Mechanics of fibrosis in 3D biomimetic extracellular matrices

 纤维化是许多重大疾病的核心组成部分，在发达国家，估计有45%的死亡与纤维化有关。纤维化的标志包括肌成纤维细胞（MF）诱导、过度MF增殖和基质合成以及细胞外基质（ECM）的硬化或收缩，但这些变化如何相互关联和反馈以加强纤维化过程尚不清楚。这部分是因为我们缺乏对细胞如何感知、机械响应以及在纤维化期间如何改变其周围环境的结构和力学的基本理解。为了填补我们知识的空白并开发针对纤维化机制的治疗方法，我们需要实验平台，使我们能够1）准确捕获组织的纤维结构和机械行为，以及2）监测驱动MF分化，增殖和随后的纤维化硬化的机械力。拟议工作的总体重点是在MF诱导和增殖的启动（K99）和强化（R 00）期间连接细胞内和细胞外力学。在K99阶段，我们将开发一种技术来测量新建立的合成ECM中的牵引力，该合成ECM是纤维状的、机械可调的，并且可以被细胞重组。使用这个平台，我们将检查TGF-β是否诱导RhoA介导的原纤维重组，以增加牵引力的产生和随后的MF诱导和增殖。了解MF诱导和增殖是如何启动的，我们将继续研究增加MF细胞密度如何加强R 00期的纤维化变化。我们将确定高密度收缩性MF产生的纤维状ECM内的张力是否刺激进一步的MF诱导。接下来，我们将研究高MF密度通过ECM合成对纤维状网络硬化的影响，并询问这是否反过来也促进MF诱导。为了了解细胞的物理环境，细胞内信号传导和细胞牵引力产生之间的相互作用，这项工作将依赖于生物材料工程，分子生物学和有限元建模方法。虽然申请人在生物材料开发方面具有丰富的经验，并且已经建立了拟用于拟议研究的合成纤维状ECM，但他需要在肌成纤维细胞生物学、机械建模和牵引力测量开发方面进行进一步培训。该奖项和组建的指导团队提供的支持和培训将为申请人提供对其未来独立研究计划至关重要的工具和专业知识。此外，这项工作将揭示伴随许多疾病的纤维化变化所共有的生物物理机制，并将为可以破坏这一过程的疗法提供试验平台。

 DESCRIPTION (provided by applicant): Project Summary Fibrosis is a central component of numerous major diseases and is implicated in an estimated 45% of all deaths in the developed world. Hallmarks of fibrosis include myofibroblast (MF) induction, excessive MF proliferation and matrix synthesis, and stiffening or contraction of the extracellular matrix (ECM), but how these changes are interrelated and feedback to reinforce the fibrotic process is not understood. This is in part because we lack a fundamental understanding of how cells sense, mechanically respond to, and in turn alter the structure and mechanics of their surroundings during fibrosis. To fill ths gap in our knowledge and develop treatments that target the mechanisms of fibrosis, we need experimental platforms that allow us to 1) accurately capture the fibrous structure and mechanical behavior of tissues, and 2) monitor the mechanical forces that drive MF differentiation, proliferation, and subsequent fibrotic stiffening. The overall focus of the proposd work is to connect intracellular and extracellular mechanics during the initiation (K99) and reinforcement (R00) of MF induction and proliferation. During the K99 phase, we will develop a technique to measure traction forces in a newly established synthetic ECM that is fibrous, mechanically tunable, and can be reorganized by cells. Using this platform, we will examine whether TGF-ß induces RhoA-mediated fibril reorganization to increase traction force generation and subsequent MF induction and proliferation. With an understanding of how MF induction and proliferation is initiated, we will go on to examine how increased MF cell density reinforces fibrotic changes during the R00 phase. We will determine whether tension within the fibrillar ECM generated by high densities of contractile MFs spurs further MF induction. Next, we will examine the effect of high MF density on fibrillar network stiffening via ECM synthesis, and query whether this in turn also promotes MF induction. To understand the interplay between the physical surroundings of the cell, intracellular signaling, and cellular traction generation, this work will rely on biomaterial engineering, molecular biology, and finite element modeling approaches. While the applicant has significant experience in biomaterial development and has already established the synthetic fibrillar ECM to be employed in the proposed studies, he requires further training in myofibroblast biology, mechanical modeling, and the development of traction force measurements. The support and training provided by both this award and the assembled mentoring team will grant the applicant tools and expertise critical to his future independent research program. Additionally, this work will shine light on biophysical mechanisms common to fibrotic changes accompanying numerous diseases, and will provide a test bed for therapeutics that can disrupt this process.