精氨酸酶I在动脉粥样硬化中的作用及机制研究
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摘要
研究背景及目的
炎症是动脉粥样硬化(AS)发生、发展过程中的一个重要因素,炎症的发生常常关系到动脉粥样硬化的进程、严重程度及其后果。炎性因子的分泌能够引起单核、巨噬细胞的趋化、迁移,进一步加重炎症反应。血管平滑肌细胞位于血管壁的中层,在正常的生理状态下这些细胞通常是“安静”的,表现出一种分化状态来维持血管的结构及其紧张度。然而,在某些条件下(如动脉粥样硬化形成),血管平滑肌细胞获得了一种“分泌”型的表型,表现为去分化状态,并能够合成、分泌一些炎性因子,从而促进了血管的病变过程。
精氨酸酶Ⅰ(Arg Ⅰ)能够促进平滑肌细胞的增殖,但其在炎症反应中的作用还未明确。L-精氨酸是精氨酸酶(Arginase)和一氧化氮合成酶(NOS)的共同催化底物。在炎症反应中,iNOS能够产生过量的一氧化氮(NO),通过与细胞内的超氧阴离子(02.-)反应产生有致炎作用的ON00-。因此,减少iNOS的底物有助于抑制细胞内的炎症。
在以往研究的基础上,我们提出如下假设：1)精氨酸酶Ⅰ具有抑制细胞内炎症作用；2)精氨酸酶Ⅰ抑制炎症的作用是通过与iNOS竞争底物实现的。
本研究正是基于以上思路,拟通过体外和体内两部分实验以验证这一假说。旨在探讨Arg Ⅰ在平滑肌细胞(SMC)炎症反应中的作用,以期寻找到与SMC相关的炎症性疾病的预防和治疗靶点。
材料与方法
1.细胞培养及模型建立：采用人主动脉平滑肌细胞(hASMCs)作为研究对象；体外以脂多糖(LPS)作为诱导剂诱导hASMCs发生炎症反应。
2.细胞内Arg Ⅰ干预：采用瞬时转染ARGI-pcDNA3.1(+)质粒和si-ARGI小干扰片段的方式实现对hASMCs细胞内Arg Ⅰ的干预。
3.动物模型的建立及干预：新西兰大白兔采用腹主动脉球囊拉伤+高脂喂养的方式建立动脉粥样硬化斑块模型,于喂养12周后按照本实验室建立的斑块内注射的方法对斑块进行ARG Ⅰ过表达及小干扰慢病毒载体转染,继续喂养四周后将动物实施安乐死后取材。
4.采用酶联免疫吸附法(ELISA):检测平滑肌细胞培养中的肿瘤坏死因子a (TNFa)的含量；
5.实时定量逆转录PCR (Real-time RT-PCR)、Western-blot及酶活性检测法：分别检测精氨酸酶I(Arg Ⅰ)和诱导型一氧化氮合成酶(iNOS)的基因表达水平、蛋白质表达水平及酶活性变化。
6.单核细胞趋化及迁移检测：采用Transwell小室构建了人单核细胞(THP1)与hASMCs的共培养体系,检测单核细胞的趋化及迁移能力。
7.细胞荧光免疫组化及组织荧光免疫组化：显示细胞内的Arg Ⅰ、iNOS、NF-κB的P65亚基、斑块内细胞成分及TNF a的表达。
8.流式细胞术、激光共聚焦显微技术：分别检测细胞内过氧亚硝基(ONOO-)、活性氧簇(ROS)及超氧阴离子(02.-)的含量。
研究结果
1.LPS对hASMCs细胞内TNFa产生的诱导具有时间-剂量依赖性：LPS (>10ng/ml)能够诱导hASMCs细胞内TNFa的释放,且TNFa释放量随LPS刺激剂量增加或时间延长而增大。细胞受LPS刺激后能引起TNFa的快速释放,但刺激超过24小时,其TNFa释放量增加幅度有所减小；刺激超过48小时,其增加幅度减小更加明显。
2.LPS通过激活iNOS增加人主动脉平滑肌细胞内TNFa的表达量：LPS能够诱导hASMCs细胞TNFa的释放,但加入iNOS抑制剂(AG或1400W)后,此过程被抑制,TNFa的分泌量减少大约70～80%,证明LPS通过激活iNOS增加人主动脉平滑肌细胞内TNFa的表达量。
3.LPS能够同时上调人主动脉平滑肌细胞内Arg Ⅰ和iNOS的表达：细胞在LPS的刺激下,无论基因还是蛋白水平Arg Ⅰ和iNOS均随LPS刺激而升高,但Arg Ⅰ的升高速度落后于iNOS。iNOS的mRNA水平在8小时达到高峰,而Arg Ⅰ的mRNA水平在12小时达到高峰,之后二者的增幅出现回落；在蛋白表达水平,iNOS在LPS的刺激下表现为持续升高,而Arg Ⅰ在LPS刺激超过48小时后其蛋白水平开始下降。
4. Arg Ⅰ与iNOS之间存在底物竞争：与上述趋势一致的是,两种酶的催化活性也随LPS刺激而升高。但与NOS活性持续升高不同的是,精氨酸酶活性在48小时后明显下降。而且,当精氨酸酶活性降低时,NOS活性反而有小幅升高。在预先向培养基中加入二者的共同底物L-精氨酸后,两种酶的催化活性在LPS作用12小时后均有所增加,这种升高趋势一直持续至72小时。
5. Arg Ⅰ能够抑制TNFa的生成和单核细胞的趋化和迁移：精氨酸酶Ⅰ能够明显抑制LPS诱导的人主动脉平滑肌细胞TNFa的释放,而精氨酸酶Ⅰ抑制或干扰则能加剧TNFa的释放。经LPS诱导后的人主动脉平滑肌细胞,单核细胞向其趋化和迁移的能力明显增加。而提高精氨酸酶Ⅰ的表达则能降低单核细胞的这种趋化和迁移能力,反之,则加剧单核细胞的迁移和趋化能力的增加。
6. Arg Ⅰ通过与iNOS竞争共同的催化底物来抑制平滑肌细胞NO的释放：免疫组化结果显示在人主动脉平滑肌细胞中精氨酸酶Ⅰ和iNOS在空间上存在很大程度的共定位；升高精氨酸酶Ⅰ能够加剧LPS诱导的NO的释放,而抑制或干扰精氨酸酶Ⅰ的表达,则能逆转这一现象；但升高精氨酸酶Ⅰ却未能对iNOS的表达量造成影响,提示精氨酸酶Ⅰ通过与iNOS竞争共同的催化底物L-精氨酸来抑制NO的释放,而非通过减少iNOS的表达量。
7. Arg Ⅰ能够减少人主动脉平滑肌细胞内ROS、02.-以及ONOO-的生成：增加人主动脉平滑肌细胞内的精氨酸酶Ⅰ表达能够抑制LPS对细胞内ROS、02.-及ON00-的生成的诱导；抑制iNOS的活性同样能够抑制细胞内02.-及ON00-的生成增加,而未能对ROS的生成产生明显影响。
8.Arg Ⅰ通过抑制NF-kB的激活降低TNFa的生成：LPS能够引起NF-κB的激活,而后者的激活则能引起细胞内TNFa的生成。精氨酸酶Ⅰ过表达能够通过抑制NF-kB的激活降低TNFa的生成。
9. Arg Ⅰ能够降低动脉粥样硬化斑块内的炎症反应：局部转染精氨酸酶Ⅰ过表达慢病毒载体能够引起斑块内TNFa、iNOS的表达量及巨噬细胞含量的降低,而抑制精氨酸酶Ⅰ则起到相反的效果。TNFa与SMC的面积比计算结果显示,精氨酸酶Ⅰ能够明显地降低该比值。
结论及意义
1.精氨酸酶Ⅰ具有抑制SMC炎症的作用；
2.精氨酸酶Ⅰ具有抑制动脉粥样硬化斑块内炎症的作用；
3.精氨酸酶Ⅰ的抑炎作用通过与具有促炎作用的iNOS竞争底物,减少了后者催化产物的生成量,从而减少ONOO-的生成,进而起到抑制炎症的作用。
4.在本实验研究中,我们对Arg Ⅰ在对入主动脉平滑肌细胞和兔动脉粥样硬化斑块炎症细胞因子分泌中的作用及其机制做了系统的阐述,以期为临床治疗此类疾病提供新的思路和治疗靶点。
研究背景及目的
动脉粥样硬化斑块破裂(Plaque Rupture)是引发心肌梗死、脑血栓等心脑血管急性事件的主要原因。而动脉粥样硬化斑块内炎症细胞的浸润及继发的炎症反应是引发动脉粥样硬化斑块不稳定的主要因素。炎症反应贯穿了整个动脉粥样硬化进程：从发生、发展直至斑块破裂、血栓形成,甚至死亡。因此,有效地抑制炎症反应的发生可以稳定动脉粥样硬化易损斑块,避免心血管急性事件的发生。
