Cytotoxic phospholipid oxidation products. Cell death from mitochondrial damage and the intrinsic caspase cascade

Abstract

Phospholipid oxidation products accumulate in the necrotic core of atherosclerotic lesions, in apoptotic cells, and circulate in oxidized low density lipoprotein. Phospholipid oxidation generates toxic products, but little is known about which specific products are cytotoxic, their receptors, or the mechanism(s) that induces cell death. We find the most common phospholipid oxidation product of oxidized low density lipoprotein, phosphatidylcholine with esterified sn-2-azelaic acid, induced apoptosis at low micromolar concentrations. The synthetic ether phospholipid hexadecyl azelaoyl phosphatidylcholine (HAzPC) was rapidly internalized, and overexpression of PLA2g7 (PAF acetylhydrolase) that specifically hydrolyzes such oxidized phospholipids suppressed apoptosis. Internalized HAzPC associated with mitochondria, and cytochrome c, and apoptosis-inducing factor escaped from mitochondria to the cytoplasm and nucleus, respectively, in cells exposed to HAzPC. Isolated mitochondria exposed to HAzPC rapidly swelled and released cytochrome c and apoptosis-inducing factor. Other phospholipid oxidation products induced swelling, but HAzPC was the most effective and was twice as effective as its diacyl homolog. Cytoplasmic cytochrome c completes the apoptosome, and activated caspase 9 and 3 were present in cells exposed to HAzPC. Irreversible inhibition of caspase 9 blocked downstream caspase 3 activation and prevented apoptosis. Mitochondrial damage initiated this apoptotic cascade, because overexpression of Bcl-X(L), an anti-apoptotic protein localized to mitochondria, blocked cytochrome c escape and apoptosis. Thus, exogenous phospholipid oxidation products target intracellular mitochondria to activate the intrinsic apoptotic cascade.

Figure 1. The oxidized phospholipid HAzPC induces apoptosis

A. HL60 cells were incubated with the stated concentrations of synthetic HAzPC for 6 h, or exposed to 1 mW/cm² UVB (not shown) for 5 min as a positive control for apoptosis. The cells were stained for surface phosphatidylserine with fluorescent Alexa Fluor₄₈₈-annexin V as described in “Methods.” Flow cytometry for the intensity of annexin V fluorescence on the x-axis vs cell number was normalized to the maximal number of cells in a channel in order to overlay the plots. This experiment is representative of three independent experiments. B. HepG2 cells were exposed to HAzPC at the stated concentrations before the level of Annexin V staining determined as in panel A.

Figure 2. HAzPC internalized by HL60 cells is cytotoxic

A. Time-dependent internalization of HAzPC. HAzPC (5 μM) was added to suspended HL60 cells and the cells recovered by centrifugation at the stated times. The cells were washed once with 0.5% human serum albumin, an [²H]PAF internal standard was added and the lipids were extracted and purified by reversed phase HPLC. HAzPC was determined by tandem mass spectrometry as the molecular cation [M+H⁺] of m/z 652 producing a phosphocholine daughter ion of m/z 184. The average of duplicate values is shown, and this experiment is representative of three experiments. B. Exogenous HAzPC accumulates in mitochondria. HL60 cells (7 × 10⁸) were incubated with or without 5 μM HAzPC for 20 min at room temperature. The cells were washed once in RPMI containing 0.5% human serum albumin and once in RPMI before the cells were suspended in 10 ml EB medium (200 mM D-mannitol, 70 mM sucrose, 20 mM Hepes (pH 7.4), 1 mM EGTA and 100 μM Pefabloc.) and then mechanically homogenized. Homogenates were centrifuged (2000 × g, 5 min) to remove unbroken cells. The resulting lysates were centrifuged (9500 × g, 10 min) and the mitochondrial pellets were resuspended in the same volume of buffer. Lipids were extracted from these duplicate samples and the content of HAzPC determined by HPLC-MS/MS as described in “Methods”. A separate experiment showed the mitochondrial fraction contained no endoplasmic reticulum associated calnexin by western blotting, and that the mitochondrial recovery, visualized by blotting adenine nucleotide translocase, was incomplete. Therefore, the relative amount of HAzPC recovered in the mitochondrial fraction is an underestimate. C. HL60 cells expressing PAF acetylhydrolase are protected from HAzPC toxicity. HL60 cells stably expressing the oxidized phospholipid phospholipase PLA2g7 (plasma PAF acetylhydrolase) or its vector control were treated with 5 μM HAzPC for 6 h and stained with Alexa Fluor₄₈₈-annexin V as in Fig. 1.

Figure 3. Mitochondria are compromised in cells exposed to HAzPC

Mitochondria were visualized with MitoTracker RED, a cationic dye accumulated in energized mitochondria, by culturing HUVEC on chamber slides, treating the cells with buffer (A) or with 5 μM HAzPC (B) for 30 min, and then adding 25 nM MitoTracker Red for an additional 30 min before the cells were fixed and visualized by fluorescence microscopy.

