Targeting durotaxis in lung injury and fibrosis

项目摘要/摘要 特发性肺纤维化(IPF)是一种以肺部进行性疤痕为特征的致命肺部疾病， 最终会阻碍呼吸能力。成纤维细胞在组织损伤部位的病理性募集 随后激活成瘢痕形成肌成纤维细胞是瘢痕疙瘩发生发展的关键步骤。 肺纤维化。相应地，指导成纤维细胞募集的分子介质的鉴定 而肌成纤维细胞的活化，不仅将进一步提高我们对肺的发病机制的认识 但也为新的抗纤维化治疗提供了合理的治疗靶点。我们和其他人有 最近发现纤维化肺中基质硬度的增加促进了成纤维细胞的机械激活。 此外，基质僵硬通过将僵硬激活的肌成纤维细胞锁定在机械支架上而放大组织纤维化。 正反馈循环，由不完全了解的机制。我们最近有证据表明，矩阵刚性 纤维化肺组织产生的梯度通过趋杜性促进成纤维细胞向组织损伤部位的募集。 细胞从低硬度区域到高硬度区域的定向迁移，这与扩散无关 化学诱导剂或底物结合的触觉线索。随着成纤维细胞对僵硬增加的区域的“硬化税”， 这些区域的僵硬会促使到达的成纤维细胞分化为肌成纤维细胞。 因此，这一提议的中心假设是：(1)成纤维细胞被招募到病灶部位 通过趋性作用造成的组织损伤，在这种机制中，细胞独立于 趋化信号；以及(2)抑制成纤维细胞趋向性有可能成为一种新的治疗方法 IPF的战略。本申请中提出的研究旨在观察成纤维细胞在体内的趋向性， 明确成纤维细胞趋化的分子机制并开发新的治疗策略以抑制 多药趋向性。具体地说，我们建议：(1)在体外使用多光子显微镜对成纤维细胞的趋向性进行成像 肺切片精切及体内靶向成纤维细胞趋化作用的研究 通过抑制机械敏感的FAK/paxlin途径建立小鼠肺纤维化模型；(2)确定 α-v-β-3/FAK/帕西林通路调节特发性肺间质纤维化中基质刚性感受和趋化作用的机制 成纤维细胞。我们将研究αvβ3整合素的生化和生物物理调节及其在 成纤维细胞趋向性；以及(3)确定肌动蛋白-微管串扰介导成纤维细胞的机制 多药趋向性。具体来说，我们将研究α-TAT1介导的微管乙酰化的机制 控制αvβ3/FAK/巴西林复合体在耐药细胞中的动态循环。我们还将测试 αTAT-1在博莱霉素肺纤维化模型中的体内成纤维细胞趋向性和肺纤维化中的作用 本实验室培育的成纤维细胞特异性αTAT-1 KO小鼠。此应用程序中建议的实验 将提供关于成纤维细胞趋化在肿瘤的发生和发展中作用的新知识。 肺纤维化，以及抗耐药疗法治疗肺纤维化的潜在疗效。

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.