血管平滑肌细胞在动脉粥样硬化发生、发展过程中发挥“双刃剑”的作用。在动脉粥样硬化形成早期,平滑肌细胞的增殖能够促进斑块的形成；在中期,平滑肌细胞的泡沫化能够促进斑块的进程；而在斑块发展晚期,平滑肌细胞的增多则有助于纤维帽的增厚,促进斑块的稳定性。以往的研究证实,精氨酸酶Ⅰ具有抑制炎症反应的作用；我们在前面的研究中还发现,精氨酸酶Ⅰ具有抑制炎症反应的作用;但该酶动脉粥样硬化斑块稳定性中的作用还未见报道。
在以往研究的基础上,我们提出如下假设：1)在动脉粥样硬化发展的晚期,高表达精氨酸酶Ⅰ能够稳定动脉粥样硬化斑块；2)精氨酸酶Ⅰ稳定动脉粥样硬化斑块的作用是通过抑制巨噬细胞炎症反应和促进血管平滑肌细胞增殖的双重机制实现的。
本研究正是基于以上思路,从体内和体外两方面设计实验以验证这一假说。旨在探讨Arg Ⅰ在动脉粥样硬化斑块稳定性中的作用,以期为稳定易损斑块、预防心血管急性事件的发生防治寻找新的契机和治疗靶点。
研究方法
1.动物模型的建立：雄性新西兰大白兔,采用高脂喂养(1%胆固醇)+球囊拉伤腹主动脉的方法,构建动脉粥样硬化模型。
2.细胞培养：分别培养人单核来源的巨噬细胞(MDM)和人主动脉平滑肌细胞(hVSMC)。
3.精氨酸酶Ⅰ的干预：采用慢病毒载体和精氨酸酶抑制剂。
4.天狼猩红、油红0染色染色：分别显示斑块的大体结构、斑块内胶原及斑块内脂质含量。
5.免疫组化检测：检测斑块组织内平滑肌细胞、巨噬细胞的含量、TNFa、IL-6的表达量、细胞增殖核抗原(PCNA)的阳性率、精氨酸酶Ⅰ与iNOS在巨噬细胞内的定位关系及细胞内ONOO-的表达量。
6.单核细胞趋化、迁移实验：采用人单核细胞与人MDM共培养体系。
7. Western Blot及生化法：分别用于检测细胞及组织内内的精氨酸酶Ⅰ及iNOS的蛋白及其活性。
8.流式细胞术：用于检测细胞内ROS及02.-的生成量。
9. ELISA检测：用于检测血清及细胞培养上清中TNFa及IL-6的含量。
10.EDU掺入实验：检测人主动平滑肌细胞的增殖能力。
研究结果
1.精氨酸酶Ⅰ能够影响动脉粥样硬化的形态结构及斑块组分：较之LacZ转染和生理盐水处理组斑块而言,精氨酸酶Ⅰ转染的斑块其结构更加致密且坏死核心较小；斑块内胶原含量和平滑肌细胞的数量增多(both p<0.01, VS. LacZ转染组),而巨噬细胞和脂质核心则明显减少(bothp<0.01, VS. LacZ转染组)；斑块易损指数明显降低(both p<0.01, VS. LacZ转染组)。
2.精氨酸酶Ⅰ能够抑制斑块内的炎症反应：局部转染精氨酸酶Ⅰ能够明显降低斑块内TNFa和IL-6的水平(both p<0.01, VS. LacZ转染组),而上述因子在血清内的浓度却未受到明显改变(p>0.05, VS. LacZ转染组)；转染精氨酸酶Ⅰ未能对斑块内IL-1β的表达产生明显影响；局部转染精氨酸酶Ⅰ增加了斑块内精氨酸酶Ⅰ的表达及总精氨酸酶的活性(both p<0.01, VS. LacZ转染组),而却降低了精氨酸酶Ⅱ的表达(p<0.05, VS. LacZ转染组)。
3.精氨酸酶Ⅰ能够抑制LPS诱导的巨噬细胞的炎症反应：对于人巨噬细胞和小鼠巨噬细胞而言,增加精氨酸酶Ⅰ的表达均能够有效降低由LPS诱导引起的TNFa和IL-6的生成量(All p<0.01, VS. LPS单独处理组)；而降低精氨酸酶Ⅰ的表达或抑制其活性则加剧了上述两种因子的释放(All p<0.01, VS. LPS单独处理组),且抑制剂使用引起的效果较精氨酸酶Ⅰ明显。进一步的实验证实,LPS能够引起单核细胞向巨噬细胞的趋化和迁移,而增加巨噬细胞内的精氨酸酶Ⅰ能够加剧单核细胞的趋化和迁移能力(Both p<0.01, VS.LPS单独处理组),而降低或抑制精氨酸酶Ⅰ则能抑制单核细胞的趋化和迁移(All p<0.01, VS. LPS单独处理组)。
4.精氨酸酶1能够通过与iNOS竞争底物发挥炎症抑制的作用：LPS对精氨酸酶和iNOS的蛋白表达及酶活性的诱导具有时间依赖性；添加L-精氨酸不但能够加剧LPS诱导的巨噬细胞内TNFa和IL-6的表达,而且能够逆转精氨酸酶Ⅰ对两种因子释放的抑制作用;而添加iNOS抑制剂则能抑制LPS诱导的两种炎性因子的释放。转染精氨酸酶Ⅰ能够增加细胞内精氨酸酶的活性,而降低iNOS的活性,且两种酶在细胞内存在很大程度的共定位。但调控精氨酸酶Ⅰ却不能改变iNOS的蛋白表达量。这些结果说明精氨酸酶Ⅰ通过与iNOS竞争底物而不是减少其表达量来达到抑制炎症的目的。
5.精氨酸酶Ⅰ能够降低巨噬细胞内的02.-, ROS以及ONOO-的生成：提高巨噬细胞内精氨酸酶Ⅰ的表达能够降低LPS诱导的02.-,ROS以及ONOO-的生成。而降低或抑制精氨酸酶Ⅰ则起到相反的效果；使用iNOS的抑制能够LPS诱导的细胞内02.-及ONOO-的生成增加,但对ROS的影响不大。
6.精氨酸酶Ⅰ能够促进平滑肌细胞的增殖：局部转染精氨酸酶Ⅰ较之LacZ转染和生理盐水处理而言,其斑块内平滑肌细胞增殖数量明显增加(p<0.01, VS. LacZ转染组)；提高精氨酸酶Ⅰ能够促进斑块内平滑肌细胞及体外培养的人主动脉平滑肌细胞的增殖,而降低或抑制精氨酸酶则起到相反的作用。
结论及意义
1.精氨酸酶Ⅰ具有促进动脉粥样硬化斑块稳定性的作用；
2.精氨酸酶Ⅰ具有抑制巨噬细胞炎症反应的作用；
3.精氨酸酶Ⅰ通过与致炎基因iNOS竞争反应底物,减少iNOS的催化底物NO的生成,进而减少ONOO-的生成,从而达到抑制炎症的目的；
4.精氨酸酶Ⅰ能够促进人主动脉平滑肌细胞的增殖；
5.精氨酸酶Ⅰ对动脉粥样硬化斑块的稳定作用,是通过抑制炎症反应及促进平滑及细胞的增殖来实现的。
6.该研究为进一步阐明AS斑块稳定性的作用机理和AS斑块破裂及相关的心血管急性事件的防治提供新的思路及有效的治疗靶点。
Backgroud
Inflammation plays an important role in the generation and development of atherosclerosis. Inflammation is often related to the development of atherosclerosis, severity and its consequences. The secretion of inflammatory factors can trigger chemotaxis and migration of monocytes and macrophage, and further to aggravate inflammatory reaction. Smooth muscle cells (SMCs) locate in the middle layer of vascular wall and keep "quiescent" under normal physiological conditions. However, these kinds of cells often acquire a state of dedifferentiation and exhibit the character of "synthetic" phenotype under atherosclerotic conditions, contributing to vascular pathological changes by synthesizing and secreting certain inflammatory factors.
