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

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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.