Role of Smooth Muscle Cells in Graft Intimal Hyperplasia

描述（由申请人提供）： 人工血管移植物广泛用于血管重建手术，但其长期通畅性有限，部分原因是吻合口内膜增生的发展，其特征在于平滑肌细胞（SMC）的积聚和细胞外基质（特别是I型胶原）的沉积。我们已经证明：1）人工移植物材料诱导单核细胞氧化LDL 2）与天然主动脉相比，兔中的高胆固醇血症增加了移植物中的胆固醇积累，3）与邻近主动脉相比，脂质氧化产物在移植物中积累，以及4）氧化LDL（oxLDL）刺激移植物SMC产生血小板衍生生长因子（PDGF）和胶原蛋白，但对主动脉SMC的影响较小。在初步的研究中，我们已经表明，活性氧（ROS）刺激SMC胶原蛋白合成非常迅速的增加，而不增加mRNA。ROS还增加细胞内游离钙（[Ca 2 +]i）浓度并激活蛋白激酶C-δ（PKC δ）。PKC 8可以磷酸化翻译起始复合物中的蛋白质。基于我们的初步研究，我们提出了一个总体假设，即合成血管移植物内形成的脂质氧化产物产生氧化应激，通过多种机制刺激SMC产生胶原蛋白，包括[Ca 2 +]i升高和由于PKC δ介导的起始因子磷酸化而增加的翻译。胶原蛋白升高有助于内膜增生的发展。我们将通过研究ROS诱导的胶原蛋白产生和移植SMC敏感性升高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。我们将探讨升高的[Ca 2 +]i在增加胶原蛋白产生中的作用，以及ROS介导的[Ca 2 +]i增加的机制，特别是TRPC 6钙通道的作用。我们将研究ROS激活的PKC δ在胶原合成增加中的作用。最后，我们将评估胶原蛋白产生的翻译调节机制，特别是起始因子磷酸化的作用，以及影响转录特异性调节前胶原mRNA翻译的细胞因子的存在。拟议的研究将确定oxLDL和ROS在合成血管移植物SMC与天然主动脉SMC相比增加胶原蛋白产生中的作用。研究还将解决抗氧化剂控制胶原蛋白生产的功效。这将导致更好地理解脂质在移植物愈合中的作用，并最终导致限制胶原蛋白合成和人工移植物内膜增生发展的方法，从而延长小直径血管移植物的通畅性。

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.