It has been demonstrated that arginase Ⅰ have the ability to promote SMCs proliferation. However, its role in inflammation has remains unknown. Both arinase and nitric oxide synthetase (NOS) catalyze L-arginine, which can be catatlyzed by induced nitric oxide synthetase (iNOS) to produce excess nitric oxide (NO) in cytoplasm. Under inflammatory conditions, the iNOS can produce excessive nitric oxide (NO), which can produce proinflammatory effects of ONOO-by reaction with intracellular superoxide anion (O2-). Then, it may inhibit the inflammation to decrease the catalyzing substrate of iNOS.
Based on the previous studies, we make the following assumptions:1) Arginase Ⅰ has the ability to inhibit inflammation;2) Arginase Ⅰ inhibits the inflammation by decreasing catalyzing substrate of pro-inflammatory iNOS.
Based on the above ideas, we design the experiments in vivo and in vitro to validate the hypothesize in the present study. We aim to investigate the role of Arg Ⅰ in inflammaiton of SMCs and try to look for new opportunities and therapeutic targets in SMC associated inflammatory disease.
Material and Methods
1. Cell Culture and Inflammatory Cell Model: Human aorta smooth muscular cells (hASMCs) were cultured and lipopolysaccharide (LPS) was used to induce inflammatory reaction in hASMCs in our experiments.
2. ARG Ⅰ Interference in hASMCs: Transient transfection with Lipofectamine2000was performed in our experiments.
3. Animal Model and ARG Ⅰ Interference: New Zealand White rabbits were subjected to balloon injury of the abdominal aorta and fed with high fat diet to establish atherosclerotic plaque model. At the end of12weeks, lentivirus vectors carried ARG Ⅰ or si-ARG Ⅰ was locally delivered to plaques as our laboratory described before. The rabbits were continued to fed with high fat food for4weeks, then were anesthetized and killed for further studies.
4. ELISAAssay: To determine the extent of cellular inflammatory reaction by detecting released TNFa in cell culture media supernatant.
5. Real-time RT-PCR, Western-blot Analysis and Enzyme Activity Measurement: To determine the effect of LPS on the leve of gene, on protein content or on catalyzing activities of Arg Ⅰ and iNOS, Real-time RT-PCR, Western-blot and enzyme activity measurement were performed, respectively.
6. Monocyte Chemotaxis and Migration: Co-culture system of THP1and hASMCs was established in Transwells to determine the effect of Arg Ⅰ on hASMCs.
7. Immunocytochemistry and Immunohistochemistry: To indicate the locations of the Arg Ⅰ and iNOS, subunit of NF-κB, components of atherosclerotic plaque and inflammatory factors in it, immunocytochemistry and immunohistochemistry were performed, respectively.
8. FCM and Laser Confocal Microscopy: To determine the content of intracellular ONOO-, ROS and O2.-, flowcytometry and laser confocal microscopy were chosen in our experiments.
Results
1. Induction of LPS on produce of TNFα in hASMCs is time-dose dependent LPS (>10ng/ml) induced TNFa release in hASMCs, and TNFα content increased as LPS stimulating dose increased or stimulating time extended. LPS caused quick release of TNFα, but the rate of quantity increasing of TNFα reduced after24hours, and the rate decreased more obviously after48hours.
2. LPS increases TNFα production via iNOS activity in hASMCs LPS induced TNFa release in hASMCs, but this course was suppressed in the presence of iNOS inhibitors (AG or1400W), and the release of TNFα was abolished by about70%to80%, proving that LPS increased TNFa production via iNOS activity in hASMCs.
3. LPS upregulates expression of Arg Ⅰ and iNOS simultaneously Expression of Arg Ⅰ and iNOS increased with the stimulus of LPS in both gene and protein level, but the increasing velocity of Arg Ⅰ lagged behind iNOS. iNOS mRNA level achieved peak after8hours, whereas Arg Ⅰ mRNA level achieved peak after12hours, and then both of their amplification declined; on protein level, iNOS continued to increase with LPS stimulus, whereas Arg Ⅰ began to decline48hours after stimulus.
4. Substrate competition between Arg Ⅰ and iNOS Accordant with above tendency, the catalytic activity of the two enzymes increased with LPS stimulus. But different with the continuous increase of iNOS activity, Arg Ⅰ activity apparently declined after48hours. Moreover, iNOS activity slightly increased as Arg Ⅰ activity began to decline. After pretreating cells with L-arginine which is the common substrate of the two enzymes, both of their catalytic activities increased after12hours and continued to increase till72hours after LPS stimulus.
5. Arg Ⅰ suppresses TNFα production and monocyte chemotaxis and migration Arg Ⅰ apparently suppressed TNFα release induced by LPS in hASMCs, and Arg Ⅰ inhibition or interference augmented TNFα release. Monocyte chemotaxis and migration to hASMCs apparently increased after hASMCs being induced by LPS, whereas Arg Ⅰ upregulation decreased Monocyte chemotaxis and migration and Arg Ⅰ downregulation increased the chemotaxis and migration.
6. Arg Ⅰ inhibits NO release without decreasing iNOS expression Immunohistochemistry showed that Arg Ⅰ and iNOS were largely colocalized in hASMCs. NO release was decreased with Arg Ⅰ elevation but increased with Arg Ⅰ inhibition or interference; whereas Arg Ⅰ elevation had no effect on iNOS expression, indicating Arg Ⅰ suppresses NO release through substrate competition other than decreasing iNOS expression.
7. Arg Ⅰ decreases ROS,O2.-, and ONOO-generation in hASMCs LPS-induced generation of ROS, O2.-, and ONOO-was attenuated by intra-hASMCs Arg Ⅰ elevation, and the increase of O2.-andONOO-generation was also attenuated by iNOS inhibition, without apparent affect on ROS generation.
8. Arg Ⅰ inhibits TNFα produce via NF-kB activation LPS induced NF-kB activation, and the latter caused TNFα generation in cells. Arg Ⅰ decreased TNFα produce via suppressing NF-kB activation.
