Homocysteine and Endothelial Cell Growth Inhibition

描述(由申请人提供)：本次修订的竞争性更新申请的总体目标是确定高同型半胱氨酸血症(HHcy)诱导内皮细胞(EC)生长抑制的分子机制。我们提供的初步证据表明，病理生理相关浓度的同型半胱氨酸(Hcy)通过与低甲基化相关的机制抑制EC的生长，但不抑制其他类型的细胞。在之前的授权期，我们发现Hcy抑制细胞周期蛋白A的转录、DNA甲基转移酶1(DNMT1)活性和DNA甲基化，并且腺病毒转导的细胞周期蛋白A和DNMT1基因的表达拯救了Hcy对EC生长的抑制作用。我们在体内的研究表明，HHcy通过激活PKC损害内皮功能和eNOS活性，并损害小鼠的再内皮化和增加新生内膜形成。我们的基本假设是，同型半胱氨酸通过抑制血管内皮细胞增殖而损害血管内皮细胞的再内皮化，并导致同型半胱氨酸的动脉粥样硬化加重。本项目将利用三个相互关联的具体目标来研究这一假设。首先，在目标1中，我们将探讨同型半胱氨酸诱导的EC细胞周期蛋白D2/D3抑制的调控机制。其次，在目标2中，我们将确定EC中同型半胱氨酸低甲基化和生长抑制的生化基础。最后，在目标3中，我们将检测EC治疗对HHcy小鼠损伤后再内皮化和新生内膜形成的影响。我们认为，完成特定的目标应该提供有价值的新信息，以建立HHcy和动脉粥样硬化之间的联系，并导致治疗优势。 公众与人类的相关性有人提出，同型半胱氨酸是肾脏疾病、糖尿病、老龄化和绝经后妇女心血管疾病患病率较高的原因，这不是传统危险因素所能解释的。然而，其潜在的机制尚不清楚，同型半胱氨酸(Hcy)诱导的内皮细胞生长抑制在CVD中的作用也不清楚。我们先前已经证明，Hcy通过一种与低甲基化相关的机制，对细胞周期蛋白A的转录和内皮细胞的生长产生高度选择性的抑制作用，从而阻止细胞周期和内皮细胞的再生。由于内皮损伤是血管疾病的早期事件，而且内皮再生决定了动脉粥样硬化的发生，我们假设Hcy通过抑制EC的增殖来损害再内皮化，从而促进动脉粥样硬化。在这项建议中，我们建议研究HHcy在改变EC代谢和EC生物学中的作用和机制，目的是通过体外和体内方法确定潜在的机制。

DESCRIPTION (provided by applicant): The overall objective of this revised competitive renewal application is to determine the molecular mechanisms responsible for Hyperhomocysteinemia (HHcy)-induced endothelial cell (EC) growth inhibition. We provided initial evidence demonstrating that pathophysiologically relevant concentrations of homocysteine (Hcy) inhibit EC growth, but not that of other cell types, through a hypomethylation related mechanism. In the previous grant period, we discovered that Hcy inhibits cyclin A transcription, DNA methyltransferase 1 (DNMT1) activity and DNA methylation in cyclin A promoter, and that adenovirus-transduced expression of cyclin A and DNMT1 genes rescued EC growth from the inhibitory effect of Hcy. Our in vivo studies indicate that HHcy impairs endothelial function and eNOS activity via PKC activation, and that HHcy impaired reendothelialization and increased neointimal formation in mice. Our basic hypothesis is that that Hcy impairs reendothelialization via inhibition of EC proliferation, and contribute, to the increased atherosclerosis in HHcy. This project will study this hypothesis utilizing three linked specific aims. First, in Aim 1, we will explore the regulatory mechanisms of Hcy-induced cyclin D2/D3 suppression in EC. Second, in Aim 2, we will Determine biochemical basis of Hcy-hypomethylation and growth inhibition in EC. Finally, in Aim 3, we will examine the effect of EC therapy in post-injury reendothelialization and neointima formation in HHcy mice. We believe that completion of the specific aims should provide valuable new information to establish the links between HHcy and atherosclerosis, and lead to therapeutic advantage. PUBLIC HUMAN RELEVANCE It has been suggested that HHcy accounts for the higher prevalence of CVD in renal disease, diabetes, ageing and in postmenopausal women that is not explained by traditional risk factors. However, the underlying mechanism is largely unknown and the role of homocysteine (Hcy)-induced endothelial growth inhibition in CVD is unclear. We have previously demonstrated that Hcy exerts highly selective inhibitory effect on cyclin A transcription and EC growth through a hypomethylation related mechanism, which blocks cell cycle progression and endothelium regeneration. Since endothelial injury is an early event in vascular disease, and since endothelial regeneration determines the onset of atherosclerosis, we hypothesize that Hcy promotes atherosclerosis by impairing reendothelialization via inhibition of EC proliferation. In this proposal, we propose to investigate the role and mechanisms of HHcy in altering EC metabolism and EC biology, with the goal of identifying the underlying mechanisms, using in vitro and in vivo approaches.