Targeting Matrix Stiffness in Lung Fibrosis Associated with Aging

衰老与人类和实验性肺纤维化的发展有关 动物。细胞外基质(ECM)硬化是肺纤维化的一个显著特征。互动 在硬化的纤维化ECM和组织纤维化的效应细胞之间，称为肌成纤维细胞(MFBs)，提供了一种 支持/放大肺纤维化的前馈机制。靶向基质僵硬以打破纤维化 反馈环是治疗持续性/进展性肺纤维化的一种有前景的策略。这个 目前的研究旨在测试一种针对肺纤维化的基质去僵化疗法的概念验证。 随着年龄的增长。先前的研究表明，晚期糖基化终末产物(AGEs)的形成 随着年龄的增长而积累，并在肺纤维化中加速发生。细胞外基质，特别是胶原蛋白 基质，由于其缓慢的周转速度，对糖基化非常敏感。糖基化终末产物推动非酶交联 胶原蛋白纤维。分子内和分子间胶原交联物的形成是硬化的关键因素。 ECM。在初步研究中，我们以AGE介导的糖基化交联为靶点，用于基质去僵化和 博莱霉素诱导的增龄相关肺纤维化的潜在抗纤维化治疗 老鼠模型。我们发现，抗衰老治疗减少了交联型肺胶原蛋白的数量， 降低肺僵硬，促进老龄小鼠实验性肺纤维化的消退。我们确认了 小鼠双分钟4(MDM4)，P53的主要内源性抑制因子，作为基质刚性调节因子 机械敏感分子。降低基质硬度可下调MDM4的表达，导致P53的去表达。 抑制/激活。P53功能增强通过上调Fas诱导肺微血管内皮细胞对细胞凋亡的敏感性 释放CX3CL1趋化因子的MFBs募集巨噬细胞并表达死亡的免疫原性转化 结构域1α(DD1α)吞噬受体以促进巨噬细胞介导的对凋亡的MFBs的吞噬。 这些发现表明，通过靶向非酶AGE交联的基质去僵化激活了 纤维化消解途径。当前项目的中心假设是矩阵去僵化传感通过 MDM4可促进肺MFB清除和逆转衰老相关的未消退肺纤维化。 具体目标是：(1)确定基质硬度调节MDM4表达的机制；(2) 确定MDM4对基质硬度的感知是否通过激活一种蛋白来介导肺微纤维的清除 P53介导的涉及Fas、CX3CL1和Dd1α的基因程序；以及(3)确定机械敏感性的作用 MDM4在小鼠肺微纤维细胞命运决定和衰老相关肺纤维化逆转中的作用。这个 拟议的研究将阐明逆转衰老相关的细胞和分子机制 靶向非酶AGE交联的肺纤维化。这一假设，如果得到证实，将建立一个证据- 抗衰老相关肺纤维化的基质去僵化治疗的概念。

Aging is associated with the development of nonresolving pulmonary fibrosis in both human and experimental animals. Stiffening of the extracellular matrix (ECM) is a prominent feature of lung fibrosis. Interactions between stiffened fibrotic ECM and effector cells of tissue fibrosis, known as myofibroblasts (MFBs), provide a feedforward mechanism that sustains/amplifies lung fibrosis. Targeting matrix stiffness to break the profibrotic feedback loop represents a promising strategy for treatment of persistent/progressive lung fibrosis. The current study aimed to test a proof-of-concept of matrix de-stiffening therapy against lung fibrosis associated with aging. Previous studies have shown that the formation of advanced glycation end-products (AGEs) accumulates with aging and occurs at an accelerated rate in lung fibrosis. The ECM, in particular collagen matrix, is highly susceptible to glycation due to its slow turnover rate. AGEs drives nonenzymatic crosslinking of collagen fibers. The formation of intra- and inter-molecular collagen crosslinks is a crucial factor that stiffens the ECM. In preliminary studies, we targeted AGE-mediated glycation crosslinking for matrix de-stiffening and potential anti-fibrotic therapy against aging-associated nonresolving lung fibrosis in a bleomycin injury-induced mouse model. We found that anti-AGE treatment reduces the amount of crosslinked lung collagens, decreases lung stiffness, and promotes the resolution of experimental lung fibrosis in aged mice. We identified mouse double minute 4 (MDM4), a major endogenous inhibitor of p53, as a matrix stiffness-regulated mechanosensitive molecule. Reducing matrix stiffness downregulates MDM4 expression, resulting in p53 de- repression/activation. Gain of p53 function sensitizes lung MFBs to apoptosis by upregulating Fas and induces immunogenic conversion of MFBs that release CX3CL1 chemokine to recruit macrophages and express Death Domain 1α (DD1α) engulfment receptor to facilitate macrophage-mediated phagocytosis of apoptotic MFBs. These findings suggest that matrix de-stiffening by targeting nonenzymatic AGE crosslinking activates a fibrosis resolution pathway. The central hypothesis of the current project is that matrix de-stiffening sensing by MDM4 promotes lung MFB clearance and the reversal of aging-associated nonresolving pulmonary fibrosis. Specific aims are: (1) determine the mechanisms by which matrix stiffness regulates MDM4 expression; (2) determine whether matrix stiffness sensing by MDM4 mediates the clearance of lung MFBs by activation of a p53-directed gene program involving Fas, CX3CL1 and DD1α; and (3) determine the role of mechanosensitive MDM4 in the fate decisions of lung MFBs and the reversal of aging-associated pulmonary fibrosis in mice. The proposed study will elucidate cellular and molecular mechanisms involved in the reversal of aging-associated lung fibrosis by targeting nonenzymatic AGE crosslinking. The hypothesis, if proven, will establish a proof-of- concept of matrix de-stiffening therapy against aging-associated nonresolving pulmonary fibrosis.