Homocysteine-induced Endothelial Cell Growth Inhibition

描述(由申请人提供)： 高同型半胱氨酸血症是心肌梗死和中风的独立危险因素，但同型半胱氨酸(Hcy)促进动脉硬化的机制尚不清楚。已报道的Hcy对血管细胞的生物学效应大多归因于氧化机制，当Hcy浓度高于1 mM或更高时，观察到的氧化机制可被另一种非致病生物硫醇半胱氨酸模拟。因此，同型半胱氨酸独特的生化机制仍有待确定。我们提出低甲基化是同型半胱氨酸引起血管损伤和导致心血管疾病的一种特殊机制。正在进行的项目和建议的项目的基本假设是，在临床相关浓度下，Hcy通过与低甲基化相关的机制选择性地抑制EC的生长。正在进行的研究旨在探讨RAS去甲基化在Hcy-EC生长中的作用，并利用细胞和动物模型剖析Hcy信号转导的机制。由于内皮细胞的损伤是动脉硬化的重要特征，因此内皮细胞的生长抑制可能是Hcy诱发动脉硬化的重要机制。在拟议的研究中，我们假设其他分子的低甲基化也可能在Hcy相关的EC生长抑制中发挥重要作用。我们增加了两个新的靶点来表征基因组DNA和蛋白质的甲基化状态，检测组蛋白甲基转移酶在同型半胱氨酸处理的EC(Aim 4)中的活性，并利用逆转录病毒介导的遗传筛选和放射性标记甲基化敏感的双向电泳蛋白质组学(Aim 5)来寻找新的功能靶基因。这两个新目标是资助项目的扩展，并将探索Hcy抑制EC生长的关键功能分子机制。这项建议的广泛和长期目标是阐明Hcy信号在EC生长抑制中的作用，并评估其在动脉粥样硬化在Hcy病理中的作用。如果我们能够确定同型半胱氨酸诱导的动脉硬化的关键事件，阻断这些步骤的遗传或生化方法可能会带来治疗优势。

DESCRIPTION (provided by applicant): Hyperhomocysteinemia is an independent risk factor for myocardial infarction and stroke, yet the mechanisms by which homocysteine (Hcy) promotes arteriosclerosis are not clear. Most of the reported biological effects of Hcy in vascular cells have been attributed to oxidative mechanisms, which were observed at Hcy concentrations higher than 1 mM or higher, and can be mimicked by cysteine, another nonpathogenic biothiol. Thus, a biochemical mechanism unique to Hcy remains to be identified. We have proposed hypomethylation as a specific mechanism by which Hcy induces vascular injury and leads to cardiovascular disease. The basic hypothesis of the ongoing and this proposed projects is that Hcy, at clinically relevant concentrations, selectively inhibits EC growth through a hypomethylation-related mechanism. The ongoing research is designed to investigate the role of Ras demethylation in Hcy-EC growth, to dissect the mechanism in Hcy signaling using cellular and animal models. Because damage to EC is a key feature of arteriosclerosis, the growth inhibition of EC may represent an important mechanism to explain Hcy-induced arteriosclerosis. In the proposed study, we hypothesize that hypomethylation of other molecules may also play an important role in Hcy-related EC growth inhibition. We added two new aims to characterize methylation status of genomic DNA and protein, to examine the activities of histone methyltransferase in Hcy-treated EC (Aim 4), and to identify new functional target genes using retrovirus-mediated genetic screening and radiolabelled methylation sensitive two-dimensional electrophoresis proteomics (Aim 5). These two new aims are expansion of the funded project and would explore key functional molecular mechanisms by which Hcy inhibit EC growth. The broad, long-term objective of this proposal is to elucidate Hcy signaling in EC growth inhibition, and to evaluate its importance in the role of atherogenesis in Hcy pathology. If we can identify the key events in Hcy-induced arteriosclerosis, genetic or biochemical approaches to block these steps could lead to therapeutic advantage.