Deciphering mechanisms of vascular disease with cell and process specific ligands

项目摘要/摘要 许多急性血管疾病的主要机制，包括支架内再狭窄、静脉移植 心脏移植物动脉病，是病理性的血管平滑肌细胞(VSMC)激活。 VSMC的激活导致损伤血管的新生内膜形成和再闭塞。预防的策略 异常的VSMC重塑，如药物洗脱支架中使用的细胞生长抑制剂，有不良的影响 损害再内皮化的影响。这种内皮细胞愈合的减弱增加了 新的动脉粥样硬化和晚期支架血栓形成。为了防止病理性VSMC生长，而不是复发 内皮化，迫切需要确定细胞表面蛋白，这些蛋白可能是药物的靶向，以 区分VSMC和内皮细胞(ECs)并调节VSMC利用的细胞过程 在不影响内皮愈合的病理重塑期间。这项提议的目的是利用 特异调控VSMC迁移、增殖和凋亡的VSMC靶向适配子定义1) 明确VSMC靶向适配子调控VSMC的细胞表面蛋白及其作用机制 而不是EC过程；以及2)确定这些细胞和过程特异的配体对新生内膜的影响 急性血管损伤后的形成和再内皮化。我们最近发现了一种VSMC特异性抗- 迁移适体，可防止VSMC迁移，但不影响EC迁移。我们决定， VSMC抗迁移适体通过拮抗PDGFR-β激活发挥作用，但不影响PDGFR-BB- 介导VSMC增殖。这些数据表明，PDGFR-β迁移和增殖的新概念 信号可能是分离的。我们现在已经确定了另外两个VSMC靶向适配子，它们可以调节 VSMC，而不是EC，通过起源于细胞表面的未知机制进行增殖和凋亡。 我们将测试VSMC细胞表面蛋白和信号通路对 急性血管损伤后的迁移、增殖和凋亡可能是细胞特异性调控的 在不改变内皮细胞再内皮化的情况下，防止新生内膜的形成。在目标1中，我们将定义机制 通过这种方式，依赖于PDGFR-β的VSMC迁移与依赖于PDGFR-β的增殖分离，并可能 被抑制，以防止新的内膜形成，而不干扰再内皮化。目标2将确定 细胞表面蛋白，使用全基因组CRISPR文库的新应用，并定义其机制 其中VSMC的增殖可能被特异性地抑制，以防止新的内膜形成，而不是 影响再内皮化。目标3将确定VSMC的机制和细胞表面蛋白 可能通过诱导细胞凋亡来限制新生内膜的形成，同时保留再内皮化。完成 这项研究将导致对调节VSMC迁移、增殖和凋亡的机制的理解 防止新的内膜形成和保存再内皮化，适用于一些 急性血管疾病。

Project Summary / Abstract The primary mechanism responsible for many acute vascular disorders, including in-stent restenosis, vein graft disease, and cardiac allograft arteriopathy, is pathological vascular smooth muscle cell (VSMC) activation. VSMC activation leads to neointimal formation and re-occlusion of injured blood vessels. Strategies to prevent abnormal VSMC remodeling, such as cell growth inhibitors used in drug eluting stents, have the undesired effect of impairing re-endothelialization. This attenuation of endothelial healing increases the risk of neoatherosclerosis and late stent thrombosis. In order to prevent pathological VSMC growth, but not re- endothelialization, there is a critical need to identify cell-surface proteins that may can be targeted by agents to distinguish between VSMCs and endothelial cells (ECs) and modulate cellular processes utilized by VSMCs during pathological remodeling that do not affect endothelium healing. The objective of this proposal is to utilize VSMC-targeting aptamers that specifically modulate VSMC migration, proliferation and apoptosis to define 1) define the cell-surface proteins and mechanism of action by which VSMC-targeting aptamers modulate VSMC but not EC processes; and 2) determine the impact of these cell- and process-specific ligands on neointimal formation and re-endothelialization following acute vascular injury. We recently identified a VSMC-specific anti- migratory aptamer that prevents VSMC migration with no effect on EC migration. We determined that the VSMC anti-migratory aptamer operates by antagonizing PDGFR-β activation, but has no effect on PDGF-BB- mediated VSMC proliferation. These data suggest the novel concept that PDGFR-β migration and proliferation signaling may be dissociated. We have now identified two additional VSMC-targeting aptamers that modulate VSMC, but not EC, proliferation and apoptosis through unknown mechanisms originating at the cell surface. We will test the overall hypothesis that VSMC cell surface proteins and signaling pathways necessary for migration, proliferation and apoptosis following acute vascular injury may be cell-specifically modulated to prevent neointimal formation without altering EC re-endothelialization. In Aim 1, we will define the mechanism by which VSMC PDGFR-β dependent migration is dissociated from PDGFR-β dependent proliferation and may be inhibited to prevent neointimal formation without interfering with re-endothelialization. Aim 2 will identify the cell-surface protein, using a novel application of a whole-genome CRISPR library, and define the mechanisms where VSMC proliferation may be specifically inhibited in VSMCs to prevent neointimal formation without impacting re-endothelialization. Aim 3 will determine the mechanism and cell surface-proteins by which VSMC apoptosis may be induced to limit neointimal formation while preserving re-endothelialization. Completion of this study will result in a mechanistic understanding of modulating VSMC migration, proliferation and apoptosis towards preventing neointimal formation and preserving re-endothelialization, with applicability to a number of acute vascular diseases.