Biomimetic Vascular Matrix for Vascular Smooth Muscle Cell Mechanobiology and Pathology

总结 动脉僵硬度是心血管疾病（CVD）事件的关键风险因素。动脉硬度变化 是血管损伤、动脉粥样硬化和冠状动脉疾病中的重要病理学， 血管壁促进血管平滑肌细胞的异常迁移和增殖 血管平滑肌细胞（VSMC）引起血管壁的新生内膜形成。然而， 病理性ECM硬度调节与病理性NE相关的VSMC增殖和迁移， 内膜形成不清楚。这项研究提案将通过探索如何改变 在动脉硬化引起VSMC行为，有助于CVD。更具体地说，这项工作借鉴了 新收集的初步数据显示，蛋白生存素作为一种关键的调节因子， 硬化介导的VSMC增殖和迁移以及动脉硬化和重塑的效应器- ing.使用小鼠和人类VSMCs，本研究将首先探索血管ECM刚度如何影响 VSMC迁移、增殖和单细胞水平的染色质组织（疾病早期 进展;目的1）;其次，确定病理性ECM硬度如何驱动新生内膜- 信息改变了体外VSMCs的局部机械环境（疾病进展的晚期阶段）， 目的2）。最后，这项研究计划将测试生存素在调节ECM产生和ECM分泌中的作用。 和动脉硬度（体内动物模型;目的2）。这些目标将通过3D细胞培养来实现。 使用一种新的基于体外猪脱细胞主动脉ECM（daECM）的纤维支架系统， 工程小鼠损伤模型。简而言之，将培养从小鼠和人睾丸中分离的VSMC， 基于daECM的纳米纤维支架的不同刚度，模拟正常和病理性的con. 身体里的位置。VSMC对病理性ECM僵硬的反应将使用先进的 显微镜观察细胞/核结构、生物力学特性和RNA的变化 体外单细胞水平的蛋白表达。最后，工程小鼠将用于研究 硬度和VSMC功能，进行组织学检查和生化检查， 动脉损伤、药物治疗或遗传因素导致僵硬后解剖组织的分析 突变该项目将首次研究分子和生物物理机制， 存活素1）介导刚度敏感性VSMC功能，和2）有助于新生内膜形成， 硬化，揭示了VSMCs和动脉粥样硬化病理学中生存素生物学的一个全新方面， 刚度总的来说，该提案在识别潜在的新治疗靶点方面是独一无二的。 心血管疾病的治疗。

SUMMARY Arterial stiffness is a key risk factor for cardiovascular disease (CVD) events. Change in arterial stiffness is a significant pathology in vascular injury, atherosclerosis, and coronary disease by which stiffening of the vessel wall promotes anomalous migration and proliferation of vascular smooth muscle cells (VSMCs) causing neointima formation of the vessel wall. Yet, the molecular mechanisms by which pathological ECM stiffness regulates VSMC proliferation and migration associated with pathological ne- ointima formation are unclear. This research proposal will address this gap by exploring how changes in arterial stiffness elicit VSMC behaviors that contribute to CVD. More specifically, this work draws upon newly collected preliminary data that show a novel role for the protein survivin as a key regulator of stiffness-mediated VSMC proliferation and migration and an effector of arterial stiffening and remodel- ing. Using mouse and human VSMCs, this study will first explore how vascular ECM stiffness impacts VSMC migration, proliferation, and chromatin organization at the single-cell level (early stage of disease progression; Aim 1); and, secondly, determine how pathological ECM stiffness drives neointima for- mation altering the local mechanical environment of VSMCs in vitro (advanced stage of disease pro- gression; Aim 2). Lastly, this research proposal will test survivin’s role in regulating both ECM production and arterial stiffness (in vivo animal model; Aim 2). These aims will be achieved using a 3D cell culture using a novel in vitro porcine decellularized aorta ECM based (daECM) fibrous scaffold system and engineered mouse injury models. Briefly, VSMCs isolated from mouse and human aortas will be cultured on daECM-based nanofibrous scaffolds of different stiffnesses that mimic normal and pathological con- ditions in the body. The VSMC responses to pathological ECM stiffness will be analyzed using advanced microscopy to observe changes in cellular/nuclear structure, biomechanical properties, and the RNA and protein expressions at the single-cell level in vitro. Finally, engineered mice will be used to study stiffness and VSMC function in intact arteries, performing a histological examination and biochemical analyses of dissected tissue after stiffness is manipulated by arterial injury, drug treatment, or genetic mutations. This project will, for the first time, study the molecular and biophysical mechanisms by which survivin 1) mediates stiffness-sensitive VSMC functions, and 2) contributes to neointima formation and stiffening, revealing a completely new aspect of survivin biology in VSMCs and in the pathology of arte- rial stiffness. Overall, this proposal is unique in its ability to identify potential new therapeutic targets for the treatment of CVDs.