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

• 批准号: 9040162
• 负责人: ROCKY S TUAN
• 金额: $ 33.85万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 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/9040162
• 关键词: 3-Dimensional; Adherence; Adhesions; Adhesives; Adopted; Adult; Affect; Atomic Force Microscopy; Attention; Behavior; Biological; Biological Assay; Bone Marrow; Brain; Caveolae; Cell Adhesion; Cell Differentiation process; Cell Fractionation; Cell Shape; Cell membrane; Cells; Cellular Morphology; Characteristics; Cholesterol; Cholesterol Homeostasis; Cytoskeletal Modeling; Electron Microscopy; Endocytosis; Event; F-Actin; Flow Cytometry; Fluorescence Polarization; Focal Adhesions; Future; Gelatin; Gene Expression; Goals; Health; Homeostasis; Human; Hyaluronan; Hydrogels; Immunofluorescence Immunologic; Integrins; Knowledge; Ligands; Mechanics; Mediating; Membrane; Membrane Fluidity; Membrane Microdomains; Mesenchymal Stem Cells; Morphology; Neurons; Osteoblasts; Osteocalcin; Osteogenesis; Outcome; Pathway interactions; Pharmaceutical Preparations; Phenotype; Population; Property; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Role; Rupture; Seeds; Shapes; Signal Pathway; Signal Transduction; Small Interfering RNA; Sucrose; Supplementation; Suspension substance; Suspensions; System; Testing; Time; Tissue Engineering; Tissues; Western Blotting; base; caveolin 1; cell behavior; chemical property; design; ethylene dimethacrylate; in vivo; inhibitor/antagonist; knock-down; osteogenic; physical property; regenerative therapy; response; scaffold; soft tissue; stem cell differentiation; tissue regeneration

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.

摘要