Homocysteine and Endothelial Cell Growth Inhibition

描述（由申请人提供）：本修订竞争性续期申请的总体目标是确定高同型半胱氨酸血症（HHcy）诱导的内皮细胞（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）通过低甲基化相关机制抑制EC的生长，但对其他类型的细胞没有作用。在之前的研究中，我们发现Hcy抑制细胞周期蛋白A的转录、DNA甲基转移酶1 （DNA methyltransferase 1, DNMT1）活性和细胞周期蛋白A启动子的DNA甲基化，并且腺病毒介导的细胞周期蛋白A和DNMT1基因的表达将EC的生长从Hcy的抑制作用中拯救出来。我们的体内研究表明，HHcy通过PKC激活损害内皮功能和eNOS活性，并且HHcy损害小鼠的再内皮化和增加新内膜形成。我们的基本假设是，Hcy通过抑制EC增殖来损害再内皮化，并导致Hcy中动脉粥样硬化的增加。本项目将利用三个相关的具体目标来研究这一假设。首先，在Aim 1中，我们将探索hcy诱导的EC中细胞周期蛋白D2/D3抑制的调控机制。其次，在Aim 2中，我们将确定EC中hcy -低甲基化和生长抑制的生化基础。最后，在Aim 3中，我们将研究EC治疗对HHcy小鼠损伤后再内皮化和新内膜形成的影响。我们相信，具体目标的完成将为建立HHcy与动脉粥样硬化之间的联系提供有价值的新信息，并带来治疗优势。

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.