9. Arg Ⅰ attenuates atherosclerotic plaque inflammation Arg Ⅰ transfection caused decrease of TNFα and iNOS expression and lessen of macrophages in plaque, and Arg Ⅰ inhibition acted conversely. The area ratio between TNFa and SMCs showed that Arg Ⅰ attenuated the ratio apparently.
Conclusion
1. Arginase Ⅰ has the ability to inhibit inflammation in SMCs.
2. Arginase Ⅰ can inhibit inflammation in atherosclerotic plaque.
3. Arginase Ⅰ inhibit inflammation by competiting common catalyzing substrate with the pro-inflammation factor iNOS and then reduce the production of toxic ONOO-generation.
Background
Atherosclerotic plaque rupture is the main cause of myocardial infarction, cerebral thrombosis and other cardiovascular or cerebral vascular acute events. Inflammatory cell infiltration and secondary inflammatory response triggered within the atherosclerotic plaque play important roles in plaque rupture. And inflammation exists through the whole process of the atherosclerosis:for occurrence, development, plaque rupture, thrombosis and even leads to death. Therefore, it can stabilize the vulnerable atherosclerotic plaque and then prevent cardiovascular or cerebral vascular acute events to suppress the inflammation effectively.
Vascular smooth muscle cells (VSMCs) act as "double-edge sword" in the generation and development of atherosclerosis. In the early stage of atherosclerosis, the proliferation of SMCs may contribute to the formation of plaque; And in the middle stage of atherosclerosis, the formation of foam cells derived from SMCs can promote the development of plaque; While in the late stage of atherosclerosis, the proliferation of SMCs may contribute to the thickness of fibrous cap and then increase the stability of the atherosclerotic plaque. It has been documented that Arg Ⅰ can promote SMCs proliferation. And our previous studies also have demonstrated the inflammation inhibited ability of Arg Ⅰ. However, the role of Arg Ⅰ in the stability of atherosclerotic plaque remains unknown.
Based on previous studies, we make the following hypothesis:1) Over expression of Arg Ⅰ can increase the stability of atherosclerotic plaque in the advanced stage of atherosclerosis;2) Arg Ⅰ promote the stability of atherosclerotic plaque through suppressing inflammation of macrophages and promoting SMCs proliferation.
Based on the above ideas, we design the experiments both in vivo and in vitro to validate the hypothesis in the present studies. Our studies try to demonstrate the role of Arg Ⅰ in the stability of atherosclerotic plaque and look for new opportunities and therapeutic target for vulnerable plaque stability and cardiovascular acute events prevention.
Meterials and Methods
1. Animal Model: Male New Zealand White rabbits were fed with high fat diet (1%cholesterol) and subjected to balloon injury to establish atherosclerosis model.
2. Cell Culture: Human mononuclear cells derived macrophage (MDM) and human aorta smooth muscle cells were cultured.
3. Arginase Ⅰ Intervention: Lentiviral vector and arginase inhibitors were both used in our experiments.
4. Sirius Red Staining and Oil Red O Staining: The methods were used to indicate macroscopic structure, collagen content and lipid content of the plaque.
5. Immunohistochemistry and Immunocytochemistry: Immunohistochemistry was used to detect the content of smooth muscle cells and macrophages, the expression of TNFα and IL-6as well as the positive rate of proliferating cell nuclear antigen (PCNA) in the plaque; immunocytochemistry was used to indicating the location and expression of aginase Ⅰ and iNOS in macrophages.
6. Monocyte Chemotaxis and Migration: Co-culture system of human monocyte and human MDM were used in our experiments.
7. Western Blot and Enzyme Activity Measurement: The methods were used to detect the expression and catalyzing activities of Arg Ⅰ and iNOS.
8. Flow Cytometry: To detect the intracellular production of ROS and O2.-,FCM were used.
9. ELISA: ELISA were used to detect the content of TNF α and IL-6in serum and in cell culture supernatant.
10. EDU Incorporation: EDU incorporation was used to evaluate the proliferation of hASMCs.
Results
1. Arginase Ⅰ affects morphosis and components of atherosclerotic plaque: Plaques transfected with arginase Ⅰ had more compact texture and less necrosis cores than plaques transfected with LacZ and treated with physiological saline; collagen content and quantity of smooth muscle cells increased(both p<0.01,VS.LacZ transfection), whereas quantity of macrophages and lipid cores significantly decreased((both p<0.01,VS.LacZ transfection); plaque vulnerability index significantly decreased(both p<0.01, VS.LacZ transfection).
2. Arginase Ⅰ suppresses inflammation in plaque: Concentration of TNF and IL-6in plaques significantly decreased by arginase Ⅰ transfection (both p<0.01, VS. LacZ transfection), whereas the concentration of the above cytokines in the serum did not significantly alter (p>0.05, VS. LacZ transfection); expression of IL-1β wasn't significantly affected by arginase Ⅰ transfection; expression of arginase Ⅰ and activity of arginase were significantly increased (both p<0.01, VS. LacZ transfection) by arginase Ⅰ transfection, while expression of arginase Ⅱ was depressed significantly(p<0.05, VS. LacZ transfection).
3. Arginase Ⅰ suppresses inflammation induced by LPS in macrophages: Production of TNFa and IL-6induced by LPS was depressed by arginase I upregulation in human macrophages and mice macrophages (All p<0.01, VS. LPS alone group); while the release of the above cytokines was increased by arginase Ⅰ downregulation or inhibition(All p<0.01, VS. LPS alone group),and inhibition acted more obviously than downregulation. Further studies validated that LPS induced monocytes chemotaxis and migration to macrophages, and upregulation of arginase Ⅰ in macrophages aggravated monocytes chemotaxis and migration (Both p<0.01, VS.LPS alone group),while arginase Ⅰ downregulation or inhibition suppressed monocytes chemotaxis and migration (All p<0.01, VS. LPS alone group)
5. Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS: Induction of LPS on protein expression and enzyme activity of arginase and iNOS appeared to be time dependent; addition of L-arginine increased the expression of TNF and IL-6induced by LPS in macrophages and inverted the suppression of arginase Ⅰ on the two cytokines; whereas iNOS inhibitor suppressed the release of the two cytokines induced by LPS. Arginase Ⅰ transfection increased intracellular arginase activity, but decreased iNOS activity, and the two enzymes colocalized largely in cells. But regulation of Arginase Ⅰ didn't change the protein expression of iNOS. The results suggested that Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS rather than reducing its expression.
6. Arginase Ⅰ decreases O2.-, ROS and ONOO-generation in macrophages: Arginase Ⅰ elevation attenuated production of O2.-, ROS and ONOO-induced by LPS, whereas arginase Ⅰ suppression or inhibition acted conversely; iNOS inhibition augmented production of O2.-andONOO-induced by LPS, but had little effect on ROS.
7. Arginase Ⅰ contributes to proliferation of smooth muscle cells: Quantity of smooth muscle cells in plaque transfected with arginase Ⅰ was significantly increased (p<0.01, VS. LacZ transfection) compared with plaque transfected with LacZ or treated with physiological saline; arginase Ⅰ elevation promoted proliferation of smooth muscle cells in plaque and cultured human aorta smooth muscle cells, while arginase Ⅰ depression or inhibition acted conversely.
Conclusion
1. Arginase Ⅰ contributes to atherosclerotic plaque stability;
2. Arginase Ⅰ has effect of suppressing inflammation;
3. Arginase Ⅰ exerts the effect of suppressing inflammation through substrate competition with the pro-inflammation factor iNOS and reducing the production of ONOO-by decreasing the catalysate NO of iNOS;
4. Arginase Ⅰ contributes to the proliferation of human aorta smooth muscle cells;
5. Arginase Ⅰ exerts the effect of stabilizing atherosclerotic plaque through suppressing inflammation and promoting proliferation of smooth muscle cells.
