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

基本信息

批准号：
10669074
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
项目类别：
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/10669074
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 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 biomaterial compatibility bisulfite sequencing blood pump cycloaddition design fabrication 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

项目摘要

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.

项目概要尽管纤维化在衰老和疾病引起的组织功能障碍中发挥着普遍的作用，但没有代表性的研究纤维化微环境的体外模型存在纤维化的特征是细胞外基质过多。 (ECM) 沉积使细胞微环境变硬，因此，为了模拟体外纤维化，需要进行细胞培养。然而，允许定量、动态调整基质力学的基质是必要的。动态水凝胶培养平台通常依赖于可能对细胞有毒或同时具有毒性的化学物质更改多个参数，使得很难分配矩阵属性之间的因果关系纤维化硬化发生在多种组织中，包括骨骼肌、肝脏、许多遗传性心肌病的特征是进行性纤维化硬化。虽然已知纤维化硬化会损害心脏泵血的能力，但它的影响心肌细胞僵硬对表型的影响仍知之甚少。建议开发一种基于动态水凝胶生物材料的组织纤维化体外模型，该模型能够实时测量细胞功能障碍，以确定进行性纤维化僵硬的痛苦程度作为一个模型系统，我们将探讨硬化对人类心肌细胞的影响。与杜氏肌营养不良症 (DMD) 患者的诱导多能干细胞分化而来。是研究由外向内机械信号传递的理想模型系统，因为 DMD 是由于缺乏肌营养不良蛋白而产生的，连接收缩细胞骨架和 ECM 的结构蛋白我们将使用开发的动态水凝胶。在这项研究中评估收缩功能障碍、机械转导信号的异常激活，以及纤维化硬化产生的“机械记忆”的新分子机制。在目标 1 中，我们将开发一种使用近红外光和生物正交的合成水凝胶系统动态硬化凝胶的反应，模拟纤维化，这些水凝胶将用于确定如何进行。收缩功能障碍是由纤维化僵硬引起的。在目标 2 中，我们将确定硬度增加如何改变。心肌细胞中的生化信号传导，重点关注通过 Rho 的“规范”机械转导 GTPases 和 YAP 信号传导以及主动收缩细胞中的新机械敏感途径，涉及活性氧 (ROS) 的机械生成、DNA 损伤和线粒体生物合成受损。在目标 3 中，我们将研究心肌细胞中“机械记忆”的第一个例子。水凝胶平台被一种波长的光硬化，随后被第二种波长软化。该系统将能够识别暴露于硬化环境的分子机制。微环境会导致持续的细胞功能障碍和逆转这种记忆的策略。开发的平台将广泛用于研究进行性遗传疾病和衰老中的纤维化。