Asokan Devarajan

Paraoxonase 2 Deficiency Alters Mitochondrial Function and Exacerbates the Development of Atherosclerosis

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.

Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis

Purpose of review During the past decade, paraoxonase 1, a HDL-associated protein, has been demonstrated to be an important contributor to the antioxidant capacity of HDL. Studies using paraoxonase 1 null mice by gene targeting and transgenic mice corroborated the hypothesis that paraoxonase 1 protects against atherosclerosis. In contrast to paraoxonase 1, the other two members of the paraoxonase gene family, namely paraoxonase 2 and paraoxonase 3, are either undetectable (paraoxonase 2) or detected at very low levels (paraoxonase 3) on HDL, and are considered to participate in intracellular antioxidant mechanisms. In this review, we summarize studies reported in the past 2 years suggesting a protective role for paraoxonase 2 and paraoxonase 3 in the development of atherosclerosis in mice. Recent findings Adenovirus-mediated expression of human paraoxonase 2 or paraoxonase 3 proteins protects against the development of atherosclerosis in apolipoprotein E-deficient mice. Paraoxonase 2-deficient mice develop significantly larger atherosclerotic lesions than their wild-type and heterozygous counterparts on an atherogenic diet despite having lower levels of apolipoprotein B-containing lipoproteins. Atherosclerotic lesions were significantly lower in male hPON3Tg/LDLR null mice than in LDLR null mice on a western diet. Summary We conclude that, in addition to paraoxonase 1, both paraoxonase 2 and paraoxonase 3 proteins are protective against the development of atherosclerosis in mice. These findings underscore the utility of all members of the paraoxonase gene family as therapeutic targets for the treatment of atherosclerosis.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3s anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Protectors or Traitors: The Roles of PON2 and PON3 in Atherosclerosis and Cancer

Cancer and atherosclerosis are major causes of death in western societies. Deregulated cell death is common to both diseases, with significant contribution of inflammatory processes and oxidative stress. These two form a vicious cycle and regulate cell death pathways in either direction. This raises interest in antioxidative systems. The human enzymes paraoxonase-2 (PON2) and PON3 are intracellular enzymes with established antioxidative effects and protective functions against atherosclerosis. Underlying molecular mechanisms, however, remained elusive until recently. Novel findings revealed that both enzymes locate to mitochondrial membranes where they interact with coenzyme Q10 and diminish oxidative stress. As a result, ROS-triggered mitochondrial apoptosis and cell death are reduced. From a cardiovascular standpoint, this is beneficial given that enhanced loss of vascular cells and macrophage death forms the basis for atherosclerotic plaque development. However, the same function has now been shown to raise chemotherapeutic resistance in several cancer cells. Intriguingly, PON2 as well as PON3 are frequently found upregulated in tumor samples. Here we review studies reporting PON2/PON3 deregulations in cancer, summarize most recent findings on their anti-oxidative and antiapoptotic mechanisms, and discuss how this could be used in putative future therapies to target atherosclerosis and cancer.

D-4F, an apoA-I mimetic peptide, inhibits proliferation and tumorigenicity of epithelial ovarian cancer cells by upregulating the antioxidant enzyme MnSOD

We recently reported that apoA‐I and apoA‐I mimetic peptides prevent the development of flank tumors in immunocompetent C57BL/6J mice. To delineate the mechanism(s) of action of apoA‐I mimetic peptides in tumor development, we examined the effect of D‐4F (an apoA‐I mimetic peptide) on the antioxidant status and on the gene expression and function of antioxidant enzymes in ID8 cells (a mouse epithelial ovarian cancer cell line) and in a mouse model. We demonstrate that D‐4F treatment significantly reduces the viability and proliferation of ID8 cells, with a concomitant improvement of the antioxidant status of ID8 cells as measured by lipid peroxidation, protein carbonyl, superoxide anion, and hydrogen peroxide levels. D‐4F treatment induces MnSOD (but not CuZnSOD) mRNA, protein, and activity. Inhibition of MnSOD in ID8 cells using shRNA vectors abrogates the inhibitory effects of D‐4F on ID8 cell viability and proliferation. Moreover, tumor development from ID8 cells carrying shRNA for MnSOD were unaffected by D‐4F treatment. Our results suggest that the inhibitory effects of D‐4F on ID8 cell proliferation and tumor development are mediated, at least in part, by the induced expression and activity of MnSOD.

