Pulmonary fibrosis inducer, bleomycin, causes redox-sensitive activation of phospholipase D and cytotoxicity through formation of bioactive lipid signal mediator, phosphatidic acid, in lung microvascular endothelial cells.
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. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating phospholipase D (PLD) and generating the bioactive lipid signal-mediator phosphatidic acid (PA) in our established bovine lung microvascular endothelial cell (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (5, 10, and 100 µg) and time-dependent (2-12 hours) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 µg) were significantly attenuated by the thiol protectant (N-acetyl-L-cysteine), 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 happen upstream of bleomycin-induced cytotoxicity in BLMVECs, which was protected by FIPI. This was also supported by our current findings that exposure of cells to exogenous PA led to internalization of PA and cytotoxicity in BLMVECs. For the first time, this study revealed novel mechanism of the bleomycin-induced redox-sensitive activation of PLD that led to the generation of PA, which was capable of inducing lung EC cytotoxicity, thus suggesting possible bioactive lipid-signaling mechanism/mechanisms of microvascular disorders encountered in IPF.
Lipid Signaling, Lipidomics, and Vasculotoxicity Laboratory, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, 43210, USA.
Rishi B
RB
Patel
Sainath R
SR
Kotha
Shariq I
SI
Sherwani
Sean M
SM
Sliman
Travis O
TO
Gurney
Brooke
B
Loar
Susan O'Connor
SO
Butler
Andrew J
AJ
Morris
Clay B
CB
Marsh
Narasimham L
NL
Parinandi
D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D001761:Bleomycin / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D004294:Domperidone / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D054990:Idiopathic Pulmonary Fibrosis / Q000139:chemically induced* / Q000201:enzymology / Q000473:pathology; D007211:Indoles / Q000494:pharmacology; D008168:Lung / Q000098:blood supply; D055806:Microvessels / Q000166:cytology / Q000187:drug effects; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010712:Phosphatidic Acids / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
C546435:5-fluoro-2-indolyldeschlorohalopemide; D000903:Antibiotics, Antineoplastic; D007211:Indoles; D010712:Phosphatidic Acids; D017382:Reactive Oxygen Species; D001761:Bleomycin; D004294:Domperidone; D010739:Phospholipase D