Cholesterol Sensitivity and Mechanisms of MSC Responses to 3D Substrate Rigidity

摘要 我们建议对成人骨髓来源的生物反应进行机制研究。 间充质干细胞利用光交联度可控的水凝胶支架对3D基质的刚性 僵硬。具体地说，我们的重点是膜胆固醇和小凹亚域的作用，以及 这些反应中的黏附信号。骨髓间充质干细胞对基质刚性的响应，特别是在3D 它们代表着对其生物活动的重要调节机制，尤其与 它们在组织工程和再生中的应用。我们在此假设，这些回应包括 整合素介导的焦点黏附信号，并对细胞胆固醇稳态具有潜在的敏感性。 因此，我们建议分析胆固醇/小窝蛋白-1(CaV-1)/小窝膜系统的影响， 已知其调节焦点黏附信号，对MSC底物刚性反应，具有特殊的 注意差异化。有关此系统对MSC行为的影响的信息可能在 更广泛的背景下，如果它在体内受到降胆固醇药物的影响，这些药物被广泛用于临床和 因此可能会影响再生疗法的结果。 我们假设细胞膜上胆固醇/Cav-1/小凹水平的升高降低了MSC 增加整合素内吞和减少病灶对底物硬度的敏感性 黏附信号。为了验证我们的假设，我们提出了使用胆固醇的三个具体目标 耗竭、补充胆固醇、下调Cav-1和药物抑制剂，以研究 MSC膜胆固醇、Cav-1、小窝和粘着斑信号在MSC刚性感知中的3D研究 实验平台。目的1：检测胆固醇/Cav-1/小窝动态平衡的扰动对 MSC膜的性能和粘接特性。这将验证对胆固醇/Cav的操纵- 1/小凹影响MSC细胞膜对底物感知的重要方面，包括整合素 表达、激活和内化，以及细胞对特定底物的黏附强度。目标2：测试 细胞内胆固醇/Cav-1/小凹稳态扰动前后间充质干细胞对 3D环境中的不同刚度。这将决定MSCs如何在3D中对不同的基质刚性做出响应 它们的形态、底物黏附、细胞骨架组织和分化，以及IF 对胆固醇/Cav-1/小窝的操纵会影响这些反应。目标3：测试整合素的活性- 在柔软和僵硬的3D基质中激活的MSCs中的焦点黏附信号通路，以及调节 胆固醇/Cav-1/Caveolae对粘着斑信号转导和下游分化的影响 由柔软和僵硬的3D脚手架。这将决定MSC底物中是否涉及焦点黏附信号 刚性反应，以及这种参与是否受到胆固醇/Cav-1/小凹的影响。

ABSTRACT We propose a mechanistic study of the biological responses of adult human bone marrow-derived mesenchymal stem cells (MSCs) to 3D substrate rigidity using hydrogel scaffolds of photocrosslink-controllable stiffness. Specifically, our focus is on the role of membrane cholesterol and caveolae subdomains, and focal adhesion signaling in these responses. The responses of MSCs to substrate rigidity, particularly in a 3D context, represent an important regulatory mechanism of their biological activities, of particular relevance to their application in tissue engineering and regeneration. We postulate here that these responses involve integrin-mediated focal adhesion signaling, and are potentially sensitive to cellular cholesterol homeostasis. We therefore propose to analyze the effects of the cholesterol/Caveolin-1 (Cav-1)/caveolae membrane system, which is known to regulate focal adhesion signaling, on MSC substrate rigidity responses, with special attention to differentiation. Information on the effects of this system on MSC behavior may be important in a wider context, if it is affected in vivo by cholesterol-modifying drugs, which are widely clinically prescribed and thus may influence outcomes of regenerative therapies. We hypothesize that elevated cell membrane levels of cholesterol/Cav-1/caveolae decrease MSC sensitivity to substrate stiffness through increasing integrin endocytosis and decreasing focal adhesion signaling. To test our hypothesis, we propose three specific aims in which we will use cholesterol depletion, cholesterol supplementation, Cav-1 knockdown, and pharmacological inhibitors, to study the roles of MSC membrane cholesterol, Cav-1, caveolae, and focal adhesion signaling in MSC rigidity sensing in our 3D experimental platform. AIM 1: Test the effects of perturbations in cholesterol/Cav-1/caveolae homeostasis on MSC membrane properties and adhesive characteristics. This will verify that manipulation of cholesterol/Cav- 1/caveolae impacts aspects of the MSC cell membrane important to substrate sensing, including integrin expression, activation and internalization, and the strength of cell adhesion to defined substrates. AIM 2: Test the responses of MSCs with and without perturbations in cellular cholesterol/Cav-1/caveolae homeostasis to varied stiffness in a 3D context. This will determine how MSCs respond to varied substrate rigidity in 3D, in terms of their morphology, substrate adhesion, cytoskeletal organization, and differentiation, and if manipulation of cholesterol/Cav-1/caveolae affects these responses. AIM 3: Test the activity of integrin- activated focal adhesion signaling pathways in MSCs within soft and stiff 3D substrates, and the regulatory effects of cholesterol/Cav-1/caveolae on focal adhesion signaling and downstream differentiation as influenced by soft and stiff 3D scaffolds. This will determine if focal adhesion signaling is involved in MSC substrate rigidity responses, and if such involvement is affected by cholesterol/Cav-1/caveolae.