炎症是动脉粥样硬化(AS)发生、发展过程中的一个重要因素,炎症的发生常常关系到动脉粥样硬化的进程、严重程度及其后果。炎性因子的分泌能够引起单核、巨噬细胞的趋化、迁移,进一步加重炎症反应。血管平滑肌细胞位于血管壁的中层,在正常的生理状态下这些细胞通常是“安静”的,表现出一种分化状态来维持血管的结构及其紧张度。然而,在某些条件下(如动脉粥样硬化形成),血管平滑肌细胞获得了一种“分泌”型的表型,表现为去分化状态,并能够合成、分泌一些炎性因子,从而促进了血管的病变过程。
精氨酸酶Ⅰ(Arg Ⅰ)能够促进平滑肌细胞的增殖,但其在炎症反应中的作用还未明确。L-精氨酸是精氨酸酶(Arginase)和一氧化氮合成酶(NOS)的共同催化底物。在炎症反应中,iNOS能够产生过量的一氧化氮(NO),通过与细胞内的超氧阴离子(02.-)反应产生有致炎作用的ON00-。因此,减少iNOS的底物有助于抑制细胞内的炎症。
在以往研究的基础上,我们提出如下假设：1)精氨酸酶Ⅰ具有抑制细胞内炎症作用；2)精氨酸酶Ⅰ抑制炎症的作用是通过与iNOS竞争底物实现的。
本研究正是基于以上思路,拟通过体外和体内两部分实验以验证这一假说。旨在探讨Arg Ⅰ在平滑肌细胞(SMC)炎症反应中的作用,以期寻找到与SMC相关的炎症性疾病的预防和治疗靶点。
材料与方法
1.细胞培养及模型建立：采用人主动脉平滑肌细胞(hASMCs)作为研究对象；体外以脂多糖(LPS)作为诱导剂诱导hASMCs发生炎症反应。
2.细胞内Arg Ⅰ干预：采用瞬时转染ARGI-pcDNA3.1(+)质粒和si-ARGI小干扰片段的方式实现对hASMCs细胞内Arg Ⅰ的干预。
3.动物模型的建立及干预：新西兰大白兔采用腹主动脉球囊拉伤+高脂喂养的方式建立动脉粥样硬化斑块模型,于喂养12周后按照本实验室建立的斑块内注射的方法对斑块进行ARG Ⅰ过表达及小干扰慢病毒载体转染,继续喂养四周后将动物实施安乐死后取材。
4.采用酶联免疫吸附法(ELISA):检测平滑肌细胞培养中的肿瘤坏死因子a (TNFa)的含量；
5.实时定量逆转录PCR (Real-time RT-PCR)、Western-blot及酶活性检测法：分别检测精氨酸酶I(Arg Ⅰ)和诱导型一氧化氮合成酶(iNOS)的基因表达水平、蛋白质表达水平及酶活性变化。
6.单核细胞趋化及迁移检测：采用Transwell小室构建了人单核细胞(THP1)与hASMCs的共培养体系,检测单核细胞的趋化及迁移能力。
7.细胞荧光免疫组化及组织荧光免疫组化：显示细胞内的Arg Ⅰ、iNOS、NF-κB的P65亚基、斑块内细胞成分及TNF a的表达。
8.流式细胞术、激光共聚焦显微技术：分别检测细胞内过氧亚硝基(ONOO-)、活性氧簇(ROS)及超氧阴离子(02.-)的含量。
研究结果
1.LPS对hASMCs细胞内TNFa产生的诱导具有时间-剂量依赖性：LPS (>10ng/ml)能够诱导hASMCs细胞内TNFa的释放,且TNFa释放量随LPS刺激剂量增加或时间延长而增大。细胞受LPS刺激后能引起TNFa的快速释放,但刺激超过24小时,其TNFa释放量增加幅度有所减小；刺激超过48小时,其增加幅度减小更加明显。
2.LPS通过激活iNOS增加人主动脉平滑肌细胞内TNFa的表达量：LPS能够诱导hASMCs细胞TNFa的释放,但加入iNOS抑制剂(AG或1400W)后,此过程被抑制,TNFa的分泌量减少大约70～80%,证明LPS通过激活iNOS增加人主动脉平滑肌细胞内TNFa的表达量。
3.LPS能够同时上调人主动脉平滑肌细胞内Arg Ⅰ和iNOS的表达：细胞在LPS的刺激下,无论基因还是蛋白水平Arg Ⅰ和iNOS均随LPS刺激而升高,但Arg Ⅰ的升高速度落后于iNOS。iNOS的mRNA水平在8小时达到高峰,而Arg Ⅰ的mRNA水平在12小时达到高峰,之后二者的增幅出现回落；在蛋白表达水平,iNOS在LPS的刺激下表现为持续升高,而Arg Ⅰ在LPS刺激超过48小时后其蛋白水平开始下降。
4. Arg Ⅰ与iNOS之间存在底物竞争：与上述趋势一致的是,两种酶的催化活性也随LPS刺激而升高。但与NOS活性持续升高不同的是,精氨酸酶活性在48小时后明显下降。而且,当精氨酸酶活性降低时,NOS活性反而有小幅升高。在预先向培养基中加入二者的共同底物L-精氨酸后,两种酶的催化活性在LPS作用12小时后均有所增加,这种升高趋势一直持续至72小时。
5. Arg Ⅰ能够抑制TNFa的生成和单核细胞的趋化和迁移：精氨酸酶Ⅰ能够明显抑制LPS诱导的人主动脉平滑肌细胞TNFa的释放,而精氨酸酶Ⅰ抑制或干扰则能加剧TNFa的释放。经LPS诱导后的人主动脉平滑肌细胞,单核细胞向其趋化和迁移的能力明显增加。而提高精氨酸酶Ⅰ的表达则能降低单核细胞的这种趋化和迁移能力,反之,则加剧单核细胞的迁移和趋化能力的增加。
6. Arg Ⅰ通过与iNOS竞争共同的催化底物来抑制平滑肌细胞NO的释放：免疫组化结果显示在人主动脉平滑肌细胞中精氨酸酶Ⅰ和iNOS在空间上存在很大程度的共定位；升高精氨酸酶Ⅰ能够加剧LPS诱导的NO的释放,而抑制或干扰精氨酸酶Ⅰ的表达,则能逆转这一现象；但升高精氨酸酶Ⅰ却未能对iNOS的表达量造成影响,提示精氨酸酶Ⅰ通过与iNOS竞争共同的催化底物L-精氨酸来抑制NO的释放,而非通过减少iNOS的表达量。
7. Arg Ⅰ能够减少人主动脉平滑肌细胞内ROS、02.-以及ONOO-的生成：增加人主动脉平滑肌细胞内的精氨酸酶Ⅰ表达能够抑制LPS对细胞内ROS、02.-及ON00-的生成的诱导；抑制iNOS的活性同样能够抑制细胞内02.-及ON00-的生成增加,而未能对ROS的生成产生明显影响。
8.Arg Ⅰ通过抑制NF-kB的激活降低TNFa的生成：LPS能够引起NF-κB的激活,而后者的激活则能引起细胞内TNFa的生成。精氨酸酶Ⅰ过表达能够通过抑制NF-kB的激活降低TNFa的生成。
9. Arg Ⅰ能够降低动脉粥样硬化斑块内的炎症反应：局部转染精氨酸酶Ⅰ过表达慢病毒载体能够引起斑块内TNFa、iNOS的表达量及巨噬细胞含量的降低,而抑制精氨酸酶Ⅰ则起到相反的效果。TNFa与SMC的面积比计算结果显示,精氨酸酶Ⅰ能够明显地降低该比值。
结论及意义
1.精氨酸酶Ⅰ具有抑制SMC炎症的作用；
2.精氨酸酶Ⅰ具有抑制动脉粥样硬化斑块内炎症的作用；
3.精氨酸酶Ⅰ的抑炎作用通过与具有促炎作用的iNOS竞争底物,减少了后者催化产物的生成量,从而减少ONOO-的生成,进而起到抑制炎症的作用。
4.在本实验研究中,我们对Arg Ⅰ在对入主动脉平滑肌细胞和兔动脉粥样硬化斑块炎症细胞因子分泌中的作用及其机制做了系统的阐述,以期为临床治疗此类疾病提供新的思路和治疗靶点。
研究背景及目的
动脉粥样硬化斑块破裂(Plaque Rupture)是引发心肌梗死、脑血栓等心脑血管急性事件的主要原因。而动脉粥样硬化斑块内炎症细胞的浸润及继发的炎症反应是引发动脉粥样硬化斑块不稳定的主要因素。炎症反应贯穿了整个动脉粥样硬化进程：从发生、发展直至斑块破裂、血栓形成,甚至死亡。因此,有效地抑制炎症反应的发生可以稳定动脉粥样硬化易损斑块,避免心血管急性事件的发生。
