Phospholipase D Signaling Mediates Reactive Oxygen Species‐Induced Lung Endothelial Barrier Dysfunction

Abstract

Reactive oxygen species (ROS) have emerged as critical players in the pathophysiology of pulmonary disorders and diseases. Earlier, we have demonstrated that ROS stimulate lung endothelial cell (EC) phospholipase D (PLD) that generates phosphatidic acid (PA), a second messenger involved in signal transduction. In the current study, we investigated the role of PLD signaling in the ROS-induced lung vascular EC barrier dysfunction. Our results demonstrated that hydrogen peroxide (H2O2), a typical physiological ROS, induced PLD activation and altered the barrier function in bovine pulmonary artery ECs (BPAECs). 1-Butanol, the quencher of PLD, generated PA leading to the formation of physiologically inactive phosphatidyl butanol but not its biologically inactive analog, 2-butanol, blocked the H2O2-mediated barrier dysfunction. Furthermore, cell permeable C2 ceramide, an inhibitor of PLD but not the C2 dihydroceramide, attenuated the H2O2-induced PLD activation and enhancement of paracellular permeability of Evans blue conjugated albumin across the BPAEC monolayers. In addition, transfection of BPAECs with adenoviral constructs of hPLD1 and mPLD2 mutants attenuated the H2O2-induced barrier dysfunction, cytoskeletal reorganization and distribution of focal adhesion proteins. For the first time, this study demonstrated that the PLD-generated intracellular bioactive lipid signal mediator, PA, played a critical role in the ROS-induced barrier dysfunction in lung vascular ECs. This study also underscores the importance of PLD signaling in vascular leak and associated tissue injury in the etiology of lung diseases among critically ill patients encountering oxygen toxicity and excess ROS production during ventilator-assisted breathing.

Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury

“…PLD1 was expressed by multiple cell types in the lung, and played important roles in processes central to the development of ALI, such as endothelial barrier function, clearance of alveolar edema, and neutrophil activation. PLD signaling results in ROS‐mediated endothelial cytoskeletal dysfunction . PLD1 activation by TGF‐beta results in internalization of the epithelial sodium channel ENaC, which prevents clearance of alveolar edema and potentiates lung injury .…”

Inhibited Insulin Signaling in Mouse Hepatocytes Is Associated with Increased Phosphatidic Acid but Not Diacylglycerol

“…Anti-IRS1, anti-Flag M2, and anti-␣-tubulin antibodies, bovine serum albumin (fatty acid free), insulin (human recombinant), sodium-D-lactate, phorbol 12-myristate 12-acetate (PMA), Avertin (2-2-2-tribromoethanol), phosphatase inhibitor cocktails 1 and 2, Percoll, ATP, and CHAPS were from Sigma. Anti-PLD2 antibody was made as described (20). Type I collagenase was from Worthington Biochemical Corporation.…”

Neutrophil‐mediated vascular barrier injury: Role of neutrophil extracellular traps

“…ROS produced by N-formylmethionyl-leucyl-phenylalanine (fMLP)-activated neutrophils increase microvessel permeability 36 ; similarly, lung vascular endothelial cells exposed to H 2 O 2 display increased paracellular permeability to albumin 37 . Conversely, neutrophil-induced endothelial barrier disruption is diminished by the application of antioxidants 38 .…”