Molecular Signaling in Oxidative Stress-induced Vascular Calcification

描述(申请人提供)：血管钙化是动脉粥样硬化的标志，动脉粥样硬化是美国死亡和发病的主要原因。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成骨过程的概念。氧化应激增加和活性氧(ROS)的产生加速了动脉粥样硬化和血管钙化的进程。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(H_2O_2)是血管细胞产生的一种重要的ROS，是细胞内信号转导的重要介质。我们发现，H_2O_2诱导培养的VSMC钙化，表现为“骨标志物”的表达增加和“VSMC标志物”的下调，同时关键的成骨转录因子Runx2的表达和活性增加。此外，在Runx2缺乏的VSMC中，过氧化氢诱导的VSMC钙化受到抑制，而Runx2单独过表达则促进了VSMC的钙化。重要的是，在晚期动脉粥样硬化病变中发现了Runx2的表达增加，但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发展尚不清楚，也不清楚Runx2调控这一过程的机制。普通Runx2缺失小鼠和Runx2 C末端消融小鼠由于骨形成缺陷而导致新生死亡，这排除了Runx2在体内血管钙化中的作用的表征。在这个方案中，我们将建立一种SMC特异性的Runx2基因消融的小鼠模型，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Runx2，而Runx2对血管钙化是必不可少的。将追求两个特定的目标来验证我们的假设：目标1：在活体中表征Runx2依赖的血管钙化。将建立具有载脂蛋白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：明确氧化应激诱导的VSMC钙化过程中的Runx2依赖信号。在氧化应激诱导的VSMC钙化过程中，Runx2依赖的阶段，负责VSMC钙化和基因调控的Runx2功能结构域，以及在过氧化氢诱导的血管钙化中Runx2调节的分子信号将被描述。了解Runx2在调节氧化应激诱导的血管钙化中的作用，将为开发成功的动脉粥样硬化和血管钙化治疗干预的新策略和新靶点提供重要的见解。

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.