Paraoxonase 2 deficiency alters mitochondrial function and exacerbates the development of atherosclerosis

Abstract

Increased production of reactive oxygen species (ROS) as a result of decreased activities of mitochondrial electron transport chain (ETC) complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. Our previous studies established that paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. The aim of the present study was to determine the mechanism by which PON2 modulates ROS production. In this report, we demonstrate that PON2-def mice on the hyperlipidemic apolipoprotein E(-/-) background (PON2-def/apolipoprotein E(-/-)) develop exacerbated atherosclerotic lesions with enhanced mitochondrial oxidative stress. We show that PON2 protein is localized to the inner mitochondrial membrane, where it is found associated with respiratory complex III. Employing surface-plasmon-resonance, we demonstrate that PON2 binds with high affinity to coenzyme Q(10), an important component of the ETC. Enhanced mitochondrial oxidative stress in PON2-def mice was accompanied by significantly reduced ETC complex I + III activities, oxygen consumption, and adenosine triphosphate levels in PON2-def mice. In contrast, overexpression of PON2 effectively protected mitochondria from antimycin- or oligomycin-mediated mitochondrial dysfunction. Our results illustrate that the antiatherogenic effects of PON2 are, in part, mediated by the role of PON2 in mitochondrial function.

FIG. 1.

Quantification of atherosclerotic lesion formation in PON2-def/apoE^−/− mice. (A) Analysis of aortic root lesions in female apoE^−/− (n = 13) and PON2-def/apoE^−/− (n = 9) mice maintained on a standard chow diet for 16 weeks. (B) Analysis of aortic root lesions in female apoE^−/− (n = 8) and PON2-def/apoE^−/− (n = 11) mice placed on a high-fat Western diet for 16 weeks. (C) En face analysis of atherosclerotic lesions of female apoE^−/− (n = 9) and PON2-def/apoE^−/− (n = 10) mice placed on a high-fat western diet. Horizontal lines represent mean values. Circles represent results obtained from individual mice. Representative aorta from apoE^−/− and PON2-def/apoE^−/− mice are shown on the right panel. *p < 0.05 relative to control. (D) PON2−def/apoE^−/− mice have elevated macrophage content in aortic root sections. Serial 10 μM aortic root sections from apoE^−/− (n = 6) and PON2-def/apoE^−/− (n = 6) mice administered a high-fat Western diet were immunostained for macrophage-specific marker CD68. Data presented as morphometric analysis of stained area of lesions from each group. *p < 0.05 relative to control. PON2, paraoxonase 2; apoE^−/−, apolipoprotein E^−/−. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

Quantification of LOOH in serum LDL and LDL modified by peritoneal macrophages. (A) LDL fractions were isolated from experimental groups, pooled, and lipid extracted with chloroform: methanol and LOOH content quantified. (B) Peritoneal macrophages isolated from apoE^−/− or PON2-def/apoE^−/− mice were incubated with 250 μg of control LDL. Supernatants were collected after 18 h and LOOH levels quantified. *p < 0.05 relative to control on indicated diet. Data represent one of three experiments. LDL, low-density lipoprotein; LOOH, lipid hydroperoxide.

FIG. 3.

Mitochondrial function in PON2-def mice. PON2-def mice and littermate controls were placed on an atherogenic diet. Mitochondria were isolated from the livers, and (A) mitochondrial complex I + III activity and (B) total ATP level was measured from the total liver extract. Values are represented as relative luminescence unit/g tissue. (C) Mitochondrial superoxide quantified. Values are represented as relative fluorescence unit (n = 6). *p < 0.05 relative to control. ATP, adenosine triphosphate; RFU, relative fluorescence units; RLU, relative luminescence units.

FIG. 4.

Mitochondrial oxidative stress in PON2-def mice. PON2-def mice and littermate controls were placed on an atherogenic diet. Mitochondria were isolated from livers. (A) MDA content and (B) reduced glutathione levels were quantified using 100 μg of liver mitochondrial protein. n = 6. *p < 0.05 relative to control. In another set of experiments mitochondria were isolated from female apoE^−/− (n = 4) and PON2-def/apoE^−/− (n = 6) mice maintained on a standard chow diet for 16 weeks. (C) MDA levels and (D) reduced glutathione levels were quantified using 100 μg of liver mitochondrial protein. *p < 0.05 relative to apoE^−/−. MDA, malondialdehyde.

FIG. 5.

