血管外膜激活促进移植性血管病新内膜形成及发展的实验研究
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摘要
移植性血管病(transplant vasculopathy, TV)是一种增殖性血管疾病,是影响移植器官长期存活的最主要因素。TV与动脉粥样硬化(atherosclerosis,AS)及血管成形术后再狭窄(restenosis, RS)有许多相同的诱发因素和病理表现。传统观点认为TV的病理表现主要包括平滑肌细胞(smooth muscle cells,SMCs)迁移、增殖,炎症细胞浸润,巨噬细胞及SMCs中脂质聚集,细胞外基质(extracellular matrix, ECM)生成增多以及新内膜形成。因此,人们对于血管疾病的研究主要集中在内皮和平滑肌功能失调,而血管外膜一直未成为关注的焦点。
动脉血管壁分为三层：内膜、中膜和外膜。每一层都有特殊的组织学、生物化学和功能特点。因此,每一层都以特定的方式来维持血管稳态,调节血管对于应激和损伤的反应。与内膜和中膜相比,外膜一直被认为仅仅是一层支撑组织,为平滑肌层提供营养。近年来,血管外膜在血管疾病如AS和RS中的作用日益受到重视。有资料提示血管外膜对于血管的结构和功能调节起着直接或间接的重要调控作用。成纤维细胞是血管外膜最主要的细胞类型,越来越多的实验研究证明外膜成纤维细胞在新内膜形成和血管重塑过程中并非是被动的旁观者,而是对多种刺激具有反应的积极的参与者。当受到细胞因子、损伤和牵拉等刺激时,外膜成纤维细胞可以被激活导致细胞表型转化、迁移、增殖以及合成细胞外基质。些重要的细胞因子如转化生长因子-β1(transforming growth factor beta 1,TGF-β1)和内皮素-1(endothelin-1, ET-1)都能诱导成纤维细胞转化为肌成纤维细胞(myofibroblast,MF),从而可以穿过破损的中膜参与新内膜的形成。血管外膜成纤维细胞也能够分泌多种细胞因子如TGF-β1、单核细胞趋化蛋白-1 (monocyte chemoattrant protein-1, MCP-1)、白细胞介素(interleukin, IL)、ET-1、肿瘤坏死因子α(tumor necrosis factor alpha, TNFα)、基质金属蛋白酶(matrix metalloproteinases, MMPs)及尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase)等。外膜反应普遍存在于各种血管疾病中。在猪的血管成形术模型中,血管周围组织炎症反应要先于新内膜的形成。我们实验室的前期研究工作也发现在载脂蛋白E基因敲除(apolipoprotein E knockout,apoE(-/-))小鼠AS模型中,外膜成纤维细胞在AS早期被激活,并且早于内膜病灶的形成,能够诱导单核细胞游走进入血管壁的MCP-1最早表达于外膜成纤维细胞。有研究表明在猪球囊损伤模型中血管外膜有大量的α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫反应阳性细胞。Li等在兔球囊损伤模型中将β-半乳糖苷酶基因转入颈动脉外膜成纤维细胞用以标记细胞,成功地在体直接证明外膜成纤维细胞能迁移到新内膜并参与新内膜的形成。在颈动脉动-静脉移植中,外膜成纤维细胞增殖,表型转化为肌成纤维细胞并向内膜迁移均早于新内膜的形成。大量研究表明仅仅是外膜损伤也能引起新内膜和／或动脉粥样硬化病灶的形成。近年来,基因疗法成为当前治疗心血管疾病的一个研究“热点”,而动脉外膜因其在血管外围更有利于药物输送和基因治疗。Dourron等设计了一种新型的含有成纤维细胞活化启动子并能表达NADPH氧化酶抑制序列gp91ds (Ad-PDGFβR-gp91ds/eGFP)的复制缺陷性腺病毒,该腺病毒转入大鼠颈总动脉外膜后有效抑制了整个血管O2-产生和新内膜形成。
尽管人们在血管疾病中对血管外膜作用机制研究取得了一些进展,但血管外膜的许多生物学机制及其与血管疾病发生的关系仍不清楚,尤其是在移植性血管病中的作用并不清楚,目前国内外的报道也非常少。
目的：
本实验利用袖套管技术将大鼠胸主动脉移植到腹主动脉建立移植性血管病模型,通过在体动物实验以及体外培养移植血管外膜成纤维细胞,观察血管外膜细胞表型转化、增殖、迁移以及多种细胞因子在mRNA和蛋白水平表达的动态变化,进一步探讨血管外膜以及外膜成纤维细胞激活在移植性血管病新内膜形成及发展中的作用机制。在以上实验基础上,本研究进一步探讨应用RNA干扰技术特异性地抑制NADPH氧化酶亚基p47phox的表达,从而下调NADPH氧化酶的活性,观察其对移植血管外膜成纤维细胞增殖和迁移的抑制作用,为移植性血管病以血管外膜成纤维细胞为作用靶点进行基因治疗提供实验依据。
本课题分以下三个部分进行研究：
第一部分：移植性血管病大鼠模型的建立
方法：
以Sprague-Dawley (SD)大鼠的胸主动脉为供体血管,利用袖套管技术移植到Wistar大鼠腹主动脉作为异系移植实验组,以SD大鼠对SD大鼠同系移植作为对照组。在血管移植后3,7,14天,取出移植的胸主动脉,进行苏木素-伊红(HE)染色及Weigert弹力纤维染色,应用Image-Pro Plus 5.0计算机图像分析系统检测移植血管的内膜和中膜增生情况及内膜／中膜厚度比值。
结果：
手术成功率为90%,移植血管的通畅率达100%。同系移植对照组在各个时间点未出现内膜的明显增生,而异系移植实验组在移植后7天出现内膜增生,术后14天内膜厚度显著增加。对照组和实验组移植血管中膜均未见明显增厚。异系移植实验组内膜／中膜厚度比值在移植后7天开始增高,14天时达到顶峰。
结论：
利用袖套管技术进行SD和Wistar大鼠间的血管移植符合移植性血管病的病理形态改变,并且操作简单,费用小,重复性好,可以用于研究移植性血管病的病理机制及干预治疗。
第二部分：在体动物实验研究血管外膜激活在移植性血管病新内膜形成及发展中的作用
方法：
在血管移植后3,7,14天,取出移植的胸主动脉。用免疫组织化学方法观察不同时间点血管外膜和内膜波形蛋白(vimentin),α平滑肌肌动蛋白(α-SMA),T细胞抗原受体CD3,细胞核增殖抗原Ki-67,核因子-κB (nuclear factorκB,NF-κB), MCP-1,细胞间粘附分子-1(intercellular adhesion molecule-1, ICAM-1),血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1),基质金属蛋白酶-7 (MMP-7), TGF-β1,硝基酪氨酸及p47phox等细胞因子蛋白水平表达的动态变化；用荧光原位杂交技术检测gp91phox mRNA的表达水平；用荧光实时定量聚合酶链反应(quantitative real-time reverse transcriptase polymerase chain reaction,QRT-PCR)检测移植血管外膜MCP-1,IL-1β,TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox和p47phox等细胞因子mRNA的表达水平。
结果：
1.免疫组织化学检测显示异系移植实验组和同系移植对照组在血管移植后不同时间点,移植血管外膜及内膜或新内膜的大部分细胞呈现vimentin阳性,中膜几乎无阳性细胞。异系移植实验组在术后3天移植血管外膜部分细胞即呈现α-SMA阳性,随着时间延长α-SMA阳性细胞数量逐渐增加,术后7天和14天新内膜细胞也开始表达α-SMA,而同系移植对照组血管外膜及内膜细胞几乎无α-SMA阳性表达；两组移植血管中膜α-SMA始终呈强阳性表达。
2.异系移植实验组和同系移植对照组在血管移植后不同时间点移植血管外膜及内膜或新内膜T细胞抗原受体CD3阳性细胞不超过4%,说明增生的细胞主要是外膜成纤维细胞,新内膜形成可能主要与外膜成纤维细胞有关。
3.Ki-67免疫组织化学结果显示在术后3天异系移植实验组外膜成纤维细胞开始出现增殖,内膜细胞仅有少量细胞增殖,在术后7天和14天外膜成纤维细胞及内膜细胞均显著增生；同系移植对照组术后外膜仅出现少量细胞增生。两组移植血管中膜平滑肌细胞未见增生细胞,中膜厚度也未见明显改变。
4.免疫组织化学结果显示在术后3天异系移植实验组外膜成纤维细胞开始出现NF-κB, MCP-1, ICAM-1,VCAM-1,MMP-7, TGF-β1,硝基酪氨酸,p47phox的表达,内膜有少量阳性细胞,在术后7天和14天外膜成纤维细胞及内膜细胞细胞因子表达均显著增加；同系移植对照组仅NF-κB, MCP-1、ICAM-1、VCAM-1、硝基酪氨酸在术后外膜出现少量阳性表达细胞,内膜未检测到阳性细胞。
5.荧光原位杂交技术显示在术后3天异系移植实验组外膜成纤维细胞检测到gp91phox mRNA表达,在术后7天和14天外膜成纤维细胞gp91phox mRNA表达显著增加,内膜仅有少量gp91phox mRNA表达；同系移植对照组未检测到gp91phoxmRNA表达。
6.QRT-PCR结果显示与同系移植对照组相比,异系移植实验组外膜成纤维细胞MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox和p47phoxmRNA均在术后3天开始表达,术后14天达到顶峰。