Figure 4. Mitochondrial proteins escape the mitochondrial compartment in cells exposed to HAzPC

AIF immunocytochemistry. HepG2 (A) or HUVEC (B) cells adhering to a glass surface were treated with 10 μM HAzPC in complete DMEM for 16 h. The cells were then permeabilized and stained with mouse anti-apoptosis inducing factor (AIF), Alexa Fluor₄₈₈ labeled anti-mouse and DAPI. The punctate mitochondrial green fluorescence of control cells becomes dispersed in the cytoplasm after HAzPC exposure, with many cells showing co-localization of AIF and the blue nuclear DAPI stain. Cytochrome C immunoblot. HL60 cells (C) or HepG2 cells (D) were treated with 5 μM HAzPC for 4 h, the cells recovered by centrifugation and mechanically lysed before cytoplasmic components were separated from mitochondria by centrifugation. Proteins in the cytoplasmic fraction were denatured, resolved by SDS-PAGE, transferred to a solid support, and then sequentially probed with anti-cytochrome C and anti-β-actin monoclonal antibodies. Each panel is representative of three independent experiments.

Figure 5. Phospholipid oxidation products cause mitochondrial swelling

A. Isolated mitochondria were incubated with the stated lipids at a concentration of 2.5 μM for 10 min and the change in light scattering determined as stated in “Methods.” The maximal swelling was defined as that induced by calcium in the presence of alamethicin. The abbreviations are: LPA, lysophosphatidic acid; PGPC, palmitoyl glutaroyl phosphatidylcholine; POVPC, palmitoyl oxovaleroyl phosphatidylcholine; c-PAF, cabamoyl-PAF; PAF, platelet-activating factor (hexadecyl acetyl phosphatidylcholine); C₄PAF, hexadecyl butyroyl phosphatidylcholine; CCCP, carbonyl cyanide 3-chlorophenylhydrazone. B. Effect of the sn-1 bond on mitochondrial swelling. Isolated mitochondria were treated with the ether lipid HAzPC or the diacyl homolog palmitoyl azelaoyl phosphatidylcholine (PAzPC) and swelling determined as in panel A. C. Cyclosporin A minimally affects HAzPC-induced swelling. Isolated mitochondria were pre-treated with 680 nM cyclosporin A at room temperature to interfere with flow through the mitochondrial permeability transition pore before swelling in response to 2.5 μM HAzPC was determined as before. D. Transmission electron microscopy. Electron micrographs of rat liver mitochondria treated with 5 μM lysophosphatidylcholine or HAzPC, or exposed to 5 mM CaCl₂ and 7 μg/ml alamethicin for 10 min. The bar is 1 μmeter in length.

Figure 6. Pro-apoptotic proteins are released from mitochondria exposed to HAzPC

Isolated mitochondria were treated or not with cyclosporin A for 5 min and then with the stated concentration of HAzPC for 10 min. Mitochondria were then separated from soluble material by centrifugation and the amount of AIF and cytochrome C in the supernatant was determined by western blotting as defined under “Methods.” The center lane, labeled “control”, was the amount of the two target proteins in detergent solubilized mitochondria at the start of the experiment.

Figure 7. HAzPC activates caspases of the intrinsic apoptotic cascade

A. Intracellular activated caspase 9. HL60 cells were exposed to 5 μM HAzPC for 4 h as before and then incubated with the fluorescent substrate and pro-irreversible caspase 9 inhibitor FAM-LEHD-fmk. The cells were cultured at 37° for an additional hour, washed and the amount of caspase 9-bound dye determined by single channel flow cytometry. B. The bar graph presents the average and standard error of three independent experiments. C. Active caspase 3 fragments accumulate in cells exposed to HAzPC. HL60 cells were exposed to the stated concentration of HAzPC for 4 h, lysed before the presence of 19 and 17 kDa caspase 3 fragments was assessed by western blotting. D. HAzPC induces caspase 3 enzymatic activity in a caspase 9-dependent fashion. HL 60 cells were treated for 1 h with buffer or the pan caspase inhibitor zVAD-fmk or zLEHD-fmk to inhibit caspase 9 and then incubated with buffer or 5 μM HAzPC for 4 h before cell lysates were prepared and assayed for caspase-3 activity. Each panel, including panel B, is representative of three independent experiments.

Figure 8. Caspases of the intrinsic apoptotic cascade are required for HAzPC-induced apoptosis

HL60 cells were treated with the caspase inhibitor zVAD-fmk or the caspase 9 specific inhibitor zLEHD-fmk, and with 5 μM HAzPC or maintained in buffer for 6 h. The cells were then stained with annexin V and the resulting cellular fluorescence analyzed by flow cytometry as in Figure 1. This experiment is representative of two other independent experiments.

Figure 9. Mitochondrial protection by Bcl-X_L blocks HAzPC-induced apoptosis

A. Bcl-X_L expression. HL60 cells stably over-expressing human Bcl-X_L were immunoblotted for total cellular Blc-X_L and actin. B. Cytochrome C loss from isolated mitochondria. Mitochondria were isolated from Bcl-X_L over-expressing cells, treated or not with HAzPC, and loss of cytochrome C to the supernatant was determined as in Figure 6. C. Bcl-X_L expression suppresses HAzPC-induced apoptosis. Surface display of phosphatidylserine was quantitated by flow cytometry using Alexa₄₈₈-conjugated annexin V as in Figure 1. Each panel is representative of three independent experiments. This is an arm of the experiment in Fig. 2B, so the buffer and HAzPC histograms are the same.