Peritoneal macrophages from PON2-def/apoE^−/− mice have impaired mitochondrial function compare to apoE^−/−. Peritoneal macrophages were isolated from PON2 def and control mice. (A) Oxygen consumption rate was measured by using an XF24 Extracellular Flux Analyzer. During this experiment, 1 μM oligomycin, 0.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mixture of 100 nM rotenone and myxothiazol were each sequentially injected as indicated, and the response was monitored. (B) Basal mitochondrial respiration was calculated by subtracting the respiration after rotenone/myxothiazol (complex I and complex III inhibitor) injection from the levels observed before any chemical treatments. (C) Respiration due to oxidative phosphorylation was calculated as the response to oligomycin (ATP synthase inhibitor). (D) Maximal mitochondrial respiratory capacity was deduced from the response to treatment with FCCP (uncoupler). Data shown are mean ± SE (n = 10). *p < 0.05 relative to control. (E, F) Mitochondrial superoxide levels were measured by flowcytometer using mitosox both wild and PON2-def/apoE^−/−results were represented (G) and quantitative data expressing mean fluorescence intensity of mitosox. p < 0.05 relative to control. Mean ± SD (n = 3). (H) ATP level was measured as described in the Materials and Methods section, and values were represented as relative luminescence unit (2 × 10⁶ cells). p < 0.05 relative to control. Mean ± SD (n = 3). (I) Mitochondrial superoxide was measured in the aortic region as described in the Materials and Methods. Mean ± SD (PON2-def/apoE^−/− n = 3, apoE^−/− n = 5). *p < 0.05 relative to control. RFU, relative fluorescence unit; RLU, relative luminescence unit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Localization of PON2 in mitochondrial and submitochondrial particles. (A) Total and mitochondrial protein extracts (50 μg) isolated from HeLa cells were subjected to SDS-PAGE. Western blot analyses were performed using anti-PON2, anti-Histone, and anti-Calnexin antibodies. Histone and Calnexin were used as markers for nuclear and endoplasmic reticulum membranes, respectively. (B) Submitochondrial particles were isolated from C57BL/6J mouse livers, and protein (50 μg) from outer and inner membrane preparations was subjected to SDS-PAGE. Western blot analyses were performed using indicated primary antibodies. VDAC and COX IV are markers for the outer and inner mitochondrial membranes, respectively. (C) Inner mitochondrial membranes isolated from PON2-def and control mice were subjected to Western blot analysis using anti-PON2 antibody. COX IV is a marker for the inner mitochondrial membrane. (D) Top panel shows Western blot analysis using anti-PON2 antibody after immunoprecipitation with complex I, II, or III from mouse liver mitochondrial extracts. Bottom panel shows western blot using antibodies for corresponding subunits. (E) Western blot analysis using anti-PON2 antibody after immunocapture with complex III immunocapture antibody (+) or with beads alone (−) from mitochondrial HeLa cell extracts (top panel) and mouse liver extracts (middle panel). Bottom panel shows Western blot analysis using antibody against Reiske protein after immunoprecipitation with (+) or without (−) complex III antibody from mouse liver extracts. (F) Complex III was immunoprecipitated from mitochondrial preparations isolated from the livers of control and PON2-def mice and analyzed by Western blot analysis using anti-PON2 antibody (top panel). Bottom panel shows Western blot analysis using anti-Rieske protein antibody after immunoprecipitation with complex III antibody.

FIG. 7.

Surface-plasmon-resonance analysis of CoQ₁₀ binding to human recombinant PON2. Surface-plasmon-resonance analysis of CoQ₁₀ binding to human recombinant PON2 is shown. (A) CoQ₁₀ was applied at the indicated concentrations. CoQ₁₀ interacted with PON2 in a concentration-dependent manner, and the K_D was determined to be 4 × 10⁻⁸M. (B) CoQ₁₀ binding to human recombinant PON2 (0.25 μg/ml) in the presence or absence of calcium. CoQ₁₀, coenzyme Q₁₀.

FIG. 8.

Overexpression of PON2 rescues induced mitochondrial dysfunction. (A) HeLa cells stably transfected with a doxycycline-inducible PON2 expression vector (HeLa-PON2) or transfected with a doxycycline-inducible empty vector (HeLa-EV) were treated with antimycin. Mitochondrial superoxide level was measured and the values were represented as relative fluorescence unit/mg mitochondrial protein (B) These transfected cells were treated with oligomycin and total ATP level was measured and values were represented as relative luminescence unit (5 × 10⁶ cells). SD (n = 3). *p < 0.05, relative to same cell line without doxycycline. RFU, relative fluorescence unit; RLU, relative luminescence unit.