Contribution of Macropinocytosis in fibroblast activation and systemic sclerosis

项目概要/摘要 这项R21提案描述了一项为期两年的研究计划，该计划将促进致力于以下方面的研究计划： 确定巨胞饮在系统性硬化症（SSc，硬皮病）和SSc相关间质性硬化症中的作用 肺疾病（ILD）。SSc是一种自身免疫性疾病，其表现为皮肤纤维化、血管病变和 内脏纤维化。估计SSc的患病率为每百万人50至300人， 美国是世界上患病率最高的国家之一，为每百万人240人。由于其 由于临床异质性，SSc是一种难以管理的疾病，导致疾病死亡率最高 在风湿性疾病中。ILD是SSc最常见的肺部并发症， 增加死亡率。高达80%的SSc患者发生ILD，25-30%的患者发生重度进展性 导致最终呼吸衰竭和死亡的SSc-ILD。知之甚少的分子 参与SSc和SSc-ILD发病机制。本项目将着重阐明 巨胞饮作用在皮肤和肺纤维化中的作用，以及其治疗靶向。巨胞饮作用是 一种肌动蛋白依赖但网格蛋白非依赖的内吞过程，介导非选择性的 细胞外内容物，如蛋白质、细胞碎片或病毒。巨胞饮作用已被证明参与 通过提供营养物（例如，游离氨基酸和多肽）， 细胞外环境然而，其在硬皮病和器官纤维化中的作用尚不清楚。我们的初步 研究结果表明，抑制巨胞饮作用会减弱真皮肌成纤维细胞的分化， 小鼠肺纤维化。此外，我们发现液泡蛋白分选34（Vps 34），一个唯一的成员， III类磷酸肌醇-3-激酶（PI 3 K）参与巨胞饮作用和成纤维细胞活化。基于 根据我们的初步研究结果，我们假设巨胞饮作用的增加促进了 SSc中真皮和肺纤维化的发展。因此，它的抑制在肝纤维化中发挥抗纤维化作用。 通过减少活化的成纤维细胞中的促纤维化反应来制备皮肤纤维化动物模型。我们也 假设Vps 34对于巨胞饮作用是必需，巨胞饮作用又赋予成纤维细胞活化。 我们将通过解决以下具体目标来测试我们的假设：具体目标#1：证明 巨胞饮抑制通过抑制成纤维细胞向肌成纤维细胞转化减轻小鼠皮肤和肺纤维化 分化、ECM产生和迁移。具体目标#2：证明Vps 34是一种关键蛋白， 大胞饮体的形成，并有助于皮肤和肺成纤维细胞的促纤维化反应。

PROJECT SUMMARY/ABSTRACT This R21 proposal describes a two-year research plan that will facilitate research program dedicated to determining the role of macropinocytosis in systemic sclerosis (SSc, scleroderma) and SSc-associated interstitial lung disease (ILD). SSc is an autoimmune disorder, which is manifested by skin fibrosis, vasculopathy and the fibrosis of internal organs. The prevalence of SSc is estimated to range from 50 to 300 per million across the world, with one of the highest prevalence rates observed in the United States at 240 per million. Because of its clinical heterogeneity, SSc is a challenging disease to manage which results in the highest disease mortality among the rheumatologic diseases. ILD is the most common lung complication of SSc and is associated with increased mortality. ILD occurs in up to 80% of patients with SSc, with 25–30% developing a severe progressive form of SSc-ILD leading to eventual respiratory failure and death. Little is known about the molecular mechanisms involved in the pathogenesis of SSc and SSc-ILD. This project will focus on elucidating the key roles of macropinocytosis in skin and lung fibrogenesis, as well as its therapeutic targeting. Macropinocytosis is an actin-dependent but clathrin-independent endocytic process that mediates the nonselective internalization of extracellular contents, such as proteins, cell debris, or viruses. Macropinocytosis has been shown to be involved in cell survival, migration and invasion by providing nutrients (e.g., free amino acids and polypeptides) from extracellular environment. However, its role in scleroderma and organ fibrogenesis is unknown. Our preliminary findings suggest that inhibition of macropinocytosis attenuates dermal myofibroblast differentiation and pulmonary fibrosis in mice. Furthermore, we found that vacuolar protein sorting 34 (Vps34), a sole member of class III phosphoinositide-3-kinase (PI3K), is involved in macropinocytosis and fibroblast activation. Based on published and our preliminary findings, we hypothesize that increased macropinocytosis promotes the development of dermal and lung fibrosis in SSc. Thus, its inhibition exerts anti-fibrotic effects in the animal model of skin fibrosis by reducing profibrotic responses in activated fibroblasts. We also hypothesize that Vps34 is essential for macropinocytosis, which in turn confers to fibroblast activation. We will test our hypotheses by addressing the following Specific Aims: Specific Aim #1: To demonstrate that macropinocytosis inhibition attenuates dermal and lung fibrosis in mice by inhibiting fibroblast to myofibroblast differentiation, ECM production, and migration. Specific Aim #2: To demonstrate that Vps34 is a key protein in macropinosome formation and contributes to profibrotic responses of dermal and lung fibroblasts.