Role of Smooth Muscle Cells in Graft Intimal Hyperplasia

描述(由申请人提供)： 人工血管广泛应用于血管重建手术，但其长期通畅性有限，部分原因是吻合口内膜增生，其特征是平滑肌细胞(SMC)堆积和细胞外基质沉积，尤其是I型胶原。我们已经证明：1)人工血管材料诱导单核细胞氧化低密度脂蛋白2)高胆固醇血症增加胆固醇在移植物中的积聚，3)与邻近的主动脉相比，脂质氧化产物在移植物中积累，4)氧化的低密度脂蛋白(OxLDL)刺激移植物SMC产生血小板衍生生长因子(PDGF)和胶原，但对主动脉SMC的影响较小。在初步研究中，我们已经表明，活性氧物种(ROS)刺激SMC胶原合成非常迅速地增加，而不增加mRNA。此外，ROS还可增加细胞内游离钙浓度([Ca~(2+)]i)，并激活蛋白激酶C-增量(PKC Delta)。PKC8可以磷酸化翻译起始复合体中的蛋白质。基于我们的初步研究，我们提出了一个总体假设，即合成血管移植物中形成的脂质氧化产物通过几种机制刺激SMC产生胶原，包括升高[Ca+]i和由于PKC Delta介导的起始因子的磷酸化而增加翻译。胶原蛋白的升高促进了内膜增生的发展。我们将通过研究ROS诱导胶原生成和移植物SMC敏感性增加的机制来验证我们的假设。我们将评估主动脉和移植物SMC中内源性抗氧化剂的水平。我们将探讨[Ca~(2+)]i升高在胶原生成增加中的作用，以及ROS介导的[Ca~(2+)]i升高的机制，特别是TRPC6钙通道的作用。我们将研究ROS激活的PKC Delta在胶原合成增加中的作用。最后，我们将评估胶原合成的翻译调控机制，特别是起始因子的磷酸化作用，以及影响前胶原mRNA转录特异性调控的细胞因素的存在。这项拟议的研究将确定oxLDL和ROS在人造血管移植物SMC与天然主动脉SMC相比增加胶原产生中的作用。研究还将讨论抗氧化剂控制胶原蛋白产生的效果。这将有助于更好地了解脂质在移植物愈合中的作用，并最终找到限制人工移植物胶原合成和内膜增生的方法，从而延长小直径血管移植物的通畅期。

DESCRIPTION (provided by applicant): Prosthetic grafts are used widely in vascular reconstructive surgery, but their long-term patency is limited, in part due to the development of anastomotic intimal hyperplasia characterized by accumulation of smooth muscle cells (SMC) and deposition of extracellular matrix, particularly collagen type I. We have shown that: 1) prosthetic graft material induces monocytic cells to oxidize LDL 2) hypercholesterolemia in rabbits increases cholesterol accumulation in graft compared to native aorta, 3) lipid oxidation products accumulate in grafts compared to adjacent aorta, and 4) oxidized LDL (oxLDL) stimulates platelet-derived growth factor (PDGF) and collagen production by graft SMC but has less effect on aortic SMC. In preliminary studies we have shown that reactive oxygen species (ROS) stimulate a very rapid increase in SMC collagen synthesis without an increase in mRNA. Also ROS increase intracellular concentration of free calcium ([Ca2+]i) and activate protein kinase C-delta (PKCdelta). PKC8 can phosphorylate proteins in the translation initiation complex. Based on our preliminary studies, we propose as an overall hypothesis that lipid oxidation products formed within synthetic vascular grafts create an oxidative stress that stimulates collagen production by SMC though several mechanisms including, a rise in [Ca2+]i and increased translation due to PKCdelta-mediated phosphorylation of initiation factors. Elevated collagen contributes to the development of intimal hyperplasia. We will test our hypothesis by investigating the mechanisms of ROS-induced collagen production and the elevated sensitivity of graft SMC. We will assess levels of endogenous antioxidants in aortic and graft SMC. We will explore the role of elevated [Ca2+]i in increased collagen production, and the mechanism of ROS mediated increase in [Ca2+]i, particularly the role of TRPC6 calcium channels. We will investigate the role of ROS-activated PKCdelta in the increased collagen synthesis. Finally, we will assess the mechanism of translational regulation of collagen production, specifically the role of phosphorylation of initiation factors, and the existence of cellular factors that influence transcript-specific regulation of procollagen mRNA translation. The proposed studies will determine the role of oxLDL and ROS in the increased collagen production by SMC from synthetic vascular grafts compared to SMC from native aorta. Studies will also address the efficacy of antioxidants to control the collagen production. This will lead to a better understanding of the role of lipids in graft healing, and ultimately, to methods limiting collagen synthesis and the development of intimal hyperplasia of prosthetic grafts, thereby prolonging patency of small-diameter vascular grafts.