血管平滑肌细胞在动脉粥样硬化发生、发展过程中发挥“双刃剑”的作用。在动脉粥样硬化形成早期,平滑肌细胞的增殖能够促进斑块的形成；在中期,平滑肌细胞的泡沫化能够促进斑块的进程；而在斑块发展晚期,平滑肌细胞的增多则有助于纤维帽的增厚,促进斑块的稳定性。以往的研究证实,精氨酸酶Ⅰ具有抑制炎症反应的作用；我们在前面的研究中还发现,精氨酸酶Ⅰ具有抑制炎症反应的作用;但该酶动脉粥样硬化斑块稳定性中的作用还未见报道。
在以往研究的基础上,我们提出如下假设：1)在动脉粥样硬化发展的晚期,高表达精氨酸酶Ⅰ能够稳定动脉粥样硬化斑块；2)精氨酸酶Ⅰ稳定动脉粥样硬化斑块的作用是通过抑制巨噬细胞炎症反应和促进血管平滑肌细胞增殖的双重机制实现的。
本研究正是基于以上思路,从体内和体外两方面设计实验以验证这一假说。旨在探讨Arg Ⅰ在动脉粥样硬化斑块稳定性中的作用,以期为稳定易损斑块、预防心血管急性事件的发生防治寻找新的契机和治疗靶点。
研究方法
1.动物模型的建立：雄性新西兰大白兔,采用高脂喂养(1%胆固醇)+球囊拉伤腹主动脉的方法,构建动脉粥样硬化模型。
2.细胞培养：分别培养人单核来源的巨噬细胞(MDM)和人主动脉平滑肌细胞(hVSMC)。
3.精氨酸酶Ⅰ的干预：采用慢病毒载体和精氨酸酶抑制剂。
4.天狼猩红、油红0染色染色：分别显示斑块的大体结构、斑块内胶原及斑块内脂质含量。
5.免疫组化检测：检测斑块组织内平滑肌细胞、巨噬细胞的含量、TNFa、IL-6的表达量、细胞增殖核抗原(PCNA)的阳性率、精氨酸酶Ⅰ与iNOS在巨噬细胞内的定位关系及细胞内ONOO-的表达量。
6.单核细胞趋化、迁移实验：采用人单核细胞与人MDM共培养体系。
7. Western Blot及生化法：分别用于检测细胞及组织内内的精氨酸酶Ⅰ及iNOS的蛋白及其活性。
8.流式细胞术：用于检测细胞内ROS及02.-的生成量。
9. ELISA检测：用于检测血清及细胞培养上清中TNFa及IL-6的含量。
10.EDU掺入实验：检测人主动平滑肌细胞的增殖能力。
研究结果
1.精氨酸酶Ⅰ能够影响动脉粥样硬化的形态结构及斑块组分：较之LacZ转染和生理盐水处理组斑块而言,精氨酸酶Ⅰ转染的斑块其结构更加致密且坏死核心较小；斑块内胶原含量和平滑肌细胞的数量增多(both p<0.01, VS. LacZ转染组),而巨噬细胞和脂质核心则明显减少(bothp<0.01, VS. LacZ转染组)；斑块易损指数明显降低(both p<0.01, VS. LacZ转染组)。
2.精氨酸酶Ⅰ能够抑制斑块内的炎症反应：局部转染精氨酸酶Ⅰ能够明显降低斑块内TNFa和IL-6的水平(both p<0.01, VS. LacZ转染组),而上述因子在血清内的浓度却未受到明显改变(p>0.05, VS. LacZ转染组)；转染精氨酸酶Ⅰ未能对斑块内IL-1β的表达产生明显影响；局部转染精氨酸酶Ⅰ增加了斑块内精氨酸酶Ⅰ的表达及总精氨酸酶的活性(both p<0.01, VS. LacZ转染组),而却降低了精氨酸酶Ⅱ的表达(p<0.05, VS. LacZ转染组)。
3.精氨酸酶Ⅰ能够抑制LPS诱导的巨噬细胞的炎症反应：对于人巨噬细胞和小鼠巨噬细胞而言,增加精氨酸酶Ⅰ的表达均能够有效降低由LPS诱导引起的TNFa和IL-6的生成量(All p<0.01, VS. LPS单独处理组)；而降低精氨酸酶Ⅰ的表达或抑制其活性则加剧了上述两种因子的释放(All p<0.01, VS. LPS单独处理组),且抑制剂使用引起的效果较精氨酸酶Ⅰ明显。进一步的实验证实,LPS能够引起单核细胞向巨噬细胞的趋化和迁移,而增加巨噬细胞内的精氨酸酶Ⅰ能够加剧单核细胞的趋化和迁移能力(Both p<0.01, VS.LPS单独处理组),而降低或抑制精氨酸酶Ⅰ则能抑制单核细胞的趋化和迁移(All p<0.01, VS. LPS单独处理组)。
4.精氨酸酶1能够通过与iNOS竞争底物发挥炎症抑制的作用：LPS对精氨酸酶和iNOS的蛋白表达及酶活性的诱导具有时间依赖性；添加L-精氨酸不但能够加剧LPS诱导的巨噬细胞内TNFa和IL-6的表达,而且能够逆转精氨酸酶Ⅰ对两种因子释放的抑制作用;而添加iNOS抑制剂则能抑制LPS诱导的两种炎性因子的释放。转染精氨酸酶Ⅰ能够增加细胞内精氨酸酶的活性,而降低iNOS的活性,且两种酶在细胞内存在很大程度的共定位。但调控精氨酸酶Ⅰ却不能改变iNOS的蛋白表达量。这些结果说明精氨酸酶Ⅰ通过与iNOS竞争底物而不是减少其表达量来达到抑制炎症的目的。
5.精氨酸酶Ⅰ能够降低巨噬细胞内的02.-, ROS以及ONOO-的生成：提高巨噬细胞内精氨酸酶Ⅰ的表达能够降低LPS诱导的02.-,ROS以及ONOO-的生成。而降低或抑制精氨酸酶Ⅰ则起到相反的效果；使用iNOS的抑制能够LPS诱导的细胞内02.-及ONOO-的生成增加,但对ROS的影响不大。
6.精氨酸酶Ⅰ能够促进平滑肌细胞的增殖：局部转染精氨酸酶Ⅰ较之LacZ转染和生理盐水处理而言,其斑块内平滑肌细胞增殖数量明显增加(p<0.01, VS. LacZ转染组)；提高精氨酸酶Ⅰ能够促进斑块内平滑肌细胞及体外培养的人主动脉平滑肌细胞的增殖,而降低或抑制精氨酸酶则起到相反的作用。
结论及意义
1.精氨酸酶Ⅰ具有促进动脉粥样硬化斑块稳定性的作用；
2.精氨酸酶Ⅰ具有抑制巨噬细胞炎症反应的作用；
3.精氨酸酶Ⅰ通过与致炎基因iNOS竞争反应底物,减少iNOS的催化底物NO的生成,进而减少ONOO-的生成,从而达到抑制炎症的目的；
4.精氨酸酶Ⅰ能够促进人主动脉平滑肌细胞的增殖；
5.精氨酸酶Ⅰ对动脉粥样硬化斑块的稳定作用,是通过抑制炎症反应及促进平滑及细胞的增殖来实现的。
6.该研究为进一步阐明AS斑块稳定性的作用机理和AS斑块破裂及相关的心血管急性事件的防治提供新的思路及有效的治疗靶点。
Backgroud
Inflammation plays an important role in the generation and development of atherosclerosis. Inflammation is often related to the development of atherosclerosis, severity and its consequences. The secretion of inflammatory factors can trigger chemotaxis and migration of monocytes and macrophage, and further to aggravate inflammatory reaction. Smooth muscle cells (SMCs) locate in the middle layer of vascular wall and keep "quiescent" under normal physiological conditions. However, these kinds of cells often acquire a state of dedifferentiation and exhibit the character of "synthetic" phenotype under atherosclerotic conditions, contributing to vascular pathological changes by synthesizing and secreting certain inflammatory factors.
