Mechanical Response and Cytoskeletal Rearrangement Following Interrupted Cholesterol Metabolism in Vascular Smooth Muscle Cell

摘要 血管平滑肌细胞(VSMCs)的表型迁移和迁移在VSMCs的进展中起关键作用 动脉粥样硬化。VSMC的表型转换伴随着整合素的差异表达， 它参与细胞-细胞外基质(ECM)的黏附和VSMC的迁移。取得了实质性进展 以促进对动脉粥样硬化中VSMC迁移机制的了解。然而，几乎没有什么 已知胆固醇如何直接影响VSMC的黏附状态和反应性 细胞外机械刺激。本研究从分子水平探讨了这一现象的机制。 细胞胆固醇和基质硬度对VSMC黏附和迁移的协同作用。我们会 检测整合素αVβ3和N-钙粘蛋白(N-Cad)的表达和活性，这两种蛋白已被报道为 参与动脉粥样硬化的发展。值得注意的是，一种与生理更相关的方法，他汀类药物， 将被用于管理细胞胆固醇，而不是环糊精，遵循美国的指导方针 心脏协会(AHA 2018)。在一项初步研究中，他汀类药物治疗可降低内源性VSMC胆固醇 与环糊精相比，不会导致显著的细胞死亡。我们还发现他汀类药物的使用 不同影响的整合素和N-Cad介导的VSMC粘连。基于这些发现，我们 假设高细胞胆固醇和细胞外基质硬化协同诱导血管紧张素转换酶 通过下调N-钙粘蛋白和整合素αVβ3介导的细胞黏附而导致动脉粥样硬化，从而增强 动脉粥样硬化中的VSMC迁移，而他汀类药物介导的胆固醇耗竭通过以下方式阻止这一过程 干扰血管平滑肌细胞的生物力学。这一假设将通过执行以下操作来验证 具体目标：(1)测试他汀类药物诱导的胆固醇消耗和底物硬化对 α、V、β3和N-Cad在VSMC中的表达和活性；(2)检测他汀类药物对VSMC的联合作用。 诱导胆固醇消耗和底物僵硬对VSMC硬度和细胞骨架三维构型的影响； (3)检测他汀类药物诱导的胆固醇耗竭和底物硬化对VSMC的联合作用 迁移。这项工作的创新之处在于将VSMC力学和细胞骨架组织与 动脉粥样硬化的发展。使用创新方法、综合共聚焦显微镜和原子 强制显微镜监测细胞力学和细胞骨架重塑，以及使用最先进的 图像处理技术，将使深入研究VSMC力学和 从细胞骨架结构到动脉粥样硬化。这项研究将加强本科生的研究 南达科他大学生物医学工程活动招收6名本科生 暑期研究(每年两次)。通过他们的参与，学生们将获得丰富的知识， 技能和生物医学研究方面的经验，这将为他们未来的职业发展提供很好的帮助。

SUMMARY Phenotypic shifting and migration of vascular smooth muscle cells (VSMCs) play key roles in the progression of atherosclerosis. Phenotypic switching in VSMCs is accompanied by the differential expression of integrins, which is involved in cell-extracellular matrix (ECM) adhesion and VSMC migration. Substantial progress has been made to advance understanding of the mechanism of VSMC migration in atherosclerosis. Little, however, is known about how cholesterol directly affects the adhesive state and responsiveness of VSMCs to extracellular mechanical stimulation. This study investigates the molecular mechanism underlying the synergistic effect of cellular cholesterol and substrate stiffness on VSMC adhesion and migration. We will examine the expression and activity of integrin αVβ3, and N-cadherin (N-Cad), which have been reported to be involved in the development of atherosclerosis. Significantly, a more physiologically-relevant approach, statin, will be employed to manage cellular cholesterol instead of cyclodextrin following the guidelines of American Heart Association (AHA 2018). In a preliminary study, statin treatment reduced endogenous VSMC cholesterol without resulting in significant cell death compared to cyclodextrin. We also found that use of statin differentially affected integrins and N-Cad mediated VSMC adhesions. Based on these findings, we hypothesize that high cellular cholesterol and ECM stiffening synergistically induces the development of atherosclerosis by down-regulating N-Cad and integrin αVβ3-mediated cell adhesions, thereby enhancing VSMC migration for atherogenesis, while statin-mediated cholesterol depletion prevents the process by interfering with the biomechanics of VSMCs. This hypothesis will be tested by carrying out the following Specific Aims: (1) test the combined effects of statin-induced cholesterol depletion and substrate stiffening on the expression and activity of integrin αVβ3 and N-Cad in VSMCs; (2) test the combined effects of statin- induced cholesterol depletion and substrate stiffening on VSMC stiffness and cytoskeleton 3D architecture; and (3) test the combined effects of statin-induced cholesterol depletion and substrate stiffening on VSMC migration. The novelty of this work lies in linking VSMC mechanics and cytoskeletal organization with the development of atherosclerosis. Use of innovative approaches, integrated confocal microscopy and atomic force microscopy to monitor cell mechanics and cytoskeletal remodeling, as well as use of a state-of-the-art image processing technique, will enable an in-depth study of the contribution of VSMC mechanics and cytoskeleton architecture to atherosclerosis. This study will strengthen undergraduate student research activities in biomedical engineering at University of South Dakota by recruiting six undergraduate students for summer research (two for each year). Through their participation, students will gain substantial knowledge, skills, and experience in biomedical research, which will serve them well for their future career development.