study the effect of Profilin-1on vascular inflammation and oxidative stress, and the relationship between oxidative stress and vascular remodeling.
3. To explore the mechanism of Profilin-1on vascular remodeling in molecular, histological and functional levels.
Methods
Five-month-old male SHRs (n=75) and Wistar-Kyoto rats (WKYs)(n=20) were obtained from Slac Laboratory Animal Co. SHRs were divided into3groups (n=25each) for treatment:profilin-1overexpression adenovirus vector (SHR-E), profilin-1miRNA adenovirus vector (SHR-I) or negative control adenovirus (SHR-C) after3days adaptation. WKYs (n=20) were treated with negative control adenovirus at the same time. The adenoviruses were injected twice into the tail vein at3x109infectious units per rat with an interval of6weeks between injections. At3weeks after the first administration,15SHRs from each of the three SHR groups (n=5each) were killed: At6weeks after the second administration of adenovirus, the remaining SHRs (n=60) and WKYs were killed.
1. Systolic blood pressure (SBP) and body weight were measured in conscious animals before the start of treatment and weekly during treatment. Systolic blood pressure (SBP) was measured by means of a tail-cuff sphygmomanometer.
2. Six days after the second injection of adenovirus, five rats in each group were killed and thoracic aortas were removed to determine transfection efficiency. Thoracic aorta specimens were sectioned at5μm for enhanced green fluorescent protein (EGFP) observation. Tissues underwent sky blue staining to shield the autofluorescence of the vascular tissues and DAPI staining was used to locate the nuclei. EGFP expression in the vascular tissues was observed by fluorescence microscopy.
3. Changes of vascular morphology and histology were measured by HE staining, Picrosirius-red-staining.
4. Nitrotyrosine is considered to be an indirect marker of oxidative stress, measured by immunohistochemical analysis.
5. Measure IL-6and NO by ELISA.
6. Western blot for the expression of p-p38, Profilin-1and iNOS, RT-PCR for mRNA expression of Profilin-1and iNOS.
Results
1. Transfection Efficiency of Adenovirus Vector
The high transfection efficiency was confirmed by observing EGFP expression under fluorescence microscopy and detecting the mRNA and protein expression of Profilin-1. EGFP expression was observed in more than90%cells for each transduction group. Profilin-1expression was higher in HUVECs and aortic tissues after profilin-1overexpression by adenovirus vector transfection, but was reduced by profilin-l miRNA vector treatment.
2. Effect of Profilin-1on Body Weight and SBP of SHRs
Body weight did not differ among the four groups during the12weeks of treatment. SBP for the three SHR groups increased with age, no significant difference between SHR-C and the other SHR-groups (p>0.05).
3. Effect of profilin-1on3-week-treatment SHRs
At3weeks after the administration of the adenovirus, we found that the lumen size (internal circumference), wall thickness and wall to lumen ratio (wall thickness: internal diameter) did not differ among the three SHRs groups. Protein level of iNOS and phosphorylation of p38were higher in SHR-E than in SHR-C tissues, but were decreased by the knockdown of profilin-1. We also found an increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E (0.530±0.028) and a decrease in SHR-I (0.072±0.016), as compared with SHR-C.
4. Profilin-1Promotes Vascular Hypertrophy and Fibrosis
This increased Profilin-1expression was accompanied by vascular remodeling in SHRs, as evidenced by a36.8%increase in wall thickness, a12%increase in wall-lumen ratio (the wall thickness:internal diameter) and increased vascular fibrosis. Compared with SHR-C, SHR-E showed significantly greater lumen size (internal circumference), wall thickness and wall-lumen ratio (wall thickness:internal diameter), accompanied by evidence of broken elastic membranes.
5. Effect of Profilin-1on Remodeling in SHR via the p38iNOS and Peroxynitrite-dependent Mechanism
Total p38protein expression did not differ among the four treatment groups, but phosphorylation of p38was higher in SHR-E than in SHR-C. In contrast, phosphorylation of p38was decreased by the knockdown of profilin-1. Thoracic aorta expression of iNOS showed a similar trend to that of p38phosphorylation.
Our results showed a significant increase in peroxynitrite expression (as determined by nitrotyrosine staining) in SHR-E tissues but a significant decrease in SHR-I tissues, as compared with SHR-C. IL-6level was significantly higher in SHR-E as compared with SHR-I and SHR-C.
Next, we investigated the production of NO in aortic tissues and found a lower level of NO in SHR-E as compared with SHR-I (1.45±0.15vs.2.34±0.16μmol/g protein nitrites, p<0.05) and as compared with SHR-C (1.88±0.14μmol/g protein nitrites). We also tested the NO levels in the serum of rats and found no significant difference among the three SHR groups, indicating that changes in NO levels are a local phenomenon rather than a systemic reaction.
Conclusions
1. Vascular remodeling of SHRs was companied with oxidative stress reactions and over expression of Profilin-1.
2. Over expression of Profilin-1could facilitate the vascular remodeling and oxidative stress reactions, and knockdown of Profilin-1reversed those changes.
3. Prohypertrophic effects of profilin-1are mediated, at least in part, by suppression of the p38-iNOS-peroxynitrite pathway.
引文
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