Biomimetic Vascular Matrix for Vascular Smooth Muscle Cell Mechanobiology and Pathology

摘要 动脉僵硬是心血管疾病(CVD)事件的关键危险因素。动脉僵硬的变化 在血管损伤、动脉粥样硬化和冠心病中是一种重要的病理学，通过它，僵硬的 血管壁促进血管平滑肌细胞的异常迁移和增殖 (VSMCs)导致血管壁新生内膜形成。然而，其分子机制 病理性ECM僵硬调节病理性新生血管内皮细胞的增殖和迁移 内膜形成尚不清楚。这项研究提案将通过探索如何改变来解决这一差距 动脉僵硬可诱发VSMC行为，从而导致CVD。更具体地说，这项工作借鉴了 最新收集的初步数据显示，Survivin蛋白作为一种关键调控因子发挥了新的作用 僵硬介导的VSMC增殖和迁移以及动脉硬化和重塑的效应器- 英。利用小鼠和人的血管平滑肌细胞，这项研究将首先探索血管细胞外基质硬度如何影响 单细胞水平(疾病早期)的VSMC迁移、增殖和染色质组织 进展；目标1)；第二，确定病理性ECM僵硬如何驱动新生内膜- 改变体外培养的VSMC的局部力学环境(疾病进展的晚期) 渐进性；目标2)。最后，这项研究提案将测试Survivin在调节两种细胞外基质产生中的作用 和动脉僵硬(活体动物模型；目标2)。这些目标将通过3D细胞培养来实现 应用一种新型的体外猪主动脉脱细胞外基质(DaECM)纤维支架系统 工程化小鼠损伤模型。简单地说，从小鼠和人的主动脉分离的VSMCs将被培养 在不同硬度的DAECM基纳米纤维支架上模拟正常和病理情况。 身体里的凹痕。VSMC对病理性ECM僵硬的响应将使用高级 显微镜观察细胞/核结构、生物力学特性和RNA的变化 以及体外单细胞水平的蛋白质表达。最后，基因工程小鼠将被用于研究 完整动脉的僵硬和VSMC功能，进行组织学检查和生化 动脉损伤、药物治疗或遗传操作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，通过这些机制 Survivin 1)介导僵硬敏感的VSMC功能，2)促进新生内膜的形成和 僵硬，揭示了Survivin生物学在VSMC和ARTE病理中的一个全新的方面。 里亚尔硬度。总体而言，这项建议的独特之处在于它能够确定潜在的新治疗靶点 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.