结论：
1.在移植性血管病早期新内膜形成前,动脉外膜首先被激活,主要表现为外膜成纤维细胞被激活,开始表达α-SMA,表型转化为肌成纤维细胞,增殖活性增强,合成释放多种活性细胞因子,促进移植性血管病新内膜形成及发展。
2.动脉外膜炎症是移植性血管病新内膜形成及发展过程中的早期事件之一,有可能促进血管新内膜的形成。
3.氧化应激贯穿于移植性血管病新内膜形成及发展整个过程中,可能是血管外膜成纤维细胞被激活的原因之一,并进而促进移植性血管病新内膜形成及发展。
第三部分：体外培养大鼠血管外膜成纤维细胞研究其在移植性血管病新内膜形成及发展中的作用
方法：
异系移植实验组和同系移植对照组在血管移植后14天分别取出移植胸主动脉,将血管外膜剥离,用组织块培养法体外培养血管外膜成纤维细胞,取3-6代细胞用于实验。用免疫细胞荧光染色检测vimentin,α-SMA及结蛋白desmin的表达；用细胞计数试剂盒(Cell Counting Kit-8, CCK-8)及5-溴-2-脱氧尿嘧啶(5'-bromo-2'-deoxyuridine, BrdU)掺入法检测细胞增殖活性的变化；用流式细胞仪PI染色法检测细胞周期；用插入式24孔Transwell小室及划痕实验检测细胞的迁移能力；用逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)检测MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gP91phox和p47phox等细胞因子mRNA的表达水平；用Western blot方法检测MCP-1,ICAM-1, TGF-β1,和p47phox等细胞因子蛋白的表达水平；设计合成靶向NADPH氧化酶亚基p47phox基因的小分子干扰RNA (small interfering RNA, siRNA),并进行筛选得到具有高效干扰效率的siRNA,转染异系移植实验组体外培养血管外膜成纤维细胞,观察其对血管外膜成纤维细胞NADPH氧化酶的抑制作用及其对外膜成纤维细胞增殖和迁移活性的抑制作用。
结果：
1.用组织块培养法培养的异系移植实验组和同系移植对照组血管外膜细胞,4～7天后可见细胞从组织块边缘游出,贴壁生长,10～14天后达到90%细胞融合。异系移植实验组血管外膜细胞呈现vimentin(+)、desmin(-)、约90%的细胞呈现α-SMA(+)；同系移植对照组血管外膜细胞呈现vimentin(+)、desmin(-)、90%以上的细胞呈现α-SMA(-)。
2.血清浓度为1%时,异系移植实验组和同系移植对照组培养血管外膜成纤维细胞细胞增殖活性无显著性差异,血清浓度为5%和10%时,异系移植实验组血管外膜成纤维细胞增殖活性显著高于同系移植对照组；血清浓度为10%时,BrdU掺入法也得到相同结果。流式细胞仪PI染色法显示异系移植实验组血管外膜成纤维细胞S期及G2／M期所占百分比显著高于同系移植对照组。
3.插入式24孔Transwell小室实验显示异系移植实验组迁移到Transwell小室PET膜下表面的成纤维细胞数明显高于同系移植对照组；划痕实验也显示异系移植实验组血管外膜成纤维细胞的愈合速率要明显高于同系移植对照组。
4.RT-PCR检测显示异系移植实验组血管外膜成纤维细胞MCP-1,IL-1β,TNFα,ICAM-1,VCAM-1,MMP-7,TGF-β1, gp91phox和p47phox等细胞因子mRNA的表达水平均明显高于同系移植对照组。
5.Western blot方法检测显示异系移植实验组血管外膜成纤维细胞MCP-1,ICAM-1,TGF-β1和p47phox等细胞因子蛋白的表达水平也明显高于同系移植对照组。
6. p47phox-siRNA-2#转染异系移植实验组体外培养血管外膜成纤维细胞48小时后可有效抑制p47phox基因在mRNA水平的表达,从而抑制NADPH氧化酶,并可以显著降低成纤维细胞的增殖和迁移活性。
结论：
1.约90%的异系移植实验组血管外膜细胞转化为肌成纤维细胞,而同系移植对照组90%以上的细胞为成纤维细胞。
2.异系移植实验组血管外膜成纤维细胞的增殖及迁移能力明显高于同系移植对照组。
3.异系移植实验组血管外膜成纤维细胞合成和释放多种活性细胞因子的能力明显高于同系移植对照组。
4.氧化应激参与血管外膜成纤维细胞的激活,激活的成纤维细胞又可以生成更多的超氧阴离子,形成正反馈效应。
5.NADPH氧化酶亚基p47phox基因siRNA可显著抑制成纤维细胞的增殖和迁移活性,从而可作为移植性血管病抑制新内膜形成的基因治疗新靶点。
Transplant vasculopathy (TV) is a vascular proliferative disease. TV occurs in organ transplants and is the most significant obstacle to successful long-term survival. TV has many causative factors and pathologic manifestations in common with atherosclerosis and post-angioplasty restenosis (RS).Conventional understanding of the pathologic manifestations of TV includes migration and proliferation of smooth muscle cells (SMCs), infiltration with inflammatory cells, lipid aggregation in macrophages and SMCs, increased production of the extracellular matrix (ECM), and neointima formation. Accordingly, studies on vascular diseases have focused on the endothelia and smooth muscle dysfunction; less attention has been paid to the vascular adventitia.
Arterial walls are heterogeneous three-layered structures composed of an intima, media, and adventitia. Each layer exhibits specific histological, biochemical, and functional characteristics, and, as such, each contributes in unique ways to maintaining vascular homeostasis and to regulating the vascular response to stress or injury. Compared with intima and media, the adventitia has been considered to be exclusively a supporting tissue that can provide nourishment to muscle layers. Recently, increasing attention has been paid to its role in a variety of vascular diseases, including AS and RS. Some studies have suggested that the vascular adventitia acts as an important regulator to modulate, directly or indirectly, the structure and function of the vascular wall. As the major cell type in the vascular adventitia, increasing studies showed that adventitial fibroblast was not a passive bystander but a active participant in response to diverse stimulations and thus contributed to neointima formation and vascular remodeling. Activation of the adventitial fibroblasts in response to cytokines, injury and stretch has been shown to stimulate phenotypic differentiation, migration, proliferation and synthesis of ECM. Well-known cytokines such as transforming