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展中起着重要作用。新出现的证据支持血管钙化类似成骨过程的概念。氧化应激的增加和活性氧（ROS）的产生加速了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（H2O2）是血管细胞产生的一种关键活性氧，是细胞内信号传导的重要介质。我们发现H2O2诱导培养VSMC钙化，表现为“骨标记物”表达增加，“VSMC标记物”表达下调，同时成骨关键转录因子Runx2表达增加，活性增强。此外，在Runx2缺乏的VSMC中，h2o2诱导的VSMC钙化受到抑制；而单独过表达Runx2可促进VSMC钙化。重要的是，在晚期动脉粥样硬化病变中发现Runx2表达增加，而在正常血管中没有发现。因此，我们认为Runx2在氧化应激诱导的血管钙化中起重要作用。然而，Runx2的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；而Runx2调节这一过程的机制尚未明确。常规Runx2缺失小鼠和Runx2 c端消融小鼠由于骨形成缺陷而致新生儿死亡，这就排除了Runx2在体内血管钙化中的作用。在本研究中，我们将建立一个smc特异性消融Runx2基因的小鼠模型，以确定Runx2在体内动脉粥样硬化和血管钙化中的作用；并进一步阐明runx2依赖的分子信号在调控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了对血管钙化至关重要的Runx2。两个特定的目标将追求，以验证我们的假设：目标1：表征Runx2依赖性血管钙化在体内。将产生具有载脂蛋白E缺乏背景的smc特异性Runx2消融小鼠，并用于表征Runx2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Runx2在氧化应激诱导的VSMC钙化中的依赖信号。氧化应激诱导VSMC钙化过程中Runx2依赖阶段、负责VSMC钙化和基因调控的Runx2功能域；以及runx2在h2o2诱导血管钙化过程中调控的分子信号。了解Runx2在调节氧化应激诱导的血管钙化中的功能，将为动脉粥样硬化和血管钙化的成功治疗干预的新策略和靶点的发展提供重要的见解。

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