Targeting durotaxis in lung injury and fibrosis

项目总结/摘要 特发性肺纤维化（IPF）是一种致命的肺部疾病，其特征是肺部进行性瘢痕形成， 最终阻碍呼吸的能力。成纤维细胞向组织损伤部位的病理性募集， 随后活化成瘢痕形成肌成纤维细胞是 肺纤维化因此，指导成纤维细胞募集的分子介质的鉴定 和肌成纤维细胞活化，不仅将进一步提高我们对肺的发病机制的认识， 纤维化，而且还为新的抗纤维化疗法提供合理的治疗靶点。我们和其他人已经 最近显示，纤维化肺中增加的基质硬度促进成纤维细胞的机械活化。 此外，基质刚度通过将刚度激活的肌成纤维细胞锁定在机械张力上而放大组织纤维化。 正反馈循环，通过不完全理解的机制。我们最近有证据表明基质硬度 在纤维化肺组织中产生的梯度促进成纤维细胞通过硬组织扩张募集到组织损伤部位， 细胞从较低硬度区域向较高硬度区域的定向迁移，其发生与扩散无关。 化学引诱物或底物结合的触触趋线索。作为成纤维细胞“durotax”增加刚度的区域， 这些区域的硬度将驱动到达的成纤维细胞分化成肌成纤维细胞。 因此，该建议的中心假设是：（1）成纤维细胞被募集到局灶性的部位， 硬旋转引起的组织损伤，这是一种细胞不依赖于 趋化性信号;和（2）成纤维细胞硬旋转抑制具有成为新的治疗剂的潜力 森林小组战略。在本申请中提出的研究被设计为在体内可视化成纤维细胞硬旋转， 确定成纤维细胞硬旋转的分子机制，并开发新的治疗策略， 硬脊膜扩张具体而言，我们提出：（1）使用多光子显微镜对成纤维细胞硬旋转进行离体成像， 精确切割肺切片，并在体内研究靶向成纤维细胞硬旋转的治疗效果。 通过抑制机械敏感性FAK/Paxillin通路建立小鼠肺纤维化模型;（2） αvβ3/FAK/Paxillin通路调节IPF中基质刚性感知和硬旋转的机制 成纤维细胞我们将研究αvβ3整合素的生物化学和生物物理调节因子及其在细胞凋亡中的作用。 成纤维细胞硬旋转;（3）确定肌动蛋白-微管串扰介导成纤维细胞的机制 硬脊膜扩张具体而言，我们将研究α-TAT 1介导的微管乙酰化的机制， 控制αvβ3/FAK/桩蛋白复合物在durotactic细胞中的动态再循环。我们还将测试 α达特-1在肺纤维化的博来霉素模型中体内成纤维细胞硬旋转和肺纤维化中的作用， 成纤维细胞特异性α达特-1 KO小鼠。本申请中提出的实验 将提供新的知识，关于成纤维细胞硬旋转的发展和进展的作用， 肺纤维化，以及抗durotactic疗法治疗肺纤维化的潜在功效。

Project Summary/Abstract Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease characterized by progressive scarring of the lungs, ultimately impeding the ability to breathe. Pathological recruitment of fibroblasts to sites of tissue injury and subsequent activation into scar-forming myofibroblasts are critical steps in the development and progression of pulmonary fibrosis. Accordingly, the identification of the molecular mediators directing fibroblast recruitment and myofibroblast activation, will not only further enhance our understanding of the pathogenesis of lung fibrosis, but also provide rational therapeutic targets for novel anti-fibrotic therapies. We and others have recently shown that increased matrix stiffness in fibrotic lungs promotes mechano-activation of fibroblasts. Further, matrix stiffness amplifies tissue fibrosis by locking stiffness-activated myofibroblasts on a mechanical positive feedback loop, by not fully understood mechanisms. We have recent evidence that matrix stiffness gradients produced in fibrotic lung tissues promote fibroblast recruitment to sites of tissue injury via durotaxis – the directed migration of cells from regions of lower to higher stiffness, which occurs independently of diffusible chemoattractants or substrate-bound haptotactic cues. As fibroblast “durotax” to regions of increased stiffness, the stiffness of these regions would drive the arriving fibroblasts to differentiate into myofibroblasts. Consequently, the central hypotheses of this proposal are that: (1) fibroblasts are recruited to sites of focal tissue injury via durotaxis, a mechanism in which cells migrate up stiffness gradients independently of chemotactic signals; and (2) that inhibition of fibroblast durotaxis has the potential to be a new therapeutic strategy for IPF. The studies proposed in this application are designed to visualize fibroblast durotaxis in vivo, to define molecular mechanisms of fibroblast durotaxis and to develop novel therapeutic strategies to inhibit durotaxis. Specifically, we propose: (1) To image fibroblast durotaxis ex vivo using multiphoton microscopy in precision cut lung slices and to investigate the therapeutic efficacy of targeting fibroblast durotaxis in vivo in a mouse model of lung fibrosis by inhibiting the mechanosensitive FAK/Paxillin pathway; (2) To define mechanisms by which the αvβ3/FAK/Paxillin pathway regulates matrix rigidity sensing and durotaxis in IPF fibroblasts. We will investigate both biochemical and biophysical regulators of αvβ3 integrin and their role in fibroblast durotaxis; and (3) To define mechanisms by which actin-microtubule crosstalk mediates fibroblast durotaxis. Specifically, we will investigate mechanisms by which α-TAT1-mediated microtubule acetylation controls dynamic recycling of αvβ3/FAK/Paxillin complexes in durotactic cells. We will also test the role of αTAT-1 in fibroblast durotaxis and pulmonary fibrosis in vivo in the bleomycin model of lung fibrosis, using fibroblasts-specific αTAT-1 KO mice generated by our laboratory. The experiments proposed in this application will provide new knowledge regarding the role of fibroblast durotaxis in the development and progression of lung fibrosis, and the potential efficacy of anti-durotactic therapy for the treatment of lung fibrosis.