growth factor beta (TGF-β1) and endothelin-1 (ET-1) can induce the transition of fibroblasts into myofibroblasts (MFs) which can traverse the ruptured media and participate in neointimal formation after balloon injury. Vascular adventitial fibroblasts can also secret a variety of cytokines, such as TGF-β1, monocyte chemoattrant protein-1 (MCP-1), interleukin (IL), ET-1, tumor necrosis factor alpha (TNFa), matrix metalloproteinases (MMPs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Adventitia reactions are common in various vascular diseases. Porcine models showed that the inflammatory response after angioplasty occurred throughout the entire perivascular tissue before neointimal formation. A previous study from our research team showed that adventitial fibroblasts were activated in the early stage of atherosclerosis, and that the earliest expression of MCP-1 which is capable of regulating the transmigration of monocytes into the vessel wall occurred in the adventitial fibroblasts before the formation of intimal lesions in apoE knockout mice. It was reported that abundant a-smooth muscle actin (a-SMA) immunoreactive cells were present in the adventitia in porcine after balloon injury. Li et al. tracked the rat carotid adventitial fibroblasts which transduced withβ-galactosidase and convincingly demonstrated directly in vivo the ability of adventitial fibroblasts to contribute to neointimal growth by migrating into the neointima over time after balloon injury. In carotid artery-vein grafts, neointima formation was proceded by proliferation, differentiation into myofibroblasts and migration into neointima of adventitial fibroblasts. Neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models. Recently, genge therapy has become a'hot spot'for preventing and treating cardiovascular diseases and the outer location of the adventitial fibroblast makes it suitable for drug delivery and gene therapy. Dourron et al. designed a novel replication-deficient adenovirus containing a fibroblast-active promoter that drives expression of the NADPH oxidase inhibitory sequence gp91ds (Ad-PDGFPR-gp91ds/eGFP). When this was delivered to the adventitia of the rat common carotid artery, it effectively reduced overall vascular 02·-and neointima formation. Though people carried out a lot of experiments and made some progress on the biological mechanism of vascular adventitia in vascular diseases, much mechanism especially in TV is still unclear. And there are few reports about it at present.
Objectives:
In the present study we constructed a successful allograft model in the rat via a cuff technique. In order to study the role of the activated vascular adventitia in the neointima formation and development in transplant vasculopathy, we observed the vascular adventitia especially the adventitial fibroblast phenotype differentiation, proliferation, migration and the mRNA and protein levels of various cytokines at the given time points after grafting both in vivo and in vitro. Furthermore, we investigated the inhibitive effect on the proliferation and migration of adventitial fibroblast by down-regulating the NADPH oxidase activity via RNA interference to silence the expression of p47phox, one subunit of NADPH oxidase. And the study will provide theory basis for gene therapy targeting at vascular adventitial fibroblast in preventing and treating the transplant vasculopathy. This study has been divided into three parts as following:
Part I:Construction of a transplant vasculopathy animal model in the rat
Method:
The procedure was carried out using an improved cuff technique. Thoracic aortas from SD rats transplanted into the abdominal aortas of Wistar rats worked as allografts, and isografts (SD to SD) were control. Grafts were removed on days 3,7, and 14 for HE staining and Weigert staining. The thicknesses of the neointima and media and intimal/medial thickness ratio were measured using a computer-assisted morphometry system (Image-Pro Plus 5.0).
