Dynamic Biomaterial Design to Probe the Cellular Response to Fibrotic Stiffening

动态生物材料设计探测细胞对纤维化硬化的反应

• 批准号: 10463822
• 负责人: Helen M Blau
• 金额: $ 39.35万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10463822
• 关键词: ATAC-seq; Aging; Architecture; Attention; Biochemical; Biocompatible Materials; Biogenesis; Biological Models; Biophysics; Cardiac Myocytes; Cardiomyopathies; Cell Culture Techniques; Cell Death; Cells; Cessation of life; Chemistry; Chromatin; Chronic; Cicatrix; Contracts; Cues; Cytoskeleton; DNA Damage; DNA Methylation; Defect; Deposition; Disease; Duchenne muscular dystrophy; Dystrophin; Engineering; Epigenetic Process; Exhibits; Exposure to; Extracellular Matrix; Failure; Family; Fibrosis; Fluorescence Microscopy; Formulation; Free Radicals; Functional disorder; Gel; Generations; Genetic Diseases; Goals; Guanosine Triphosphate Phosphohydrolases; Heart; Heart failure; Heritability; Human; Hydrogels; Impairment; In Situ; In Vitro; Individual; Inflammation; Intervention; Knowledge; Lead; Light; Liver; Lung; Measures; Mechanics; Mediating; Memory; Microscopy; Mitochondria; Modeling; Modification; Molecular; Mutation; Organ failure; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Pattern; Phenotype; Production; Property; Proteins; Reaction; Reactive Oxygen Species; Research; Research Proposals; Role; Sarcomeres; Signal Transduction; Skeletal Muscle; Stimulus; Stress; Structural Protein; System; Therapeutic Intervention; Time; Tissue Model; Tissues; Traction Force Microscopy; Work; base; biomaterial compatibility; bisulfite sequencing; blood pump; cycloaddition; design; imprint; in vitro Model; induced pluripotent stem cell; inherited cardiomyopathy; insight; link protein; mechanotransduction; novel; novel strategies; response; rho; rho GTP-Binding Proteins; temporal measurement; tissue regeneration; tissue repair; wound healing

PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including the skeletal muscles, liver, lungs, and heart. Numerous genetic cardiomyopathies are characterized by progressive fibrotic stiffening that precedes heart failure. While fibrotic stiffening is known to impair the heart’s ability to pump blood, the impact of stiffening on the phenotype of individual cardiomyocytes remains poorly understood. The goal of this research proposal is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials that enables real time measurement of cellular dysfunction to determine how progressive fibrotic stiffening detrimentally impacts cell fate. As a model system, we will interrogate the effects of stiffening on human cardiomyocytes differentiated from induced pluripotent stem cells from Duchenne muscular dystrophy (DMD) patients. DMD is an ideal model system for studying outside-in mechanosignaling, as DMD arises from a lack of dystrophin, a structural protein linking the contractile cytoskeleton to the ECM. We will use the dynamic hydrogels developed during this research to assess contractile dysfunction, aberrant activation of mechanotransduction signaling, and novel molecular mechanisms of “mechanical memory” arising from fibrotic stiffening. In Aim 1, we will develop a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to determine how contractile dysfunction arises from fibrotic stiffening. In Aim 2, we will determine how increased stiffness alters biochemical signaling in cardiomyocytes, focusing both on “canonical” mechanotransduction through Rho GTPases and YAP signaling and on a new mechanosensitive pathway in actively contracting cells that involves mechanical generation of reactive oxygen species (ROS), DNA damage, and impaired mitochondrial biogenesis. In Aim 3, we will investigate the first example of “mechanical memory” in cardiomyocytes. We will develop a hydrogel platform that is stiffened by one wavelength of light and subsequently softened by a second wavelength. This system will enable identification of molecular mechanisms by which exposure to a stiffened microenvironment causes persistent cellular dysfunction and strategies to reverse this memory. The engineered platforms developed will be broadly useful for studying fibrosis in progressive genetic diseases as well as aging.

项目摘要 尽管纤维化在衰老和疾病引起的组织功能障碍中无处不在，但没有代表性 存在纤维化微环境的体外模型。纤维化的特征是细胞外基质过多 （ECM）沉积会使细胞微环境变硬。因此，在体外模拟纤维化，细胞培养 允许定量的，动态调整基质力学的底物是必要的。但是，存在 动态水凝胶培养平台通常依赖于可能对细胞有毒或简单的化学物质 更改多个参数，因此很难在更改的矩阵属性之间分配因果关系 和细胞脂肪变化。纤维化僵硬发生在各种组织中，包括骨骼肌，肝脏， 肺和心。许多遗传性心肌病的特征是进行性纤维化僵硬， 在心力衰竭之前。虽然已知纤维化僵硬会损害心脏泵血的能力，但影响 在单个心肌细胞的表型上僵硬仍然了解不足。这项研究的目标 建议是基于动态水凝胶生物材料开发组织纤维化的体外模型 细胞功能障碍的实时测量，以确定渐进的纤维化僵硬如何有害 影响细胞命运。作为模型系统，我们将询问僵硬对人心肌细胞的影响 与杜钦肌营养不良（DMD）患者的诱导多能干细胞区分开。 DMD是 DMD的理想模型系统，用于研究外部机械信号，因为DMD缺乏肌营养不良蛋白，A 将收缩细胞骨架与ECM联系起来的结构蛋白。我们将使用开发的动态水凝胶 在评估收缩功能障碍的这项研究中，机械转导信号的异常激活和 纤维化僵硬引起的“机械记忆”的新型分子机制。 在AIM 1中，我们将开发一种合成水凝胶系统，该系统使用近红外的光和生物正交 动态加强凝胶的反应，模仿纤维化。这些水凝胶将用于确定如何 收缩功能障碍是由纤维化僵硬引起的。在AIM 2中，我们将确定刚度增加如何改变 心肌细胞中的生化信号传导，重点是通过Rho的“规范”机械转移 GTPases和YAP信号传导以及在积极收缩细胞的新机械敏感途径上 活性氧（ROS），DNA损伤和线粒体生物发生受损的机械产生。 在AIM 3中，我们将研究心肌细胞中“机械记忆”的第一个例子。我们将发展一个 水凝胶平台通过一个波长的光线加强，随后通过第二波长软化。 该系统将实现鉴定分子机制，通过该机制暴露于僵硬 微环境会导致持续的细胞功能障碍和策略扭转这种记忆。设计的 开发的平台对于研究进行性遗传疾病和衰老的纤维化将非常有用。