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。动脉的变化 僵硬是血管损伤、动脉粥样硬化和冠状动脉疾病的重要病理改变。变硬 血管壁的收缩促进血管平滑肌细胞的异常迁移和增殖 (VSMCs)，导致血管壁上新生内膜形成。然而，目前还不清楚 细胞外基质(ECM)影响这些病理过程。这项研究提案将解决 这是通过探索动脉僵硬的变化如何引发VSMC行为来促进心血管的 疾病。具体地说，这项工作利用了初步数据，揭示了Survivin蛋白是一个关键 僵硬介导的VSMC增殖和迁移调节因子及动脉硬化效应因子 和改建。利用小鼠和人的VSMCs，我们将首先探索血管细胞外基质僵硬是如何 在单细胞水平(早期)影响VSMC的迁移、增殖和染色质组织 疾病进展；目的1)第二，确定病理性ECM僵硬如何驱动新生内膜 VSMC在体外形成，改变局部机械环境(疾病晚期 进展；目标2)。最后，我们将确认Survivin在调节ECM产生和动脉血中的作用 刚性(活体动物模型；目标2)。这些目标将通过使用3D细胞培养和一种新的 体外猪主动脉脱细胞细胞外基质纤维支架系统及小鼠损伤模型。简单地说， 从小鼠和人的动脉中分离VSMCs，将其培养在不同种类的纳米纤维支架上。 模拟身体正常和病理状态的僵硬和结构。VSMC 对病理性ECM僵硬的反应将使用先进的显微镜进行观察 体外细胞/核结构和生物力学性质的变化以及RNA和蛋白质 表达将在单细胞水平上进行评估。最后，动脉僵硬和VSMC功能将 在受损小鼠的完整动脉中进行研究；将对解剖的组织进行组织学和生化分析 在动脉僵硬被动脉损伤、药物治疗或遗传因素操纵后进行 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(I)介导僵硬敏感的VSMC功能，(Ii)促进新生内膜形成和 僵硬，揭示了VSMC中Survivin生物学的一个全新的方面，并在VSMC的病理学中 动脉僵硬。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。 用于治疗心血管疾病。

SUMMARY Arterial stiffness is a key risk factor for cardiovascular disease (CVD) events. A change in arterial stiffness is a significant pathology in vascular injury, atherosclerosis, and coronary disease. Stiffening of the vessel wall promotes anomalous migration and proliferation of vascular smooth muscle cells (VSMCs), leading to neointima formation on the vessel wall. It is not clear, however, how the extracellular matrix (ECM) influences these pathological processes. This research proposal will address this by exploring how changes in arterial stiffness elicit VSMC behaviors that contribute to cardiovascular disease. Specifically, this work draws upon preliminary data revealing that the protein survivin is a key regulator of stiffness-mediated VSMC proliferation and migration and an effector of arterial stiffening and remodeling. Using mouse and human VSMCs, we 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, second, determine how pathological ECM stiffness drives neointima formation, altering the local mechanical environment of VSMCs in vitro (advanced stage of disease progression; Aim 2). Lastly, we will confirm survivin’s role in regulating both ECM production and arterial stiffness (in vivo animal model; Aim 2). These aims will be achieved using 3D cell culture with a novel in vitro porcine decellularized aorta ECM-based fibrous scaffold system and mouse injury models. Briefly, VSMCs isolated from mouse and human aortas will be cultured on nanofibrous scaffolds of different stiffnesses and structures that mimic normal and pathological conditions in the body. The VSMC responses to pathological ECM stiffness will be analyzed using advanced microscopy to observe changes in cellular/nuclear structure and biomechanical properties in vitro, and the RNA and protein expression will be assessed at the single-cell level. Finally, arterial stiffness and VSMC function will be studied in intact arteries of injured mice; histology and biochemical analyses of dissected tissues will be conducted after arterial stiffness has been 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 (i) mediates stiffness-sensitive VSMC functions and (ii) contributes to neointima formation and stiffening, revealing a completely new aspect of survivin biology in VSMCs and in the pathology of arterial stiffness. Overall, this proposal is unique in its ability to identify potential new therapeutic targets for the treatment of CVDs.