Result:
After transplantation, the animal survival rate was about 90%and the patency rate was 100%. No detectable neointimal hyperplasia was found in 3-,7-and 14-day isografts as well as 3-day allografts. The neointima was initially found in 7-day allografts and significantly increased in 14-day allografts. The intimal thickness and intimal/medial thickness ratio were increased in 7-day allografts and peaked in 14-day allografts. Markedly reduced medial thickness in the process was not observed.
Conclusion:
We constructed a successful transplant vasculopathy animal model in the rat. It was characterized by simple operation, less cost and favorable repeatability. Thus, it can be used to study allograft pathologic mechanism.
PartⅡ:The study on the role of the vascular adventitial activation in the neointima formation and development in transplant vasculopathy in vivo
Method:
At given time points, allografts and isografts were removed. Paraffin-embedded sections were stained with immunohistochemistry to observe the changes in the expression of vimentin,α-SMA, Ki-67, NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox in the vascular adventitia and intima/neointima. Fluorescence in situ hybridization (FISH) was performed to examine the expression of the mRNA for gp91phox. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the grafts adventitia were quantified by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR) using SYBR Green technology.
Result:
1. Vimentin-positive cells were mainly in the adventitia and intima or neointima in both isografts and allografts. a-SMA was expressed only in the media and was barely detectable in the adventitia or intima in isografts. In allografts, expression of a-SMA in the media was similar to that in isografts, but a-SMA-positive cells were first found mainly in adventitial fibroblasts after 3 days and gradually increased till 14 days. The neointima began to express a-SMA in 7-and 14-day allografts.
2. In our experiment, no more than 4%of CD3-positive T cells were found in the adventitia in both isografts and allografts in any given time points after grafting, indicating that most of the proliferative cells were adventitial fibroblasts which might contribute to the neointima formation.
3. Immunostaing for Ki-67 showed that in allografts, about 20%of adventitial fibroblasts and only a few intimal endothelial cells were proliferative after 3 days, and the number of proliferative cells was significantly increased in the adventitia and neointima after 7 and 14 days. Proliferative cells were barely detectable in all three layers in isografts. Surprisingly, there were neither obvious proliferative cells nor evident changes in the thickness in the media during the TV development.
4. In the allografts, the expression of NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox were first found in the adventitia and intima/neointima 3 days after grafting, and significantly increased in the adventitia and neointima after 7 and 14 days. In the isografts, only a few cells positive for NF-κB, MCP-1, ICAM-1, VCAM-1 and nitrotyrosine were found in the adventitia and positive cells were barely detectable in the intima or neointima.
5.In the allografts, the expression of gp91phox mRNA were first found in the adventitia 3 days after grafting, and significantly increased after 7 and 14 days. Only a few positive cells were observed in the intima. However, the expression of gp91phox mRNA were barely detectable in the isografts.
6. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox in the adventitia were increased 3 days after grafting, and peaked at 14 days.
Conclusion:
1. In the early stage of TV, vascular adventitia especially the adventitial fibroblast was activated before the neointima formation. This process was characterized by the phenotypic differentiation into myofibroblast withα-SMA expression, the increased capacity of proliferation and synthesis of various active cytokines. Thus, we hypothesized that adventitial fibroblast might contribute to the neointima formation after its activation in the development of TV.
2. Vascular adventitial inflammation is an early event in the neointima formation and development in TV.
3. Oxidative stress occured through the development of TV might be one of the causes of the activation of vascular adventitia especially the adventitial fibroblast and be involved in the neointima formation and development in TV.
PartⅢ:The study on the role of the adventitial fibroblasts in the neointima formation and development in transplant vasculopathy in vitro
Method:
Allografts and isografts were removed 14 days after grafting. Adventitia was carefully stripped from isografts and allografts, and then cultured by the tissue explant method, respectively. Cells at passages 3 to 6 were used for further study. Immunofluorescence staining for vimentin, a-SMA and desmin was used to identify the cultured adventitial cells.The proliferation of cells was examined by CCK-8 kit and BrdU incorporation assay. PI staining by flow cytometry was used to examine the cell cycle of cultured cells. Cell migration assay was carried out using 24 well ThinCertTM cell culture inserts and scratch method. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the cultured adventitial cells were quantified by reverse transcriptase polymerase chain reaction (RT-PCR) and the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox were quantified by Western blot. We designed and synthesized siRNAs targeting p47phox, one subunit of NADPH oxidase, and then screened one siRNA with high efficiency. p47phox siRNA was transfected into cultured adventitial fibroblasts from allografts to observe its inhibition effect on NADPH oxidase activity and cell proliferation and migration capacity.
Result:
1. Adventitia was cultured by the tissue explant method. About 4-7 days, small amounts of cells started to immigrate out of the explants and adhere to the flask bottom and for 10～14 days they reached 90%confluence. More than 90%cells from isografts are vimentin (+)，desmin (-) andα-SMA (-), while about 90%cells from allografts are vimentin (+), desmin (-) andα-SMA(+).
2. Compared with isografts, there is no significant difference between the two groups in the proliferation capacity of the cultured adventitial cells when cultured with 1%serum, but the proliferation capacity of the cultured adventitial cells from allografts were significantly increased when cultured with 5%or 10%serum. The same result was gained by the method of BrdU incorporation assay when cultured with 10%serum. PI staining by flow cytometry also showed the percentage of S and G2/M phase of the cultured adventitial cells from allografts was much higher than that from isografts.
3. Both 24 well ThinCertTM cell culture inserts and scratch method demonstrated that the migration capacity of the cultured adventitial cells from allografts were significantly increased compared with that from isografts.
4. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1,gp91phox and p47phox in the cultured adventitial cells from allografts were significantly increased.
5. Compared with isografts, the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox in the cultured adventitial cells from allografts were significantly increased.
6. After 48 hours of the transfection of p47phox-siRNA-2#, the mRNA level of p47phox was effectively inhibited. Furthermore, the NADPH oxidase activity and the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly decreased.
Conclusion:
1. About 90%cells from allografts differentiated into myofibroblasts while more than 90%cells from isografts were fibroblasts.
2. Compared with isografts, the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly increased.
3. Compared with isografts, the synthesis and release of various active cytokines by the cultured adventitial cells from allografts were significantly increased.
