Biosynthesis of oxidized lipid mediators via lipoprotein-associated phospholipase A2 hydrolysis of extracellular cardiolipin induces endothelial toxicity

Abstract

We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.

Keywords: cardiolipin; lineolic acid; lipoprotein-associated phospholipase A2; octadecadienoic acid; pulmonary endothelium.

Fig. 1.

Alternative biosynthesis of lipid mediators. The canonical pathway of eicosanoids and other known lipid mediator biosynthesis by PLA₂ hydrolysis followed by enzymatic oxidation of the released free fatty acids (left) is presented in contrast to the alternative pathway (right) of first oxidation of an unsaturated lipid (CL in this example) followed by enzymatic hydrolysis. This alternative pathway represents biosynthesis of lipid mediators through pathways of nontraditional enzymatic targets. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol.

Fig. 2.

In vitro oxidation and hydrolysis of tetralinoleoyl CL. A cell-free in vitro system was developed with CL oxidation by the azo initiator, AAPH. Subsequently the CLox was hydrolyzed by the Ca⁺⁺-independent Lp-PLA₂, producing monolyso- and dilyso- (not shown) CL species, as well as oxygenated free fatty acids.

Fig. 3.

EPR reveals the presence of free ascorbyl radicals generated by HBSS in a cell-free system. EPR spectra shown of a cell-free in vitro system containing HBSS were recorded 10 and 20 s after addition of 25 μM ascorbate (A), then metal chelator, DTPA (100 μM; B). C shows the addition of the free radical generator AAPH (5 mM) to 25 μM ascorbate, then addition of 100 μM DTPA (D), followed by the addition of 100 μM BHT (E).

Fig. 4.

BHCL in the presence of AAPH produced multiple oxidation products as shown by LC-MS/MS. LC-MS/MS was performed on the reduced form on BHCL (A) and BHCL + AAPH (B). A indicates 2 CL clusters at m/z 1,469 (minor), 1,447 (major). B indicates multiple CL clusters, the largest at m/z 1,447, 1,469, 1,479, 1,495 and 1,511, 1,527, 1,543, and 1,559. There was a decrease in the major m/z 1,447 CL species and increases in species at the other m/z displayed in gray representing various oxidized forms of CL with or without adducts. Each of these oxidized forms of CL having an additional oxygen molecule added was determined by the m/z increase of 16.

Fig. 5.

Lp-PLA₂ hydrolyzes BHCL only after oxidation. LC-MS profiles of monolysoCL and dilysoCL species in experimental conditions of BHCL with or without AAPH and subsequent hydrolysis by Lp-PLA₂ are shown (top). LC-MS comparison of molar percentage of intact tetralinoleoyl CL (bottom, left) and monolysoCL (MCL) species, dilysoCL (DCL) species, and free fatty acids (FFA) (bottom, right) are shown for experimental conditions of BHCL with or without AAPH and subsequent hydrolysis by Lp-PLA₂ (bottom, left).

Fig. 6.

Identification of free fatty acid products. LC-MS was performed on experimental conditions of BHCL with or without AAPH and subsequent hydrolysis by Lp-PLA₂. Molar percentages of the various free fatty acids products are shown (top). LC-MS base profiles of only a few of the formed oxygenated free fatty acids, m/z 327, 311, 295, and 293 are shown in the conditions of BHCL + AAPH and BHCL + AAPH with addition of Lp-PLA₂ after treatment with DTPA and BHT to quench Fe and AAPH free radicals (bottom).

Fig. 7.

AAPH toxicity to BPAECs. BPAEC survival after exposure to 0–10 mM AAPH. Viability of 10,000 BPAECs was determined by alamarBlue assay.

Fig. 8.

Survival of BPAECs after exposure to BHCL, AAPH, and Lp-PLA₂. BPAECs (10,000 cells) were exposed to BHCL with or without Lp-PLA₂ plus 5 mM of AAPH and subsequent addition of Lp-PLA₂ in HBSS for 4 h, and cell viability was determined by alamarBlue assay.

Fig. 9.

Lp-PLA₂ exacerbates necrotic cell death in the presence of oxidized CL. Representative flow cytometry plots of annexin V and propidium iodide staining for cell death are shown for control and 2-h exposure of 10 nmol/100 μl doses of BHCL, Lp-PLA₂, CLox, and CLox + Lp-PLA₂ in BPAECs (top). Bar graph representation of percentage of cells in Q1 (viable) and Q3 (necrotic) for all experiments (bottom). FACS, fluorescence-activated cell sorting.

Fig. 10.

Lp-PLA₂ exacerbated the AAPH-treated BHCL decrease in transendothelial cell electrical resistance (increase monolayer permeability). BPAECs (30,000 cells) were plated and grown to a confluent monolayer and exposed to BHCL in HBSS for 1 h, and permeability was determined by ECIS array experiments run with the 8W10E+ polyethylene terephthalate sterile disposable electrode array, with or without a nontoxic dose of AAPH with or without 2,000 ng/ml Lp-PLA₂. Thrombin was used as a positive control for an increase in monolayer permeability (green line). Data from each trial were averaged at specific time points and then plotted vs. time with ± error bars reflecting standard errors from the trials. *P < 0.05.

Fig. 11.

Lp-PLA₂ hydrolysis products cause decrease in transendothelial cell electrical resistance (increase monolayer permeability). ECIS experiments for a few of the commercially available Lp-PLA₂ hydrolysis products we identified (purple, orange, and blue lines) were performed. *P < 0.05.

Fig. 12.

CLox and monolysoCL are present in Pseudomonas aeruginosa infection. LC-MS profiles of plasma from control and mice 4 h after Pseudomonas aeruginosa infection are shown for CL (A), CLox (B), and monolysoCL (C) with chemical structures of the hypothesized Lp-PLA₂ hydrolysis pathway on the left.

Fig. 13.

CLox molar percentage is increased in Pseudomonas aeruginosa infection. Molar percentage of CLox/(CLox + CL) was calculated from plasma of control and mice 4 h after Pseudomonas aeruginosa infection.