Pulmonary fibrosis inducer, bleomycin, activates phospholipase D and generates bioactive lipid signal mediator, phosphatidic acid in lung microvascular endothelial cells

Abstract

The mechanisms of lung microvascular complications and pulmonary hypertension known to be associated with idiopathic pulmonary fibrosis (IPF), a debilitating lung disease, are not known. The bioactive lipid signal mediator, phosphatidic acid (PA), generated by phospholipase D (PLD), is emerging as a key player in vascular endothelial dysfunctions. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating PLD and generating PA in our established bovine lung microvascular EC (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (0-100 μg) and time-dependent (2-12 h) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 μg) were significantly attenuated by the thiol protectant (N-acetylcysteine), antioxidants, and iron chelators, suggesting the role of reactive oxygen species (ROS), lipid peroxidation, and iron therein. Furthermore, our study demonstrated the formation of ROS and loss of glutathione (GSH) in cells following bleomycin treatment, confirming oxidative stress as a key player in the bleomycin-induced PLD activation and PA generation in ECs. More noticeably, PLD activation and PA generation were observed to occur upstream of cytotoxic response (cell morphology alterations or lactate dehydrogenase release) in BLMVECs, which was protected by FIPI. Overall, this study revealed novel information that bleomycin induced the redox-sensitive activation of PLD leading to the generation of PA, capable of inducing lung EC cytotoxicity, thus suggesting possible mechanism(s) of microvascular disorders encountered in IPF.