4. Oxidative stress was involved in the activation of adventitial fibroblasts, while activated adventitial fibroblasts might produce more superoxide anion and thus a positive feedback might be formed between them.
5. p47phox siRNA effectively inhibited the proliferation and migration capacity of adventitial fibroblasts, and thus might be a new target for the gene therapy to attenuate the neointima formation in TV.
动脉血管壁分为三层：内膜、中膜和外膜。每一层都有特殊的组织学、生物化学和功能特点。因此,每一层都以特定的方式来维持血管稳态,调节血管对于应激和损伤的反应。与内膜和中膜相比,外膜一直被认为仅仅是一层支撑组织,为平滑肌层提供营养。近年来,血管外膜在血管疾病如AS和RS中的作用日益受到重视。有资料提示血管外膜对于血管的结构和功能调节起着直接或间接的重要调控作用。成纤维细胞是血管外膜最主要的细胞类型,越来越多的实验研究证明外膜成纤维细胞在新内膜形成和血管重塑过程中并非是被动的旁观者,而是对多种刺激具有反应的积极的参与者。当受到细胞因子、损伤和牵拉等刺激时,外膜成纤维细胞可以被激活导致细胞表型转化、迁移、增殖以及合成细胞外基质。些重要的细胞因子如转化生长因子-β1(transforming growth factor beta 1,TGF-β1)和内皮素-1(endothelin-1, ET-1)都能诱导成纤维细胞转化为肌成纤维细胞(myofibroblast,MF),从而可以穿过破损的中膜参与新内膜的形成。血管外膜成纤维细胞也能够分泌多种细胞因子如TGF-β1、单核细胞趋化蛋白-1 (monocyte chemoattrant protein-1, MCP-1)、白细胞介素(interleukin, IL)、ET-1、肿瘤坏死因子α(tumor necrosis factor alpha, TNFα)、基质金属蛋白酶(matrix metalloproteinases, MMPs)及尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase)等。外膜反应普遍存在于各种血管疾病中。在猪的血管成形术模型中,血管周围组织炎症反应要先于新内膜的形成。我们实验室的前期研究工作也发现在载脂蛋白E基因敲除(apolipoprotein E knockout,apoE(-/-))小鼠AS模型中,外膜成纤维细胞在AS早期被激活,并且早于内膜病灶的形成,能够诱导单核细胞游走进入血管壁的MCP-1最早表达于外膜成纤维细胞。有研究表明在猪球囊损伤模型中血管外膜有大量的α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫反应阳性细胞。Li等在兔球囊损伤模型中将β-半乳糖苷酶基因转入颈动脉外膜成纤维细胞用以标记细胞,成功地在体直接证明外膜成纤维细胞能迁移到新内膜并参与新内膜的形成。在颈动脉动-静脉移植中,外膜成纤维细胞增殖,表型转化为肌成纤维细胞并向内膜迁移均早于新内膜的形成。大量研究表明仅仅是外膜损伤也能引起新内膜和／或动脉粥样硬化病灶的形成。近年来,基因疗法成为当前治疗心血管疾病的一个研究“热点”,而动脉外膜因其在血管外围更有利于药物输送和基因治疗。Dourron等设计了一种新型的含有成纤维细胞活化启动子并能表达NADPH氧化酶抑制序列gp91ds (Ad-PDGFβR-gp91ds/eGFP)的复制缺陷性腺病毒,该腺病毒转入大鼠颈总动脉外膜后有效抑制了整个血管O2-产生和新内膜形成。
尽管人们在血管疾病中对血管外膜作用机制研究取得了一些进展,但血管外膜的许多生物学机制及其与血管疾病发生的关系仍不清楚,尤其是在移植性血管病中的作用并不清楚,目前国内外的报道也非常少。
目的：
本实验利用袖套管技术将大鼠胸主动脉移植到腹主动脉建立移植性血管病模型,通过在体动物实验以及体外培养移植血管外膜成纤维细胞,观察血管外膜细胞表型转化、增殖、迁移以及多种细胞因子在mRNA和蛋白水平表达的动态变化,进一步探讨血管外膜以及外膜成纤维细胞激活在移植性血管病新内膜形成及发展中的作用机制。在以上实验基础上,本研究进一步探讨应用RNA干扰技术特异性地抑制NADPH氧化酶亚基p47phox的表达,从而下调NADPH氧化酶的活性,观察其对移植血管外膜成纤维细胞增殖和迁移的抑制作用,为移植性血管病以血管外膜成纤维细胞为作用靶点进行基因治疗提供实验依据。
本课题分以下三个部分进行研究：
第一部分：移植性血管病大鼠模型的建立
方法：
以Sprague-Dawley (SD)大鼠的胸主动脉为供体血管,利用袖套管技术移植到Wistar大鼠腹主动脉作为异系移植实验组,以SD大鼠对SD大鼠同系移植作为对照组。在血管移植后3,7,14天,取出移植的胸主动脉,进行苏木素-伊红(HE)染色及Weigert弹力纤维染色,应用Image-Pro Plus 5.0计算机图像分析系统检测移植血管的内膜和中膜增生情况及内膜／中膜厚度比值。
结果：
手术成功率为90%,移植血管的通畅率达100%。同系移植对照组在各个时间点未出现内膜的明显增生,而异系移植实验组在移植后7天出现内膜增生,术后14天内膜厚度显著增加。对照组和实验组移植血管中膜均未见明显增厚。异系移植实验组内膜／中膜厚度比值在移植后7天开始增高,14天时达到顶峰。
结论：
利用袖套管技术进行SD和Wistar大鼠间的血管移植符合移植性血管病的病理形态改变,并且操作简单,费用小,重复性好,可以用于研究移植性血管病的病理机制及干预治疗。
第二部分：在体动物实验研究血管外膜激活在移植性血管病新内膜形成及发展中的作用
方法：
在血管移植后3,7,14天,取出移植的胸主动脉。用免疫组织化学方法观察不同时间点血管外膜和内膜波形蛋白(vimentin),α平滑肌肌动蛋白(α-SMA),T细胞抗原受体CD3,细胞核增殖抗原Ki-67,核因子-κB (nuclear factorκB,NF-κB), MCP-1,细胞间粘附分子-1(intercellular adhesion molecule-1, ICAM-1),血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1),基质金属蛋白酶-7 (MMP-7), TGF-β1,硝基酪氨酸及p47phox等细胞因子蛋白水平表达的动态变化；用荧光原位杂交技术检测gp91phox mRNA的表达水平；用荧光实时定量聚合酶链反应(quantitative real-time reverse transcriptase polymerase chain reaction,QRT-PCR)检测移植血管外膜MCP-1,IL-1β,TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox和p47phox等细胞因子mRNA的表达水平。
结果：
1.免疫组织化学检测显示异系移植实验组和同系移植对照组在血管移植后不同时间点,移植血管外膜及内膜或新内膜的大部分细胞呈现vimentin阳性,中膜几乎无阳性细胞。异系移植实验组在术后3天移植血管外膜部分细胞即呈现α-SMA阳性,随着时间延长α-SMA阳性细胞数量逐渐增加,术后7天和14天新内膜细胞也开始表达α-SMA,而同系移植对照组血管外膜及内膜细胞几乎无α-SMA阳性表达；两组移植血管中膜α-SMA始终呈强阳性表达。