It has been demonstrated that arginase Ⅰ have the ability to promote SMCs proliferation. However, its role in inflammation has remains unknown. Both arinase and nitric oxide synthetase (NOS) catalyze L-arginine, which can be catatlyzed by induced nitric oxide synthetase (iNOS) to produce excess nitric oxide (NO) in cytoplasm. Under inflammatory conditions, the iNOS can produce excessive nitric oxide (NO), which can produce proinflammatory effects of ONOO-by reaction with intracellular superoxide anion (O2-). Then, it may inhibit the inflammation to decrease the catalyzing substrate of iNOS.
Based on the previous studies, we make the following assumptions:1) Arginase Ⅰ has the ability to inhibit inflammation;2) Arginase Ⅰ inhibits the inflammation by decreasing catalyzing substrate of pro-inflammatory iNOS.
Based on the above ideas, we design the experiments in vivo and in vitro to validate the hypothesize in the present study. We aim to investigate the role of Arg Ⅰ in inflammaiton of SMCs and try to look for new opportunities and therapeutic targets in SMC associated inflammatory disease.
Material and Methods
1. Cell Culture and Inflammatory Cell Model: Human aorta smooth muscular cells (hASMCs) were cultured and lipopolysaccharide (LPS) was used to induce inflammatory reaction in hASMCs in our experiments.
2. ARG Ⅰ Interference in hASMCs: Transient transfection with Lipofectamine2000was performed in our experiments.
3. Animal Model and ARG Ⅰ Interference: New Zealand White rabbits were subjected to balloon injury of the abdominal aorta and fed with high fat diet to establish atherosclerotic plaque model. At the end of12weeks, lentivirus vectors carried ARG Ⅰ or si-ARG Ⅰ was locally delivered to plaques as our laboratory described before. The rabbits were continued to fed with high fat food for4weeks, then were anesthetized and killed for further studies.
4. ELISAAssay: To determine the extent of cellular inflammatory reaction by detecting released TNFa in cell culture media supernatant.
5. Real-time RT-PCR, Western-blot Analysis and Enzyme Activity Measurement: To determine the effect of LPS on the leve of gene, on protein content or on catalyzing activities of Arg Ⅰ and iNOS, Real-time RT-PCR, Western-blot and enzyme activity measurement were performed, respectively.
6. Monocyte Chemotaxis and Migration: Co-culture system of THP1and hASMCs was established in Transwells to determine the effect of Arg Ⅰ on hASMCs.
7. Immunocytochemistry and Immunohistochemistry: To indicate the locations of the Arg Ⅰ and iNOS, subunit of NF-κB, components of atherosclerotic plaque and inflammatory factors in it, immunocytochemistry and immunohistochemistry were performed, respectively.
8. FCM and Laser Confocal Microscopy: To determine the content of intracellular ONOO-, ROS and O2.-, flowcytometry and laser confocal microscopy were chosen in our experiments.
Results
1. Induction of LPS on produce of TNFα in hASMCs is time-dose dependent LPS (>10ng/ml) induced TNFa release in hASMCs, and TNFα content increased as LPS stimulating dose increased or stimulating time extended. LPS caused quick release of TNFα, but the rate of quantity increasing of TNFα reduced after24hours, and the rate decreased more obviously after48hours.
2. LPS increases TNFα production via iNOS activity in hASMCs LPS induced TNFa release in hASMCs, but this course was suppressed in the presence of iNOS inhibitors (AG or1400W), and the release of TNFα was abolished by about70%to80%, proving that LPS increased TNFa production via iNOS activity in hASMCs.
3. LPS upregulates expression of Arg Ⅰ and iNOS simultaneously Expression of Arg Ⅰ and iNOS increased with the stimulus of LPS in both gene and protein level, but the increasing velocity of Arg Ⅰ lagged behind iNOS. iNOS mRNA level achieved peak after8hours, whereas Arg Ⅰ mRNA level achieved peak after12hours, and then both of their amplification declined; on protein level, iNOS continued to increase with LPS stimulus, whereas Arg Ⅰ began to decline48hours after stimulus.
4. Substrate competition between Arg Ⅰ and iNOS Accordant with above tendency, the catalytic activity of the two enzymes increased with LPS stimulus. But different with the continuous increase of iNOS activity, Arg Ⅰ activity apparently declined after48hours. Moreover, iNOS activity slightly increased as Arg Ⅰ activity began to decline. After pretreating cells with L-arginine which is the common substrate of the two enzymes, both of their catalytic activities increased after12hours and continued to increase till72hours after LPS stimulus.
5. Arg Ⅰ suppresses TNFα production and monocyte chemotaxis and migration Arg Ⅰ apparently suppressed TNFα release induced by LPS in hASMCs, and Arg Ⅰ inhibition or interference augmented TNFα release. Monocyte chemotaxis and migration to hASMCs apparently increased after hASMCs being induced by LPS, whereas Arg Ⅰ upregulation decreased Monocyte chemotaxis and migration and Arg Ⅰ downregulation increased the chemotaxis and migration.
6. Arg Ⅰ inhibits NO release without decreasing iNOS expression Immunohistochemistry showed that Arg Ⅰ and iNOS were largely colocalized in hASMCs. NO release was decreased with Arg Ⅰ elevation but increased with Arg Ⅰ inhibition or interference; whereas Arg Ⅰ elevation had no effect on iNOS expression, indicating Arg Ⅰ suppresses NO release through substrate competition other than decreasing iNOS expression.
7. Arg Ⅰ decreases ROS,O2.-, and ONOO-generation in hASMCs LPS-induced generation of ROS, O2.-, and ONOO-was attenuated by intra-hASMCs Arg Ⅰ elevation, and the increase of O2.-andONOO-generation was also attenuated by iNOS inhibition, without apparent affect on ROS generation.
8. Arg Ⅰ inhibits TNFα produce via NF-kB activation LPS induced NF-kB activation, and the latter caused TNFα generation in cells. Arg Ⅰ decreased TNFα produce via suppressing NF-kB activation.
