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核心作用。我们证明神经酰胺是细胞凋亡、氧化应激和自身合成的近端放大中枢，并且是诱导肺泡扩大和肺弹性功能降低的必要条件和充分条件，而肺弹性是肺气肿的基本特征。在这项应用中，我们提出了一个新的概念，即香烟烟雾(CS)暴露破坏了肺中鞘磷脂的动态平衡，从而产生不同的急性和慢性神经酰胺反应，导致结构性肺泡上皮细胞和内皮细胞死亡，抑制肺泡巨噬细胞对凋亡细胞的清除，以及维持肺气肿肺不可逆破坏的细胞修复受损。我们推测，CS诱导的神经酰胺引起肺泡细胞死亡，损害了凋亡小体的正常清除，并由于酸性鞘磷脂酶的顺序激活而扰乱了细胞修复，随后又合成了神经酰胺。我们将通过使用体内转基因方法，辅以靶标酶的药物抑制，并通过评估香烟烟雾诱导的肺气肿的结构和功能终点来检验这一假说。鞘磷脂的测量及其对肺泡细胞凋亡和凋亡细胞清除的影响将分别用串联质谱仪和活体/时移双光子显微镜进行研究。其具体目的是：1)确定CS激活的酸性鞘磷脂酶能触发小鼠肺泡细胞的凋亡并导致肺泡扩张。2)证实CS和细胞旁神经酰胺可激活肺泡巨噬细胞的从头通路，从而抑制凋亡体的清除。3)阐明新生神经酰胺合成的旁分泌激活是否导致肺泡细胞凋亡，从而降低CS所致的肺损伤修复。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，并将为开发COPD患者的治疗策略提供理论基础和基础。

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.