2.异系移植实验组和同系移植对照组在血管移植后不同时间点移植血管外膜及内膜或新内膜T细胞抗原受体CD3阳性细胞不超过4%,说明增生的细胞主要是外膜成纤维细胞,新内膜形成可能主要与外膜成纤维细胞有关。
3.Ki-67免疫组织化学结果显示在术后3天异系移植实验组外膜成纤维细胞开始出现增殖,内膜细胞仅有少量细胞增殖,在术后7天和14天外膜成纤维细胞及内膜细胞均显著增生；同系移植对照组术后外膜仅出现少量细胞增生。两组移植血管中膜平滑肌细胞未见增生细胞,中膜厚度也未见明显改变。
4.免疫组织化学结果显示在术后3天异系移植实验组外膜成纤维细胞开始出现NF-κB, MCP-1, ICAM-1,VCAM-1,MMP-7, TGF-β1,硝基酪氨酸,p47phox的表达,内膜有少量阳性细胞,在术后7天和14天外膜成纤维细胞及内膜细胞细胞因子表达均显著增加；同系移植对照组仅NF-κB, MCP-1、ICAM-1、VCAM-1、硝基酪氨酸在术后外膜出现少量阳性表达细胞,内膜未检测到阳性细胞。
5.荧光原位杂交技术显示在术后3天异系移植实验组外膜成纤维细胞检测到gp91phox mRNA表达,在术后7天和14天外膜成纤维细胞gp91phox mRNA表达显著增加,内膜仅有少量gp91phox mRNA表达；同系移植对照组未检测到gp91phoxmRNA表达。
6.QRT-PCR结果显示与同系移植对照组相比,异系移植实验组外膜成纤维细胞MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox和p47phoxmRNA均在术后3天开始表达,术后14天达到顶峰。
结论：
1.在移植性血管病早期新内膜形成前,动脉外膜首先被激活,主要表现为外膜成纤维细胞被激活,开始表达α-SMA,表型转化为肌成纤维细胞,增殖活性增强,合成释放多种活性细胞因子,促进移植性血管病新内膜形成及发展。
2.动脉外膜炎症是移植性血管病新内膜形成及发展过程中的早期事件之一,有可能促进血管新内膜的形成。
3.氧化应激贯穿于移植性血管病新内膜形成及发展整个过程中,可能是血管外膜成纤维细胞被激活的原因之一,并进而促进移植性血管病新内膜形成及发展。
第三部分：体外培养大鼠血管外膜成纤维细胞研究其在移植性血管病新内膜形成及发展中的作用
方法：
异系移植实验组和同系移植对照组在血管移植后14天分别取出移植胸主动脉,将血管外膜剥离,用组织块培养法体外培养血管外膜成纤维细胞,取3-6代细胞用于实验。用免疫细胞荧光染色检测vimentin,α-SMA及结蛋白desmin的表达；用细胞计数试剂盒(Cell Counting Kit-8, CCK-8)及5-溴-2-脱氧尿嘧啶(5'-bromo-2'-deoxyuridine, BrdU)掺入法检测细胞增殖活性的变化；用流式细胞仪PI染色法检测细胞周期；用插入式24孔Transwell小室及划痕实验检测细胞的迁移能力；用逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)检测MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gP91phox和p47phox等细胞因子mRNA的表达水平；用Western blot方法检测MCP-1,ICAM-1, TGF-β1,和p47phox等细胞因子蛋白的表达水平；设计合成靶向NADPH氧化酶亚基p47phox基因的小分子干扰RNA (small interfering RNA, siRNA),并进行筛选得到具有高效干扰效率的siRNA,转染异系移植实验组体外培养血管外膜成纤维细胞,观察其对血管外膜成纤维细胞NADPH氧化酶的抑制作用及其对外膜成纤维细胞增殖和迁移活性的抑制作用。
结果：
1.用组织块培养法培养的异系移植实验组和同系移植对照组血管外膜细胞,4～7天后可见细胞从组织块边缘游出,贴壁生长,10～14天后达到90%细胞融合。异系移植实验组血管外膜细胞呈现vimentin(+)、desmin(-)、约90%的细胞呈现α-SMA(+)；同系移植对照组血管外膜细胞呈现vimentin(+)、desmin(-)、90%以上的细胞呈现α-SMA(-)。
2.血清浓度为1%时,异系移植实验组和同系移植对照组培养血管外膜成纤维细胞细胞增殖活性无显著性差异,血清浓度为5%和10%时,异系移植实验组血管外膜成纤维细胞增殖活性显著高于同系移植对照组；血清浓度为10%时,BrdU掺入法也得到相同结果。流式细胞仪PI染色法显示异系移植实验组血管外膜成纤维细胞S期及G2／M期所占百分比显著高于同系移植对照组。
3.插入式24孔Transwell小室实验显示异系移植实验组迁移到Transwell小室PET膜下表面的成纤维细胞数明显高于同系移植对照组；划痕实验也显示异系移植实验组血管外膜成纤维细胞的愈合速率要明显高于同系移植对照组。
4.RT-PCR检测显示异系移植实验组血管外膜成纤维细胞MCP-1,IL-1β,TNFα,ICAM-1,VCAM-1,MMP-7,TGF-β1, gp91phox和p47phox等细胞因子mRNA的表达水平均明显高于同系移植对照组。
5.Western blot方法检测显示异系移植实验组血管外膜成纤维细胞MCP-1,ICAM-1,TGF-β1和p47phox等细胞因子蛋白的表达水平也明显高于同系移植对照组。
6. p47phox-siRNA-2#转染异系移植实验组体外培养血管外膜成纤维细胞48小时后可有效抑制p47phox基因在mRNA水平的表达,从而抑制NADPH氧化酶,并可以显著降低成纤维细胞的增殖和迁移活性。
结论：
1.约90%的异系移植实验组血管外膜细胞转化为肌成纤维细胞,而同系移植对照组90%以上的细胞为成纤维细胞。
2.异系移植实验组血管外膜成纤维细胞的增殖及迁移能力明显高于同系移植对照组。
3.异系移植实验组血管外膜成纤维细胞合成和释放多种活性细胞因子的能力明显高于同系移植对照组。
4.氧化应激参与血管外膜成纤维细胞的激活,激活的成纤维细胞又可以生成更多的超氧阴离子,形成正反馈效应。
5.NADPH氧化酶亚基p47phox基因siRNA可显著抑制成纤维细胞的增殖和迁移活性,从而可作为移植性血管病抑制新内膜形成的基因治疗新靶点。
Transplant vasculopathy (TV) is a vascular proliferative disease. TV occurs in organ transplants and is the most significant obstacle to successful long-term survival. TV has many causative factors and pathologic manifestations in common with atherosclerosis and post-angioplasty restenosis (RS).Conventional understanding of the pathologic manifestations of TV includes migration and proliferation of smooth muscle cells (SMCs), infiltration with inflammatory cells, lipid aggregation in macrophages and SMCs, increased production of the extracellular matrix (ECM), and neointima formation. Accordingly, studies on vascular diseases have focused on the endothelia and smooth muscle dysfunction; less attention has been paid to the vascular adventitia.
