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

胆固醇敏感性和 MSC 对 3D 基质刚性的响应机制

基本信息

批准号：
9240628
负责人：
ROCKY S TUAN
金额：
$ 33.83万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9240628
关键词：
3-Dimensional Adherence Adhesions Adhesives Adopted Adult Affect Atomic Force Microscopy Attention Behavior Biological Biological Assay Bone Marrow Brain Caveolae Caveolins Cell Adhesion Cell Differentiation process Cell Fractionation Cell Shape Cell membrane Cells Cellular Morphology Characteristics Cholesterol Cholesterol Homeostasis Clinical Cytoskeletal Modeling Electron Microscopy Endocytosis Event F-Actin Flow Cytometry Fluorescence Polarization Focal Adhesions Future Gelatin Gene Expression Profiling Goals Health Homeostasis Human Hyaluronan Hydrogels Hypersensitivity Immunofluorescence Immunologic Integrins Knowledge Ligands Mediating Membrane Membrane Fluidity Membrane Microdomains Mesenchymal Stem Cells Methacrylates Morphology Neurons Osteoblasts Osteocalcin Osteogenesis Outcome Pathway interactions Pharmaceutical Preparations Pharmacology Phenotype Plasticizers Population Property Reverse Transcriptase Polymerase Chain Reaction Role Rupture Seeds Shapes Signal Pathway Signal Transduction Small Interfering RNA Sucrose Supplementation Suspensions System Testing Time Tissue Engineering Tissues Western Blotting base caveolin 1 cell behavior chemical property design ethylene dimethacrylate in vivo in vivo regeneration inhibitor/antagonist knock-down mechanical properties mechanotransduction osteogenic physical property regenerative therapy response scaffold soft tissue tissue regeneration

项目摘要

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.

抽象的我们提出对成人骨髓来源的生物反应进行机制研究使用光交联可控的水凝胶支架将间充质干细胞 (MSC) 转化为 3D 基质刚性刚性。具体来说，我们的重点是膜胆固醇和小窝子域的作用，以及焦点这些反应中的粘附信号传导。 MSC 对基质刚性的反应，特别是在 3D 中背景下，代表其生物活性的重要调节机制，特别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 膜系统的影响，众所周知，它可以调节 MSC 基质刚性反应上的粘着斑信号传导，具有特殊的作用注重差异化。关于该系统对 MSC 行为的影响的信息可能很重要更广泛的背景，如果它在体内受到临床上广泛处方的胆固醇调节药物的影响，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/小窝水平升高会降低 MSC 通过增加整合素内吞作用和减少焦点对基质硬度的敏感性粘附信号。为了检验我们的假设，我们提出了三个使用胆固醇的具体目标消耗、胆固醇补充、Cav-1 敲低和药物抑制剂，以研究 3D 中 MSC 刚性传感中的 MSC 膜胆固醇、Cav-1、小凹和粘着斑信号传导实验平台。目标 1：测试胆固醇/Cav-1/小窝稳态的扰动对 MSC 膜特性和粘合剂特性。这将验证胆固醇/Cav-的操纵 1/小窝影响 MSC 细胞膜对底物传感重要的各个方面，包括整合素表达、激活和内化，以及细胞对特定基质的粘附强度。目标 2：测试 MSCs 在细胞胆固醇/Cav-1/caveolae 稳态有或没有扰动的情况下的反应 3D 环境中的不同刚度。这将决定 MSC 如何响应 3D 中不同的基质刚性就它们的形态、基质粘附、细胞骨架组织和分化而言，如果胆固醇/Cav-1/小窝的操纵会影响这些反应。目标 3：测试整合素的活性激活软和硬 3D 基质内 MSC 中的粘着斑信号通路，以及调节胆固醇/Cav-1/小窝对粘着斑信号传导和下游分化的影响由柔软和坚硬的 3D 支架组成。这将确定 MSC 基质中是否涉及粘着斑信号传导僵化反应，以及这种参与是否受到胆固醇/Cav-1/小窝的影响。