Dynamic Biomaterial Design to Probe the Cellular Response to Fibrotic Stiffening

项目总结 尽管纤维化在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 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)使细胞微环境变硬的沉积。因此，要在体外建立肝纤维化模型，细胞培养 允许定量、动态地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对细胞有害的化学物质 更改多个参数，使得在更改的矩阵属性之间分配因果关系变得困难 细胞的命运就会改变。纤维性硬化发生在广泛的组织中，包括骨骼肌、肝脏、 肺，还有心脏。许多遗传性心肌病的特点是进行性纤维化僵硬， 先于心力衰竭。虽然纤维化硬化已知会损害心脏泵血的能力，但 对单个心肌细胞表型的僵硬仍然知之甚少。这项研究的目的是 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，使 实时测量细胞功能障碍以确定进行性纤维化硬化是如何有害的 影响细胞的命运。作为一个模型系统，我们将询问僵直对人类心肌细胞的影响 从Duchenne肌营养不良症(DMD)患者的诱导多能干细胞分化而来。DMD是 一个研究自外向内机械信号的理想模型系统，因为DMD是由于缺乏dystrophin， 连接收缩细胞骨架和细胞外基质的结构蛋白。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 纤维性硬化引起的“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。 反应使凝胶动态变硬，模仿纤维化。这些水凝胶将被用来确定 收缩功能障碍是由纤维硬化引起的。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，两者都集中在通过Rho的“典型”机械转导 GTP酶和YAP信号，并在主动收缩细胞中的一条新的机械敏感途径上，涉及 机械产生的活性氧物种(ROS)，DNA损伤，并损害线粒体的生物发生。 在目标3中，我们将研究心肌细胞中的第一个“机械记忆”例子。我们将开发一种 一种水凝胶平台，被一种波长的光加固，随后被另一种波长软化。 该系统将能够识别暴露于僵硬的 微环境导致持续的细胞功能障碍和逆转这种记忆的策略。精心设计的 开发的平台将广泛用于研究进行性遗传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.