A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse Lung

Abstract

1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A 2 (PLA 2 ) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA 2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02-0.5 mmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67-87% of the injected dose for i.t. and 23-42% for i.v. administration at 4 hours postinjection. PLA 2 activity of lung homogenates was markedly inhibited (.85%) at 4 hours postadministration. Both MJ33 content and PLA 2 activity gradually returned to near control levels over the subsequent 24-72 hours. Mice treated with MJ33 at 12.5-25 mmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30-to 50-day observation period. Thus, the toxic dose of MJ33 was .25 mmol/kg, whereas the PLA 2 inhibitory dose was approximately 0.02 mmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium

“…plus horseradish peroxidase (HRP; 50 g/ml) was added to the perfusate; this fluorophore does not permeate the cell membrane and thus detects extracellular H 2O2. ANG II (50 M) was added to the lung perfusate, aliquots of the perfusate were removed during 15-to 30-min intervals, fluorescence intensity of the samples was measured (excitation/emission, 545/610) using a spectrofluorimeter (Photon Technology, Birmingham, NJ), and H 2O2 concentration was expressed as arbitrary fluorescence units (7,26). ROS generation was measured with WT and PECAM-1 Ϫ/Ϫ lungs; NOX2 Ϫ/Ϫ lungs were used as a negative (no ROS) control.…”

NOX2 Activation of Natural Killer T Cells Is Blocked by the Adenosine A2A Receptor to Inhibit Lung Ischemia–Reperfusion Injury

“…Ischemia-reperfusion (IR) injury after lung transplantation imposes a significant threat to graft and recipient survival (1). IR injury involves a complex cascade of events, including oxidative stress, inflammation, and cellular interactions that can lead to primary graft dysfunction, which significantly increases the risk for rejection and long-term graft dysfunction (2).…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…Thus Prdx6 appeared to have a major role in NOX2 activation. To evaluate the role of Prdx6 in cells, mouse alveolar macrophages obtained by lung lavage were stimulated with N-formyl-methionyl-leucyl-phenylalanine, pulmonary microvascular endothelial cells (PMVEC) were stimulated with ANG II, and PMN isolated from bone marrow were stimulated with concanavalin A, all resulting in ROS generation with wild-type cells, but markedly attenuated ROS production in cells isolated from Prdx6 null mice (15,57). To confirm that the PLA 2 activity of Prdx6 was responsible for activation of NOX2, null PMVEC were transfected with constructs that lead to 1) expression of both activities of Prdx6 (essentially recreating the wild type); 2) expression of only the PLA 2 activity; or 3) expression of only the peroxidase activity of Prdx6.…”

“…MJ33 would seem ideal for this purpose, since it inhibits the PLA 2 activity of Prdx6 but has no effect on its important anti-oxidant peroxidase activity. We determined that treatment of perfused lung or isolated cells (PMVEC, alveolar macrophage, PMN) with MJ33 essentially prevented ROS production in response to their respective agonists for NOX2 activation (15,57). To establish dosimetry for mice, MJ33 was administrated in mixed unilamellar liposomes, either intratracheally or intravenously (57); 4 nmol in a 20-g mouse was sufficient to inhibit lung aiPLA 2 activity by 80% for 24 h (Fig.…”

“…MJ33 is essentially a nonreactive compound: it inhibits PLA 2 activity by binding to the enzyme, not by chemical reactivity. We administered ϳ2,000-fold the dose of MJ33 that effectively inhibits lung aiPLA 2 activity and evaluated mice during a 7-wk observation period (57). There was no significant effect on physical activity level, body weight, hematocrit, or histology of the lung, liver, or kidney.…”