Effect of Lipids on Vascular Graft Healing

描述(申请人提供)：假体移植物在血管重建手术中被广泛使用，但其长期通畅性受到其血栓形成和内膜增生发展的限制。氧化的低密度脂蛋白和低密度脂蛋白氧化产物溶血磷脂酰胆碱(LysoPC)在移植物中积累，改变细胞功能。我们研究的长期目标是通过促进移植物表面的内皮细胞(EC)愈合来改善血管移植物的通畅性。在体外，LysoPC可抑制EC的迁移，高胆固醇血症可减少EC向受损动脉和移植物的迁移。OLD和LYSOPC可增加细胞产生的活性氧，增加细胞膜的流动性，并开放离子通道。这些作用可以抑制EC的迁移。具体地说，LysoPC激活了典型的瞬时受体电位(TRPC)离子通道TRPC6，该通道通过一个独特的TRPC激活级联反应打开TRPC5，导致细胞内游离钙离子浓度([Ca~(2+)]i)持续上升。[Ca~(2+)]i升高可通过激活钙调蛋白来抑制EC的迁移，该酶可分解迁移所必需的细胞骨架蛋白。这一建议提出的假设是，合成血管内形成的脂质氧化产物部分通过激活TRPC6和TRPC5通道来抑制其EC迁移，从而限制体内移植物的内皮化。该项目的目标是确定脂质氧化产物激活TRPC6和TRPC5通道的机制，并确定抵消这一作用的方法。为了实现这些目标，将探索脂质氧化产物激活TRPC6的机制，特别是Src激酶和磷脂酶C-31的作用。此外，还将研究TRPC6激活TRPC5的机制，重点是细胞内钙和肌球蛋白轻链激酶的作用。还将探讨活性氧物种和膜流动性的变化在这些作用中的作用。最后，我们将评估apoA-I模拟物或高密度脂蛋白在体外阻止TRPC6-TRPC5激活级联反应、改善小鼠动脉损伤区域的EC迁移以及移植到正常和高胆固醇血症兔假体移植物上的能力。这项拟议的研究将探讨脂质氧化产物限制血管损伤和人造血管移植物EC愈合的机制。研究还将探讨高密度脂蛋白促进EC愈合的能力。这些研究将有助于更好地了解脂质在移植物衰竭的病理生理学中的作用，并为开发阻断TRPC6-TRPC5激活级联反应的TRPC6通道抑制剂或药物提供动力。这些基于机制的疗法将促进血管成形术部位和假体移植物的内皮愈合，以延长其通畅性，使所有接受心血管干预的人受益。 公共卫生相关性：我们研究的长期目标是改善球囊血管成形术和支架术后搭桥移植物或动脉的愈合。我们将研究氧化脂质如何阻止内皮细胞(正常情况下排列血管的细胞)进入损伤区域或进入搭桥移植物。具体地说，我们将研究某些离子通道(TRPC通道)的作用，当消除这些通道时，会在小鼠模型中消除高胆固醇的抑制作用。这些研究结果将为血管干预后促进内皮细胞愈合的治疗方法的发展提供方向。例如，TRPC6抑制剂可以增加内皮细胞迁移，促进动脉损伤和血管移植的愈合，使所有需要心血管干预的患者受益。

DESCRIPTION (provided by applicant): Prosthetic grafts are used widely in vascular reconstructive surgery, but their long-term patency is limited by their thrombogenicity and the development of intimal hyperplasia. Oxidized LDL and lysophosphatidylcholine (lysoPC), a product of LDL oxidation, accumulate in grafts and alter cell function. The long-term goal of our research is to improve the patency of vascular grafts by promoting endothelial cell (EC) healing of graft surfaces. LysoPC inhibits EC migration in vitro, and hypercholesterolemia reduces EC migration into injured arteries and onto grafts. Old and lysoPC increase cellular production of reactive oxygen species, increase cell membrane fluidity, and open ion channels. These effects can inhibit EC migration. Specifically, lysoPC activates a canonical transient receptor potential (TRPC) ion channel, TRPC6, which opens TRPC5 through a unique TRPC activation cascade, leading to a prolonged rise in intracellular free calcium ion concentration ([Ca2+]i). Increased [Ca2+]i inhibits EC migration by activation of calpains that breakdown cytoskeletal proteins essential for migration. This proposal addresses the hypothesis that lipid oxidation products formed within synthetic vascular grafts inhibit their EC migration, in part through activation of TRPC6 and TRPC5 channels, and thereby limit endothelialization of grafts in vivo. The goals of this project are to identify mechanisms by which lipid oxidation products activate TRPC6 and TRPC5 channels and identify ways to counteract this. To accomplish these goals, the mechanism by which lipid oxidation products activate TRPC6, specifically the roles of Src kinases and phospholipase C-31, will be explored. In addition, and the mechanism by which TRPC6 activates TRPC5 will be studied, focusing on the role of intracellular calcium and myosin light chain kinase. The role of reactive oxygen species and changes in membrane fluidity in these actions will also be explored. Finally, the ability of an apoA-I mimetic or HDL, which we have shown to block the TRPC6-TRPC5 activation cascade in vitro, to improve EC migration in areas of arterial injury in mice and onto prosthetic grafts implanted in normal and hypercholesterolemic rabbits will be assessed. The proposed studies will investigate a mechanism by which lipid oxidation products limit EC healing of vascular injuries and synthetic vascular grafts. Studies will also address the ability of HDL to promote EC healing. These studies will lead to a better understanding of the role of lipids in the pathophysiology of graft failure, and provide impetus for development of TRPC6 channel inhibitors or agents that interrupt the TRPC6- TRPC5 activation cascade. These mechanism-based therapies will promote endothelial healing of angioplasty sites and prosthetic grafts to prolong their patency for the benefit of all people undergoing cardiovascular interventions. PUBLIC HEALTH RELEVANCE: The long-term goal of our research is improve the healing of bypass grafts or arteries after balloon angioplasty and stenting. We will investigate how oxidized lipids block the movement of endothelial cells (cells that normally line blood vessels) into an area of injury or onto a bypass graft. Specifically, we will study the role of certain ion channels (TRPC channels) that when eliminated abolish the inhibitory effect of high cholesterol in a mouse model. The results of these studies will provide direction in the development of treatments to promote endothelial cell healing after vascular interventions. For example, a TRPC6 inhibitor could increase endothelial cell migration and promote healing of arterial injuries and vascular grafts for the benefit all patients who require cardiovascular interventions.