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.
Int J Toxicol. 2011 Feb;30(1):69-90. doi: 10.1177/1091581810388850. Epub 2010 Dec 3.
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.
肺微血管并发症和肺动脉高压的机制与特发性肺纤维化（IPF）有关，特发性肺纤维化是一种使人虚弱的肺部疾病，但目前尚不清楚这些机制。因此，我们研究了广泛用于特发性肺纤维化诱导的博来霉素是否能够在我们建立的牛肺微血管内皮细胞（BLMVEC）模型中激活磷酯酶 D（PLD）并产生生物活性脂质信号介质磷酸酰基肌醇（PA）。我们的结果表明，博来霉素以剂量依赖性（5、10 和 100μg）和时间依赖性（2-12 小时）方式诱导 PLD 激活和 PA 生成，PLD 特异性抑制剂 5-氟-2-吲哚基去氯哈罗匹肽（FIPI）显著减弱了这种作用。博来霉素（5μg）诱导的 PLD 激活和 PA 生成明显被巯基保护剂（N-乙酰-L-半胱氨酸）、抗氧化剂和铁螯合剂减弱，这表明活性氧（ROS）、脂质过氧化和铁在其中的作用。此外，我们的研究表明，博来霉素处理后细胞中形成了 ROS 并损失了谷胱甘肽（GSH），证实氧化应激是博来霉素诱导的 PLD 激活和 EC 中 PA 生成的关键因素。更值得注意的是，在 BLMVEC 中，PLD 激活和 PA 生成发生在博来霉素诱导的细胞毒性之前，而 FIPI 可以保护其免受博来霉素的影响。这也得到了我们目前的发现的支持，即细胞暴露于外源性 PA 会导致 PA 的内化和 BLMVEC 中的细胞毒性。本研究首次揭示了博来霉素诱导的氧化还原敏感的 PLD 激活的新机制，导致 PA 的产生，PA 能够诱导肺 EC 毒性，从而提示了特发性肺纤维化中微血管障碍可能涉及的生物活性脂质信号机制/机制。
