Novel Protective Antiapoptotic Action of Alpha 1-Antitrypsin In Emphysema

项目1：肺泡细胞凋亡代表了一个主要的病理生物学过程， 肺气肿中肺泡隔的丧失和组织破坏。我们的总体概念是 SCCOR的建议是，炎症和凋亡细胞破坏的相互作用，由于破坏了细胞周期， 肺细胞的维持决定了慢性阻塞性肺疾病中肺破坏的程度 疾病（COPD）。尽管α-1型糖尿病患者肺气肿发展的流行模式 抗胰蛋白酶（A1 AT）缺乏强调A1 AT作为弹性蛋白酶抑制剂、丝氨酸蛋白酶抑制剂 丝氨酸蛋白酶抑制剂（Serpins），包括A1 AT，具有超出其作为蛋白酶的经典作用的更广泛的生物学作用 抑制剂的我们的目标是确定A1 AT的新生物学作用，这可能会增强我们对A1 AT的理解。 肺泡细胞损伤导致肺气肿的发病机制。我们假设A1 AT阻止了 肺气肿通过结合和激活caspase-3，导致肺泡保护， 细胞凋亡是肺气肿肺泡破坏的关键步骤。这个基础科学项目依靠一个 涉及肺气肿和腺相关血管内皮生长因子受体阻断模型的综合方法 体内人A1 AT的病毒转导，以及使用细胞培养物和细胞- 自由系统来探测A1 AT和caspase-3的相互作用 肺气肿肺的实验评估，基于标准化的形态测量学，肺成像， 肺功能检查（在分子病理生理学核心D的支持下），以及与以下相关的终点 凋亡、氧化应激和促凋亡脂质神经酰胺。我们的具体目标是：（1）证明 A1 AT通过阻断细胞凋亡来防止小鼠肺气肿的发展， 氧化应激和神经酰胺水平。(2)为了确定A1 AT是否保护肺内皮细胞， 通过直接细胞内抑制caspase-3的活化来防止细胞凋亡;以及（3）为了鉴定是否在细胞凋亡中起作用， 肺气肿患者，A1 AT的翻译后改变（氧化、亚硝基化或聚合） 削弱A1 AT的抗凋亡作用。发现新的细胞内进入机制和活性 α-1抗胰蛋白酶，并可能提供一个机会，改善我们的治疗方法，在α 1 抗胰蛋白酶缺乏和吸烟引起的肺气肿。我们将与项目4密切合作， 确定发育中的肺生命中的香烟烟雾肺损伤是否损害了 A1AT。我们计划将我们对A1 AT新的抗凋亡作用的机制见解转化为对A1 AT的研究。 COPD患者A1 AT翻译后修饰的研究（项目2）和暴露 环境颗粒（与项目5），其可使A1 AT对活性半胱天冬酶-3无活性。

PROJECT 1: Alveolar cell apoptosis represents one of the major pathobiological processes that account for the loss of alveolar septae and tissue destruction in emphysema. Our overall concept underlying this SCCOR proposal is that the interaction of inflammation and apoptotic cell destruction due to disruption of lung cellular maintenance determines the magnitude of lung destruction in chronic obstructive pulmonary disease (COPD). Although the prevailing paradigm of emphysema development in patients with alpha-1 antitrypsin (A1AT) deficiency emphasizes the role of A1AT as an elastase inhibitor, serine protease inhibitors (Serpins), including A1 AT, have broader biological effects beyond their classical action as protease inhibitors. It is our goal to identify novel biological roles of A1 AT that might enhance our understanding of the pathogenesis of alveolar cell injury leading to emphysema. We hypothesize that A1 AT prevents emphysema by binding to and inhibitingactive caspase-3, leading to alveolar protection against apoptosis, a critical step of alveolar destruction in emphysema. This basic science project relies onan integrated approach involving the VEGF receptor blockade model of emphysema and adeno-associated virus transduction of human A1AT in vivo, and on focused mechanistic studies using cell cultures and cell- free systems to probe for the interaction of A1 AT and caspase-3.We have developed state of the art experimental assessment of emphysema lungs, based on standardized morphometry, lung imaging, pulmonary function tests (with the support of Molecular Pathophysiology Core D), and end points related to apoptosis, oxidative stress, and the proapoptotic lipid ceramide. Our specific aims are (1) To demonstrate that A1AT prevents the development of emphysema in mice by blocking apoptosis, and thus reducing oxidative stress and ceramide levels. (2) To determine whether A1 AT protects pulmonary endothelial cells from apoptosis by a direct intracellular inhibition of caspase-3 activation; and (3) To identify whether in patients with emphysema, post-translational alterations in A1AT (oxidation, nitrosylation, or polymerization) impair the anti-apoptotic effect of A1 AT. Discovery of novel mechanisms of intracellular entry and activity of alpha-1 antritrypsin and may provide an opportunity improve our therapeutic approaches in alpha 1 antitrypsin deficiency- and smoking-induced emphysema. We will collaborate closely with Project 4 to determine whether cigarette smoke lung injury in the developing lung life impairs antiapoptotic functions of A1AT. We plan to translate our mechanistic insights of the novel antiapoptotic actions of A1AT in the investigation of posttranslational modifications of A1AT in patients with COPD (with Project 2) and exposure to environmental particulates (with Project 5) that may render A1 AT inactive against active caspase-3.