Dynamic Biomaterial Design to Probe the Cellular Response to Fibrotic Stiffening

项目摘要 尽管纤维化在由衰老和疾病引起的组织功能障碍中普遍存在，但没有代表性的研究表明， 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量 (ECM)沉积，使细胞微环境变硬。因此，为了在体外建立纤维化模型，细胞培养 需要允许定量、动态调节基质力学的基质。但现有 动态水凝胶培养平台通常依赖于可能对细胞有毒或同时 改变多个参数，使得难以在改变的基质属性之间分配因果关系 细胞命运也会改变纤维化硬化发生在广泛的组织中，包括骨骼肌，肝脏， 肺和心脏许多遗传性心肌病的特征在于进行性纤维化硬化， 会导致心力衰竭虽然已知纤维化硬化会损害心脏泵血的能力，但 个体心肌细胞表型的硬化仍然知之甚少。本研究的目的 一项提案是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， 细胞功能障碍的真实的时间测量，以确定渐进性纤维化硬化是如何渐进性地 影响细胞命运作为一个模型系统，我们将探讨硬化对人类心肌细胞的影响 从来自杜氏肌营养不良症（DMD）患者的诱导多能干细胞分化。DMD是 一个理想的模型系统，用于研究由外向内的机械信号，因为DMD是由于缺乏肌营养不良蛋白， 连接收缩性细胞骨架和ECM的结构蛋白。我们将使用动态水凝胶开发 在这项研究中，评估收缩功能障碍，机械传导信号的异常激活， 纤维化硬化引起的“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， 动态地使凝胶变性的反应，模拟纤维化。这些水凝胶将用于确定如何 收缩功能障碍由纤维化硬化引起。在目标2中，我们将确定增加的刚度如何改变 心肌细胞中的生化信号传导，集中在通过Rho的“典型”机械转导 GTPases和雅普信号传导以及在主动收缩细胞中的一种新的机械敏感性途径， 活性氧（ROS）的机械产生、DNA损伤和受损的线粒体生物发生。 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。我们将开发一个 水凝胶平台通过一种波长的光硬化，随后通过第二种波长软化。 该系统将能够识别分子机制，通过该机制，暴露于硬化的 微环境导致持续的细胞功能障碍和逆转这种记忆的策略。述工程化 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。

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.