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

摘要 我们提出了一个机制研究的生物反应，成人骨髓源性 使用光交联可控的水凝胶支架将间充质干细胞（MSC）固定到3D基底刚性 刚度具体来说，我们的重点是膜胆固醇和小窝亚结构域的作用，以及局灶性 这些反应中的粘附信号。MSC对基底刚性的反应，特别是在3D中， 背景，代表了其生物活性的重要调节机制，特别是与 它们在组织工程和再生中的应用。我们在这里假设，这些反应涉及 整合素介导的粘着斑信号传导，并且对细胞胆固醇稳态潜在敏感。 因此，我们建议分析胆固醇/Caveolin-1（Cav-1）/Caveolae膜系统的作用， 已知其调节MSC基质刚性反应上的粘着斑信号传导， 注意区分。关于该系统对MSC行为的影响的信息在本领域中可能是重要的。 更广泛的背景下，如果它在体内受到胆固醇修饰药物的影响，这些药物在临床上广泛使用， 因此可能会影响再生疗法的结果。 我们假设升高的细胞膜胆固醇/Cav-1/小窝水平降低了MSC 通过增加整联蛋白内吞作用和减少局部细胞毒性， 粘附信号为了验证我们的假设，我们提出了三个具体的目标，我们将使用胆固醇 消耗，胆固醇补充，Cav-1敲低和药理学抑制剂，以研究 MSC刚性感知中的MSC膜胆固醇、Cav-1、小窝和粘着斑信号传导 实验平台目的1：测试胆固醇/Cav-1/小窝稳态的扰动对 MSC膜的性能和粘合特性。这将证实胆固醇/Cav的操纵- 1/小窝影响MSC细胞膜对底物传感重要的方面，包括整合素 表达、活化和内化，以及细胞粘附于限定底物的强度。目标2：测试 在细胞胆固醇/Cav-1/小窝稳态受到干扰和没有受到干扰的情况下， 在3D环境中改变刚度。这将确定MSC如何在3D中响应于不同的基底刚性， 它们的形态、基质粘附、细胞骨架组织和分化，以及 操纵胆固醇/Cav-1/小窝影响这些反应。目的3：检测整合素的活性。 在软质和硬质3D基质中激活MSC中的粘着斑信号传导通路， 胆固醇/Cav-1/小窝对粘着斑信号传导和下游分化的影响 通过软硬的3D支架。这将确定粘着斑信号传导是否参与MSC基质 强直反应，以及这种参与是否受到胆固醇/Cav-1/小窝的影响。

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.