Arterial walls are heterogeneous three-layered structures composed of an intima, media, and adventitia. Each layer exhibits specific histological, biochemical, and functional characteristics, and, as such, each contributes in unique ways to maintaining vascular homeostasis and to regulating the vascular response to stress or injury. Compared with intima and media, the adventitia has been considered to be exclusively a supporting tissue that can provide nourishment to muscle layers. Recently, increasing attention has been paid to its role in a variety of vascular diseases, including AS and RS. Some studies have suggested that the vascular adventitia acts as an important regulator to modulate, directly or indirectly, the structure and function of the vascular wall. As the major cell type in the vascular adventitia, increasing studies showed that adventitial fibroblast was not a passive bystander but a active participant in response to diverse stimulations and thus contributed to neointima formation and vascular remodeling. Activation of the adventitial fibroblasts in response to cytokines, injury and stretch has been shown to stimulate phenotypic differentiation, migration, proliferation and synthesis of ECM. Well-known cytokines such as transforming growth factor beta (TGF-β1) and endothelin-1 (ET-1) can induce the transition of fibroblasts into myofibroblasts (MFs) which can traverse the ruptured media and participate in neointimal formation after balloon injury. Vascular adventitial fibroblasts can also secret a variety of cytokines, such as TGF-β1, monocyte chemoattrant protein-1 (MCP-1), interleukin (IL), ET-1, tumor necrosis factor alpha (TNFa), matrix metalloproteinases (MMPs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Adventitia reactions are common in various vascular diseases. Porcine models showed that the inflammatory response after angioplasty occurred throughout the entire perivascular tissue before neointimal formation. A previous study from our research team showed that adventitial fibroblasts were activated in the early stage of atherosclerosis, and that the earliest expression of MCP-1 which is capable of regulating the transmigration of monocytes into the vessel wall occurred in the adventitial fibroblasts before the formation of intimal lesions in apoE knockout mice. It was reported that abundant a-smooth muscle actin (a-SMA) immunoreactive cells were present in the adventitia in porcine after balloon injury. Li et al. tracked the rat carotid adventitial fibroblasts which transduced withβ-galactosidase and convincingly demonstrated directly in vivo the ability of adventitial fibroblasts to contribute to neointimal growth by migrating into the neointima over time after balloon injury. In carotid artery-vein grafts, neointima formation was proceded by proliferation, differentiation into myofibroblasts and migration into neointima of adventitial fibroblasts. Neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models. Recently, genge therapy has become a'hot spot'for preventing and treating cardiovascular diseases and the outer location of the adventitial fibroblast makes it suitable for drug delivery and gene therapy. Dourron et al. designed a novel replication-deficient adenovirus containing a fibroblast-active promoter that drives expression of the NADPH oxidase inhibitory sequence gp91ds (Ad-PDGFPR-gp91ds/eGFP). When this was delivered to the adventitia of the rat common carotid artery, it effectively reduced overall vascular 02·-and neointima formation. Though people carried out a lot of experiments and made some progress on the biological mechanism of vascular adventitia in vascular diseases, much mechanism especially in TV is still unclear. And there are few reports about it at present.
Objectives:
In the present study we constructed a successful allograft model in the rat via a cuff technique. In order to study the role of the activated vascular adventitia in the neointima formation and development in transplant vasculopathy, we observed the vascular adventitia especially the adventitial fibroblast phenotype differentiation, proliferation, migration and the mRNA and protein levels of various cytokines at the given time points after grafting both in vivo and in vitro. Furthermore, we investigated the inhibitive effect on the proliferation and migration of adventitial fibroblast by down-regulating the NADPH oxidase activity via RNA interference to silence the expression of p47phox, one subunit of NADPH oxidase. And the study will provide theory basis for gene therapy targeting at vascular adventitial fibroblast in preventing and treating the transplant vasculopathy. This study has been divided into three parts as following:
Part I:Construction of a transplant vasculopathy animal model in the rat
Method:
The procedure was carried out using an improved cuff technique. Thoracic aortas from SD rats transplanted into the abdominal aortas of Wistar rats worked as allografts, and isografts (SD to SD) were control. Grafts were removed on days 3,7, and 14 for HE staining and Weigert staining. The thicknesses of the neointima and media and intimal/medial thickness ratio were measured using a computer-assisted morphometry system (Image-Pro Plus 5.0).
Result:
After transplantation, the animal survival rate was about 90%and the patency rate was 100%. No detectable neointimal hyperplasia was found in 3-,7-and 14-day isografts as well as 3-day allografts. The neointima was initially found in 7-day allografts and significantly increased in 14-day allografts. The intimal thickness and intimal/medial thickness ratio were increased in 7-day allografts and peaked in 14-day allografts. Markedly reduced medial thickness in the process was not observed.
