The Role of TGF-Beta in Acute Lung Injury

描述(申请人提供)：我们希望确定导致急性肺损伤(ALI)发生的分子机制。这种破坏性的临床综合征的特征是形成肺水肿--肺泡内充满富含蛋白质的渗出物。最近，我们实验室已确定细胞因子转化生长因子-β(TGF-β)可能是ALI的关键介质；抑制转化生长因子-β可阻止小鼠肺水肿的发展。其他研究人员已经表明，转化生长因子-β在体外直接增加内皮细胞的通透性。我们已经发现，转化生长因子-β通过与细胞内谷胱甘肽(GSH)的耗竭有关的机制直接增加上皮的通透性。我们推测局部激活的转化生长因子-β通过对肺泡内皮细胞和内皮细胞的特异性作用而介导了与ALI相关的肺水肿的发展。此外，我们假设转化生长因子-β通过改变细胞内硫醇氧化还原状态来改变内皮细胞和上皮细胞连接蛋白的磷酸化状态。连接蛋白的磷酸化会影响连接的完整性，从而影响细胞旁通透性的程度。我们将使用转化生长因子-β绿色荧光蛋白报告鼠在体内识别转化生长因子-β刺激的单个常驻上皮细胞和内皮细胞。上皮细胞和内皮细胞单层将被用来评估转化生长因子-β对细胞旁通透性的影响。将测定还原型和氧化型谷胱甘肽，以确定硫醇氧化还原状态。将测定上皮和内皮连接蛋白的磷酸化状态，并鉴定特定蛋白。与大多数其他潜在的治疗ALT的方法不同，只有在损伤开始之前才能有效地使用转化生长因子-β抑制，即使在损伤开始后也能有效地降低ALT(以前在我们的实验室中显示)。因此，识别转化生长因子-β影响上皮和/或内皮细胞的特定分子机制可能导致治疗ALI的新的和改进的策略，ALI是一种常见的临床综合征，通常是致命的。拟议的研究还将帮助我发展和获得必要的技能，以实现我成为一名成功和独立的肺部疾病科学家的最终目标。

DESCRIPTION (provided by applicant): We wish to identify molecular mechanisms leading to the development of acute lung injury (ALI). This devastating clinical syndrome is characterized by formation of pulmonary edema--alveolar flooding with a protein-rich exudate. Recently, our laboratory has identified the cytokine transforming growth factor-Beta (TGF-Beta) as a possible critical mediator in ALI; inhibition of TGF-Beta prevents the development of pulmonary edema in murine models. Other investigators have shown that TGF-Beta directly increases endothelial permeability in vitro. We have found that TGF-Beta directly increases epithelial permeability by a mechanism that is associated with depletion of intracellular glutathione (GSH). We hypothesize that locally activated TGF-Beta mediates the development of pulmonary edema associated with ALI by exerting specific effects on resident epithelial and endothelial cells. Furthermore, we hypothesize that TGF-Beta alters the phosphorylation state of endothelial and epithelial cell junction proteins by mechanisms involving changes in intracellular thiol redox status. Junction protein phosphorylation would affect junction integrity, and therefore, the degree of paracellular permeability. We will use TGF-Beta green fluorescent protein reporter mice to identify individual resident epithelial and endothelial cells that are stimulated by TGF-Beta in vivo. Epithelial and endothelial cell monolayers will be used to assess TGF-Beta effect on paracellular permeability. Reduced and oxidized GSH will be measured to determine thiol redox status. Epithelial and endothelial junction protein phosphorylation state will be determined and specific proteins identified. In contrast to most other potential treatments for ALT, which are effective only if administered before the onset of injury, TGF-Beta inhibition effectively attenuates ALT even after the onset of injury (previously shown in our laboratory). Therefore, identification of specific molecular mechanisms by which TGF-Beta affects epithelial and/or endothelial cells could lead to novel and improved strategies to treat ALI, a common and often lethal clinical syndrome. The proposed studies will also help me develop and acquire the necessary skills to achieve my ultimate goal of becoming a successful and independent scientist in pulmonary disease.