Molecular Signaling in Oxidative Stress-induced Vascular Calcification

描述（由申请人提供）：血管钙化是动脉粥样硬化的标志，是美国死亡率和发病率的主要原因。血管平滑肌细胞（VSMC）在动脉粥样硬化和血管钙化的发生发展中起重要作用。新出现的证据支持血管钙化类似于骨生成过程的概念。增加的氧化应激和活性氧（ROS）的产生加速动脉粥样硬化和血管钙化的进展。然而，氧化应激诱导血管钙化的分子机制尚未完全阐明。过氧化氢（H2 O2）是血管细胞产生的一种重要活性氧，是细胞内信号传导的重要介质。我们发现H2 O2诱导培养的VSMC钙化，其特征是“骨标记物”表达增加和“VSMC标记物”表达下调，同时增加Runx 2的表达和活性增强，以及关键的成骨转录因子。此外，H2 O2诱导的VSMC钙化在Runx 2缺陷的VSMC中被抑制;而Runx 2单独过表达促进VSMC钙化。重要的是，Runx 2的表达增加已被发现在先进的动脉粥样硬化病变，而不是在正常血管。因此，我们得出结论，Runx 2在氧化应激诱导的血管钙化中起着重要作用。然而，Runx 2表达是否有助于动脉粥样硬化和血管钙化的发生或进展尚不清楚; Runx 2调节该过程的机制尚未确定。一般Runx 2缺失小鼠和Runx 2 C-末端消融小鼠由于骨形成缺陷是新生儿致死的，这排除了Runx 2在体内血管钙化中作用的表征。在本研究中，我们将建立SMC特异性消融Runx 2基因的小鼠模型，以确定Runx 2在体内动脉粥样硬化和血管钙化中的作用;并进一步阐明Runx 2依赖的分子信号在调节氧化应激诱导的血管钙化中的作用。我们假设氧化应激诱导Runx 2，这是必不可少的血管钙化。将追求两个具体目标来检验我们的假设：目标1：表征体内Runx 2依赖性血管钙化。将产生具有载脂蛋白E缺乏背景的SMC特异性Runx 2消融小鼠，并用于表征Runx 2在体内动脉粥样硬化和血管钙化进展中的作用。目的2：定义氧化应激诱导的VSMC钙化中的Runx 2依赖性信号。氧化应激诱导的VSMC钙化过程中的Runx 2依赖性阶段，负责VSMC钙化和基因调控的Runx 2功能域;以及H2 O2诱导的血管钙化中的Runx 2调节分子信号将被表征。了解Runx 2在调节氧化应激诱导的血管钙化中的功能，将为开发新的策略和靶点以成功治疗动脉粥样硬化和血管钙化提供重要的见解。 公共卫生相关性：动脉粥样硬化斑块中的血管钙化是动脉粥样硬化的显著特征，是美国死亡率和发病率的主要原因。我们已经证明了Runx 2在体外血管钙化中的重要作用。本研究将进一步研究Runx 2在体内血管钙化中的作用，并探讨Runx 2在氧化应激诱导的血管钙化中调节的分子信号。这些研究将为深入了解动脉粥样硬化的发病机制提供重要的分子信息，从而为开发新的预防或治疗动脉粥样硬化的策略或药物提供重要的指导。PHS 398/2590（2004年9月修订，2006年4月重新印发）

DESCRIPTION (provided by applicant): Vascular calcification is a hallmark of atherosclerosis, a major cause of mortality and morbidity in the United States. Vascular smooth muscle cells (VSMC) contribute significantly to the development of atherosclerosis and vascular calcification. Emerging evidence supports the concept that vascular calcification resembles the process of osteogenesis. Increased oxidative stress and production of reactive oxygen species (ROS) accelerate the progression of atherosclerosis and vascular calcification. However, the molecular mechanisms underlying oxidative stress-induced vascular calcification have not been fully elucidated. Hydrogen peroxides (H2O2), a key ROS produced by vascular cells, has emerged as an important mediator of intracellular signaling. We found that H2O2 induced VSMC calcification in culture, featuring increased expression of "bone markers" and downregulation of "VSMC markers", concurrent with increased expression and enhanced activity of Runx2, and the key osteogenic transcription factor. Further, H2O2-induced VSMC calcification was inhibited in Runx2 deficient VSMC; while overexpression of Runx2 alone promoted VSMC calcification. Importantly, increased expression of Runx2 has been found in advanced atherosclerosis lesions, but not in normal vessels. Therefore, we conclude that Runx2 plays an important role in oxidative stress-induced vascular calcification. However, whether Runx2 expression contributes to onset or progression of atherosclerosis and vascular calcification is not known; and the mechanisms whereby Runx2 in regulating the process have not been defined. General Runx2 null mice and Runx2 C-terminal ablation mice are neonatal lethal due to defective bone formation, which precludes the characterization of the role of Runx2 in vascular calcification in vivo. In this proposal, we will generate a mouse model with SMC-specific ablation of Runx2 gene to determine the role of Runx2 in atherosclerosis and vascular calcification in vivo; and further elucidate Runx2-dependent molecular signals in regulating oxidative stress-induced vascular calcification. We hypothesize that oxidative stress induces Runx2 that is essential for vascular calcification. Two Specific Aims will be pursued to test our hypothesis: Aim 1: Characterize Runx2 Dependent Vascular Calcification In Vivo. SMC-specific Runx2 ablation mice with apolipoprotein E deficiency background will be generated and used to characterize the role of Runx2 in the progression of atherosclerosis and vascular calcification in vivo. Aim 2: Define Runx2 Dependent Signals in Oxidative Stress-induced VSMC Calcification. Runx2-dependent stages during oxidative stress-induced VSMC calcification, Runx2 functional domains responsible for VSMC calcification and gene regulation; and Runx2-regualted molecular signals in H2O2-induced vascular calcification will be characterized. Understanding the function of Runx2 in regulating oxidative stress-induced vascular calcification will provide important insights into the development of novel strategies and targets for successful therapeutic interventions for atherosclerosis and vascular calcification. PUBLIC HEALTH RELEVANCE: Vascular calcification in atherosclerotic plaques is a prominent feature of atherosclerosis, a major cause of mortality and morbidity in the United States. We have demonstrated an essential role of Runx2 in vascular calcification in vitro. This proposal will further characterize role of Runx2 in vascular calcification in vivo and investigate the Runx2-regulated molecular signals in oxidative stress-induced vascular calcification. These studies will provide important molecular insights into understanding the pathogenesis of atherosclerosis, which will leads to development of novel strategy or drugs for atherosclerosis prevention or therapy. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page