Conclusion:
We constructed a successful transplant vasculopathy animal model in the rat. It was characterized by simple operation, less cost and favorable repeatability. Thus, it can be used to study allograft pathologic mechanism.
PartⅡ:The study on the role of the vascular adventitial activation in the neointima formation and development in transplant vasculopathy in vivo
Method:
At given time points, allografts and isografts were removed. Paraffin-embedded sections were stained with immunohistochemistry to observe the changes in the expression of vimentin,α-SMA, Ki-67, NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox in the vascular adventitia and intima/neointima. Fluorescence in situ hybridization (FISH) was performed to examine the expression of the mRNA for gp91phox. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the grafts adventitia were quantified by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR) using SYBR Green technology.
Result:
1. Vimentin-positive cells were mainly in the adventitia and intima or neointima in both isografts and allografts. a-SMA was expressed only in the media and was barely detectable in the adventitia or intima in isografts. In allografts, expression of a-SMA in the media was similar to that in isografts, but a-SMA-positive cells were first found mainly in adventitial fibroblasts after 3 days and gradually increased till 14 days. The neointima began to express a-SMA in 7-and 14-day allografts.
2. In our experiment, no more than 4%of CD3-positive T cells were found in the adventitia in both isografts and allografts in any given time points after grafting, indicating that most of the proliferative cells were adventitial fibroblasts which might contribute to the neointima formation.
3. Immunostaing for Ki-67 showed that in allografts, about 20%of adventitial fibroblasts and only a few intimal endothelial cells were proliferative after 3 days, and the number of proliferative cells was significantly increased in the adventitia and neointima after 7 and 14 days. Proliferative cells were barely detectable in all three layers in isografts. Surprisingly, there were neither obvious proliferative cells nor evident changes in the thickness in the media during the TV development.
4. In the allografts, the expression of NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox were first found in the adventitia and intima/neointima 3 days after grafting, and significantly increased in the adventitia and neointima after 7 and 14 days. In the isografts, only a few cells positive for NF-κB, MCP-1, ICAM-1, VCAM-1 and nitrotyrosine were found in the adventitia and positive cells were barely detectable in the intima or neointima.
5.In the allografts, the expression of gp91phox mRNA were first found in the adventitia 3 days after grafting, and significantly increased after 7 and 14 days. Only a few positive cells were observed in the intima. However, the expression of gp91phox mRNA were barely detectable in the isografts.
6. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox in the adventitia were increased 3 days after grafting, and peaked at 14 days.
Conclusion:
1. In the early stage of TV, vascular adventitia especially the adventitial fibroblast was activated before the neointima formation. This process was characterized by the phenotypic differentiation into myofibroblast withα-SMA expression, the increased capacity of proliferation and synthesis of various active cytokines. Thus, we hypothesized that adventitial fibroblast might contribute to the neointima formation after its activation in the development of TV.
2. Vascular adventitial inflammation is an early event in the neointima formation and development in TV.
3. Oxidative stress occured through the development of TV might be one of the causes of the activation of vascular adventitia especially the adventitial fibroblast and be involved in the neointima formation and development in TV.
PartⅢ:The study on the role of the adventitial fibroblasts in the neointima formation and development in transplant vasculopathy in vitro
Method:
Allografts and isografts were removed 14 days after grafting. Adventitia was carefully stripped from isografts and allografts, and then cultured by the tissue explant method, respectively. Cells at passages 3 to 6 were used for further study. Immunofluorescence staining for vimentin, a-SMA and desmin was used to identify the cultured adventitial cells.The proliferation of cells was examined by CCK-8 kit and BrdU incorporation assay. PI staining by flow cytometry was used to examine the cell cycle of cultured cells. Cell migration assay was carried out using 24 well ThinCertTM cell culture inserts and scratch method. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the cultured adventitial cells were quantified by reverse transcriptase polymerase chain reaction (RT-PCR) and the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox were quantified by Western blot. We designed and synthesized siRNAs targeting p47phox, one subunit of NADPH oxidase, and then screened one siRNA with high efficiency. p47phox siRNA was transfected into cultured adventitial fibroblasts from allografts to observe its inhibition effect on NADPH oxidase activity and cell proliferation and migration capacity.
Result:
1. Adventitia was cultured by the tissue explant method. About 4-7 days, small amounts of cells started to immigrate out of the explants and adhere to the flask bottom and for 10～14 days they reached 90%confluence. More than 90%cells from isografts are vimentin (+)，desmin (-) andα-SMA (-), while about 90%cells from allografts are vimentin (+), desmin (-) andα-SMA(+).
2. Compared with isografts, there is no significant difference between the two groups in the proliferation capacity of the cultured adventitial cells when cultured with 1%serum, but the proliferation capacity of the cultured adventitial cells from allografts were significantly increased when cultured with 5%or 10%serum. The same result was gained by the method of BrdU incorporation assay when cultured with 10%serum. PI staining by flow cytometry also showed the percentage of S and G2/M phase of the cultured adventitial cells from allografts was much higher than that from isografts.
3. Both 24 well ThinCertTM cell culture inserts and scratch method demonstrated that the migration capacity of the cultured adventitial cells from allografts were significantly increased compared with that from isografts.
4. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1,gp91phox and p47phox in the cultured adventitial cells from allografts were significantly increased.
5. Compared with isografts, the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox in the cultured adventitial cells from allografts were significantly increased.
6. After 48 hours of the transfection of p47phox-siRNA-2#, the mRNA level of p47phox was effectively inhibited. Furthermore, the NADPH oxidase activity and the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly decreased.
Conclusion:
1. About 90%cells from allografts differentiated into myofibroblasts while more than 90%cells from isografts were fibroblasts.
2. Compared with isografts, the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly increased.
3. Compared with isografts, the synthesis and release of various active cytokines by the cultured adventitial cells from allografts were significantly increased.
4. Oxidative stress was involved in the activation of adventitial fibroblasts, while activated adventitial fibroblasts might produce more superoxide anion and thus a positive feedback might be formed between them.
5. p47phox siRNA effectively inhibited the proliferation and migration capacity of adventitial fibroblasts, and thus might be a new target for the gene therapy to attenuate the neointima formation in TV.
引文
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