Injury of blood brain and alveolar-endothelial barriers caused by alcohol and electronic cigarettes via purinergic receptor signaling

多药滥用(尤其是酒精使用障碍、AUD和吸烟)是已知的个体 损害肺泡-内皮屏障(AEB)和血脑屏障(BBB)。非常 关于电子烟(e-cig)对肺和大脑的损害，人们知之甚少 与澳元相结合。E-Cig已经变得流行，但非常有限的数据表明，他们 导致内皮功能障碍并导致巨噬细胞的促炎表型 肺内皮细胞。虽然众所周知，e-Cig会上瘾，但它们对大脑和 认知本质上是未知的。我们的数据显示，小鼠的慢性e-Cig暴露 血脑屏障通透性增强和神经炎症，一个键的表达减弱 脑内皮细胞葡萄糖转运蛋白和紧密连接蛋白与认知功能受损。 初步数据显示，酒精/e-Cig在体内的联合暴露导致 AEB通透性增强，神经炎症/血脑屏障受损。我们发现 E-Cig和酒精通过包括线粒体在内的相同机制损伤AEB和BBB 功能障碍、钙蓄积和三磷酸腺苷胞外释放 AEB/BBB细胞成分中的嘌呤能受体，即P2X7。使用创新的体外3D AEB和BBB系统以及相关的动物模型，我们检验了BBB和BBB的假设 E-Cig/酒精暴露引起的AEB损伤是通过P2X7受体介导的。在目标1中，我们将 通过以下方法筛选损伤的程度(线粒体功能障碍、钙离子增加和ATP释放) 不同类型e-Cig联合酒精对人脑、肺内皮细胞和肺组织的影响 上皮细胞。然后，我们将使用创新的3D在体外确定死亡的机制 肺和脑微血管构筑、功能分析、线粒体评估 支持BBB和AEB的关键分子的功能和表达。我们将调查 嘌呤能P2X7受体激活在e-Cig/酒精诱导的BBB/AEB中的作用 功能障碍。第二个目标是研究慢性饮酒和e-cig后的体内肺损伤。 Vaping评估AEB通透性、屏障支持分子表达、炎症 反应(免疫组织化学、蛋白/信使核糖核酸、支气管肺泡灌洗)。P2X7基因敲除 (KO)动物将允许解剖这种受体在肺功能障碍中的作用。3号 目的将破译结合体内血脑屏障功能、表达屏障的调节作用 分子和神经炎症。在P2X7 KO小鼠身上进行的相同实验将 确定该通路在中枢神经系统损伤中的重要性。肺损伤标志物与血脑屏障 损害将在血液中进行测量，并与终末器官病理的迹象相关联。

Polydrug abuse (especially alcohol use disorder, AUD, and smoking) are known individually to compromise the lung alveolar-endothelial barrier (AEB) and the blood brain barrier (BBB). Very limited knowledge exists regarding damage in lung and brain due to electronic cigarettes (e-Cig) in combination with AUD. E-Cig have become popular, yet very limited data indicate that they cause endothelial dysfunction and result in a pro-inflammatory phenotype in macrophages and endothelium in lungs. While e-Cig are known to be addictive, their effects on the brain and cognition are essentially unknown. Our data show that chronic e-Cig exposure in mice enhanced permeability of the BBB and neuroinflammation, diminished expression of a key glucose transporter and tight junction protein on brain endothelium, and impaired cognition. Preliminary data indicate that the combination of alcohol/e-Cig exposure in vivo caused enhanced AEB permeability and amplified neuroinflammation/BBB compromise. We found that e-Cig and alcohol impair AEB and BBB via the same mechanism including mitochondrial dysfunction, Ca2+ accumulation, and ATP extracellular release, potentially mediated by purinergic receptor, P2X7, in cellular components of AEB/BBB. Using innovative in vitro 3D systems of AEB and BBB and relevant animal models, we test the hypothesis that BBB and AEB injury in e-Cig/alcohol exposure are mediated through the P2X7 receptor. In aim 1, we will screen the magnitude of injury (mitochondrial dysfunction, Ca2+ increase and ATP release) by various types of e-Cig in combination with alcohol on human brain and lung endothelial and lung epithelial cells. Then, we will define mechanisms of demise using innovative 3D in vitro constructs of lung and brain microvasculature, functional assays, assessment of mitochondrial functions and expression of key molecules supporting BBB and AEB. We will investigate the contribution of activation of the purinergic P2X7 receptor in e-Cig/alcohol induced BBB/AEB dysfunction. The 2nd aim will study in vivo lung injury after chronic alcohol feeding and e-Cig vaping evaluating AEB permeability, expression of barrier supporting molecules, inflammatory responses (immunohistochemistry, protein/mRNA, bronchoalveolar lavage). P2X7 knockout (KO) animals will allow dissection of the role of this receptor in pulmonary dysfunction. The 3rd aim will decipher combined in vivo effects of BBB function, expression of barrier-mediating molecules, and neuroinflammation. The same experiments performed in P2X7 KO mice will determine the importance of this pathway in CNS injury. Markers of lung injury and BBB damage will be measured in blood and correlated with signs of end-organ pathology.