Ceramide-induced lung destruction in emphysema

描述（由申请人提供）：我们的实验室以前发现神经酰胺在肺气肿的发展中起着重要作用。我们证明，神经酰胺是细胞凋亡、氧化应激及其自身合成的放大的近端枢纽，并且是诱导肺气肿的基本特征-肺空间扩大和肺弹性功能降低的必要和充分条件。在本申请中，我们提出了新的概念，即香烟烟雾（CS）暴露破坏肺中的鞘脂体内平衡以产生不同的急性和慢性神经酰胺反应，这导致结构性肺泡上皮细胞和内皮细胞的死亡、肺泡巨噬细胞对凋亡细胞的清除的抑制以及维持肺气肿中不可逆的肺破坏的细胞修复的损害。我们假设CS诱导的神经酰胺引发肺泡细胞死亡，损害凋亡小体的适当去除，并破坏细胞修复，由于顺序激活酸性鞘磷脂酶，然后从头神经酰胺合成。我们将通过使用转基因体内方法辅以靶酶的药理学抑制，并通过评估表征香烟烟雾诱导的肺气肿的结构和功能终点来验证这一假设。鞘脂测量及其对肺泡细胞凋亡和凋亡细胞清除的影响将分别通过串联质谱和活体/延时双光子显微镜进行研究。具体目标是：1）确定CS激活的酸性鞘磷脂酶引发小鼠肺泡细胞凋亡并导致肺泡腔扩大。2)确定CS和细胞旁神经酰胺激活肺泡巨噬细胞中的从头途径，从而抑制凋亡小体的清除。3)阐明CS是否通过旁分泌激活从头神经酰胺合成导致肺泡细胞凋亡和肺修复减少。我们的实验问题和方法将解决肺气肿发病机制的基本问题，并将为COPD患者制定治疗策略提供理论依据和基础。

DESCRIPTION (provided by applicant): Our laboratory previously discovered a central role for ceramide in the development of emphysema. We demonstrated that ceramide is a proximal hub of amplification for apoptosis, oxidative stress, and for its own synthesis, and is both necessary and sufficient to induce airspace enlargement and functional decreases of lung elastance, fundamental characteristics of emphysema. In this application we address the novel concept that cigarette smoke (CS) exposure disrupts sphingolipid homeostasis in the lung to generate distinct acute and chronic ceramide responses, responsible for the death of structural alveolar epithelial and endothelial cells, inhibition of clearance of apoptotic cells by alveolar macrophages, and impairment of cell repair that sustain an irreversible lung destruction in emphysema. We hypothesize that CS-induced ceramides trigger alveolar cell death, impair proper removal of apoptotic bodies, and disrupt cell repair due to sequential activation of acid sphingomyelinase followed by de novo ceramide synthesis. We will test this hypothesis by using transgenic in vivo approaches complemented with pharmacological inhibition of target enzymes and by assessing structural and functional endpoints that characterize cigarette smoke-induced emphysema. Sphingolipid measurements and their effect on alveolar apoptosis and clearance of apoptotic cells will be studied by tandem mass spectrometry and intravital/time-lapse two-photon microscopy, respectively. The specific aims are: 1) To determine that the CS-activated acid sphingomyelinase triggers alveolar cell apoptosis and causes airspace enlargement in mice. 2) To establish that CS and paracellular ceramides activate the de novo pathway in alveolar macrophages causing inhibition of apoptotic body removal. 3) To elucidate if the paracrine activation of de novo ceramide synthesis causes alveolar cell apoptosis and decreased lung repair by CS. Our experimental questions and approach will address fundamental questions of emphysema pathogenesis and will provide the rationale and basis for developing a therapeutic strategy for patients with COPD.