9. Arg Ⅰ attenuates atherosclerotic plaque inflammation Arg Ⅰ transfection caused decrease of TNFα and iNOS expression and lessen of macrophages in plaque, and Arg Ⅰ inhibition acted conversely. The area ratio between TNFa and SMCs showed that Arg Ⅰ attenuated the ratio apparently.
Conclusion
1. Arginase Ⅰ has the ability to inhibit inflammation in SMCs.
2. Arginase Ⅰ can inhibit inflammation in atherosclerotic plaque.
3. Arginase Ⅰ inhibit inflammation by competiting common catalyzing substrate with the pro-inflammation factor iNOS and then reduce the production of toxic ONOO-generation.
Background
Atherosclerotic plaque rupture is the main cause of myocardial infarction, cerebral thrombosis and other cardiovascular or cerebral vascular acute events. Inflammatory cell infiltration and secondary inflammatory response triggered within the atherosclerotic plaque play important roles in plaque rupture. And inflammation exists through the whole process of the atherosclerosis:for occurrence, development, plaque rupture, thrombosis and even leads to death. Therefore, it can stabilize the vulnerable atherosclerotic plaque and then prevent cardiovascular or cerebral vascular acute events to suppress the inflammation effectively.
Vascular smooth muscle cells (VSMCs) act as "double-edge sword" in the generation and development of atherosclerosis. In the early stage of atherosclerosis, the proliferation of SMCs may contribute to the formation of plaque; And in the middle stage of atherosclerosis, the formation of foam cells derived from SMCs can promote the development of plaque; While in the late stage of atherosclerosis, the proliferation of SMCs may contribute to the thickness of fibrous cap and then increase the stability of the atherosclerotic plaque. It has been documented that Arg Ⅰ can promote SMCs proliferation. And our previous studies also have demonstrated the inflammation inhibited ability of Arg Ⅰ. However, the role of Arg Ⅰ in the stability of atherosclerotic plaque remains unknown.
Based on previous studies, we make the following hypothesis:1) Over expression of Arg Ⅰ can increase the stability of atherosclerotic plaque in the advanced stage of atherosclerosis;2) Arg Ⅰ promote the stability of atherosclerotic plaque through suppressing inflammation of macrophages and promoting SMCs proliferation.
Based on the above ideas, we design the experiments both in vivo and in vitro to validate the hypothesis in the present studies. Our studies try to demonstrate the role of Arg Ⅰ in the stability of atherosclerotic plaque and look for new opportunities and therapeutic target for vulnerable plaque stability and cardiovascular acute events prevention.
Meterials and Methods
1. Animal Model: Male New Zealand White rabbits were fed with high fat diet (1%cholesterol) and subjected to balloon injury to establish atherosclerosis model.
2. Cell Culture: Human mononuclear cells derived macrophage (MDM) and human aorta smooth muscle cells were cultured.
3. Arginase Ⅰ Intervention: Lentiviral vector and arginase inhibitors were both used in our experiments.
4. Sirius Red Staining and Oil Red O Staining: The methods were used to indicate macroscopic structure, collagen content and lipid content of the plaque.
5. Immunohistochemistry and Immunocytochemistry: Immunohistochemistry was used to detect the content of smooth muscle cells and macrophages, the expression of TNFα and IL-6as well as the positive rate of proliferating cell nuclear antigen (PCNA) in the plaque; immunocytochemistry was used to indicating the location and expression of aginase Ⅰ and iNOS in macrophages.
6. Monocyte Chemotaxis and Migration: Co-culture system of human monocyte and human MDM were used in our experiments.
7. Western Blot and Enzyme Activity Measurement: The methods were used to detect the expression and catalyzing activities of Arg Ⅰ and iNOS.
8. Flow Cytometry: To detect the intracellular production of ROS and O2.-,FCM were used.
9. ELISA: ELISA were used to detect the content of TNF α and IL-6in serum and in cell culture supernatant.
10. EDU Incorporation: EDU incorporation was used to evaluate the proliferation of hASMCs.
Results
1. Arginase Ⅰ affects morphosis and components of atherosclerotic plaque: Plaques transfected with arginase Ⅰ had more compact texture and less necrosis cores than plaques transfected with LacZ and treated with physiological saline; collagen content and quantity of smooth muscle cells increased(both p<0.01,VS.LacZ transfection), whereas quantity of macrophages and lipid cores significantly decreased((both p<0.01,VS.LacZ transfection); plaque vulnerability index significantly decreased(both p<0.01, VS.LacZ transfection).
2. Arginase Ⅰ suppresses inflammation in plaque: Concentration of TNF and IL-6in plaques significantly decreased by arginase Ⅰ transfection (both p<0.01, VS. LacZ transfection), whereas the concentration of the above cytokines in the serum did not significantly alter (p>0.05, VS. LacZ transfection); expression of IL-1β wasn't significantly affected by arginase Ⅰ transfection; expression of arginase Ⅰ and activity of arginase were significantly increased (both p<0.01, VS. LacZ transfection) by arginase Ⅰ transfection, while expression of arginase Ⅱ was depressed significantly(p<0.05, VS. LacZ transfection).
3. Arginase Ⅰ suppresses inflammation induced by LPS in macrophages: Production of TNFa and IL-6induced by LPS was depressed by arginase I upregulation in human macrophages and mice macrophages (All p<0.01, VS. LPS alone group); while the release of the above cytokines was increased by arginase Ⅰ downregulation or inhibition(All p<0.01, VS. LPS alone group),and inhibition acted more obviously than downregulation. Further studies validated that LPS induced monocytes chemotaxis and migration to macrophages, and upregulation of arginase Ⅰ in macrophages aggravated monocytes chemotaxis and migration (Both p<0.01, VS.LPS alone group),while arginase Ⅰ downregulation or inhibition suppressed monocytes chemotaxis and migration (All p<0.01, VS. LPS alone group)
5. Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS: Induction of LPS on protein expression and enzyme activity of arginase and iNOS appeared to be time dependent; addition of L-arginine increased the expression of TNF and IL-6induced by LPS in macrophages and inverted the suppression of arginase Ⅰ on the two cytokines; whereas iNOS inhibitor suppressed the release of the two cytokines induced by LPS. Arginase Ⅰ transfection increased intracellular arginase activity, but decreased iNOS activity, and the two enzymes colocalized largely in cells. But regulation of Arginase Ⅰ didn't change the protein expression of iNOS. The results suggested that Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS rather than reducing its expression.
6. Arginase Ⅰ decreases O2.-, ROS and ONOO-generation in macrophages: Arginase Ⅰ elevation attenuated production of O2.-, ROS and ONOO-induced by LPS, whereas arginase Ⅰ suppression or inhibition acted conversely; iNOS inhibition augmented production of O2.-andONOO-induced by LPS, but had little effect on ROS.
7. Arginase Ⅰ contributes to proliferation of smooth muscle cells: Quantity of smooth muscle cells in plaque transfected with arginase Ⅰ was significantly increased (p<0.01, VS. LacZ transfection) compared with plaque transfected with LacZ or treated with physiological saline; arginase Ⅰ elevation promoted proliferation of smooth muscle cells in plaque and cultured human aorta smooth muscle cells, while arginase Ⅰ depression or inhibition acted conversely.
Conclusion
1. Arginase Ⅰ contributes to atherosclerotic plaque stability;
2. Arginase Ⅰ has effect of suppressing inflammation;
3. Arginase Ⅰ exerts the effect of suppressing inflammation through substrate competition with the pro-inflammation factor iNOS and reducing the production of ONOO-by decreasing the catalysate NO of iNOS;
4. Arginase Ⅰ contributes to the proliferation of human aorta smooth muscle cells;
5. Arginase Ⅰ exerts the effect of stabilizing atherosclerotic plaque through suppressing inflammation and promoting proliferation of smooth muscle cells.
引文
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