Alcohol and E-Cigarette Activate P2X7R in Lung Epithelial Cells, Stimulating Release of Plasminogen Activator in Extracellular Vesicles Injuring Blood-Brain Barrier

Chronic exposure to ethanol (ETH) and e-cigarette vapor activates the P2X7 receptor (P2X7R) in human pulmonary alveolar epithelial cells (hPAEpiCs), promoting extracellular vesicle (EV) release. Proteomic analysis of this EVs identified enrichment of urokinase-type plasminogen activator (uPA), a key extracellular matrix remodeling enzyme. Treatment of hPAEpiCs with ETH, acetaldehyde (ALD), and e-cigarette vapor, with or without nicotine, induced NLRP3 inflammasome activation, IL-1β secretion, and increased incorporation of uPA into EVs. Exposure of human brain microvascular endothelial cells (hBMVECs) to this EVs enhanced plasmin conversion, activated MMP-9 and TGF-β1, resulting in reduced transendothelial electrical resistance (TEER), indicating blood-brain barrier (BBB) dysfunction. Pharmacological inhibition of P2X7R using ‘A804598’ significantly reduced EV-associated uPA release and attenuated endothelial injury. In vivo, chronic ETH vapor exposure increased expression of uPA, uPAR, MMP-2, and MMP-9 while decreasing SERPIN1 and TIMP1 in murine brain microvessels, changes associated with BBB disruption and IgG leakage into brain parenchyma. P2X7R inhibition normalized these effects. Collectively, these findings identify EV-associated uPA as a mediator of lung-to-brain signaling and BBB injury following ETH and e-cigarette exposure.