Dynamic Biomaterial Design to Probe the Cellular Response to Fibrotic Stiffening

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 存在纤维化微环境的体外模型。纤维化的特点是细胞外基质过多 (ECM) 沉积使细胞微环境变硬。因此，为了在体外模拟纤维化，细胞培养 允许定量、动态调整基质力学的基质是必要的。然而，现有的 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，使得很难在更改的矩阵属性之间分配因果关系 和细胞命运的改变。纤维化硬化发生在多种组织中，包括骨骼肌、肝脏、 肺和心脏。许多遗传性心肌病的特征是进行性纤维化硬化， 先于心力衰竭。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。本研究的目标 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如何产生不利影响 影响细胞命运。作为模型系统，我们将探讨硬化对人类心肌细胞的影响 与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 是研究由外向内机械信号传导的理想模型系统，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硬化引起的。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，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 损伤和线粒体生物发生受损。 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。我们将开发一个 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。工程化的 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.