Blockade of CXCR2 signalling: A potential therapeutic target for preventing neutrophil-mediated inflammatory diseases

Polymorphonuclear neutrophils (PMN) play a key role in host innate immune responses by migrating to the sites of inflammation. Furthermore, PMN recruitment also plays a significant role in the pathophysiology of a plethora of inflammatory disorders such as chronic obstructive pulmonary disease (COPD), gram negative sepsis, inflammatory bowel disease (IBD), lung injury, and arthritis. Of note, chemokine-dependent signalling is implicated in the amplification of immune responses by virtue of its role in PMN chemotaxis in most of the inflammatory diseases. It has been clinically established that impediment of PMN recruitment ameliorates disease severity and provides relief in majority of other immune-associated disorders. This review focuses on different novel approaches clinically proven to be effective in blocking chemokine signalling associated with PMN recruitment that includes CXCR2 antagonists, chemokine analogs, anti-CXCR2 monoclonal antibodies, and CXCR2 knock-out models. It also highlights the significance of the utility of nanoparticles in drugs used for blocking migration of PMN to the sites of inflammation.

Inflammation, Infection, Cancer and All That…The Role of Paraoxonases

The paraoxonase (PON) gene family consists of three members, PON1, PON2 and PON3. All PON proteins possess antioxidant properties and lipo-lactonase activities, and are implicated in the pathogenesis of several inflammatory diseases including atherosclerosis, Alzheimers, Parkinsons, diabetes and cancer. Despite the role of PON proteins in critical cellular functions and associated pathologies, the physiological substrates and molecular mechanisms by which PON proteins function as anti-inflammatory proteins remain largely unknown. PON1 is found exclusively extracellular and associated solely with high-density lipoprotein (HDL) particles in the circulation, and, in part, confers the anti-oxidant and anti-inflammatory properties associated with HDL. Recent studies demonstrated that the intracellular PON proteins; PON2 and PON3 (i) are associated with mitochondria and mitochondria-associated membranes, (ii) modulate mitochondria-dependent superoxide production, and (iii) prevent apoptosis. Overexpression of PON2 and PON3 genes protected (i) mitochondria from antimycin or oligomycin mediated mitochondrial dysfunction and (ii) ER stress and ER stress mediated mitochondrial dysfunction. These studies illustrate that the anti-inflammatory effects of PON2 and PON3 may, in part, be mediated by their role in mitochondrial and associated organelle function. Since oxidative stress as a result of mitochondrial dysfunction is implicated in the development of inflammatory diseases including atherosclerosis and cancer, these recent studies on PON2 and PON3 proteins may provide a mechanism for the scores of epidemiological studies that show a link between PON genes and numerous inflammatory diseases. Understanding such mechanisms will provide novel routes of intervention in the treatment of diseases associated with pro-inflammatory oxidative stress.

Role of PON2 in innate immune response in an acute infection model.

N-(3-oxododecanoyl)-l-homoserine lactone (3OC(12)-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC(12)-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC(12)-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC(12)-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC(12)-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC(12)-HSL. PON2 expression (message and protein) is not altered in response to 3OC(12)-HSL in macrophages. 3OC(12)-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6 × 10(7) CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.

Macrophage paraoxonase 2 regulates calcium homeostasis and cell survival under endoplasmic reticulum stress conditions and is sufficient to prevent the development of aggravated atherosclerosis in paraoxonase 2 deficiency/apoE−/− mice on a Western diet

Paraoxonase 2 deficiency (PON2-def) alters mitochondrial function and exacerbates the development of atherosclerosis in mice. PON2 overexpression protects against ER stress in cell culture. In this paper, we examined the role of PON2 in the unexplored link between ER stress and mitochondrial dysfunction and tested whether restoration of PON2 in macrophages is sufficient to reduce aggravated atherosclerosis in PON2-def/apoE(-/-) mice on a Western diet. ER stress response genes, intracellular calcium levels, and apoptotic nuclei were significantly elevated in PON2-def/apoE(-/-) macrophages compared to apoE(-/-) macrophages in response to ER stressors, but not at the basal level. In contrast, PON2-def/apoE(-/-) macrophages exhibited greater mitochondrial stress at the basal level, which was further worsened in response to ER stressors. There was no difference in ER stress response genes and apoptotic nuclei between apoE(-/-) and PON2-def/apoE(-/-) macrophages when pretreated with xestospongin (which blocks the release of calcium from ER) suggesting that PON2 modulates cell survival and ER stress by maintaining calcium homeostasis. Treatment with a mitochondrial calcium uptake inhibitor, RU360, attenuated ER stressor mediated mitochondrial dysfunction in PON2-def/apoE(-/-) macrophages. CHOP expression (ER stress marker) and apoptotic nuclei were significantly higher in aortic lesions of PON2-def/apoE(-/-) mice compared to apoE(-/-) mice fed a Western diet. Restoration of PON2 in macrophages reduced ER stress, mitochondrial dysfunction and apoptosis in response to ER stressors. Furthermore, restoration of PON2 in macrophages reduced lesional apoptosis and atherosclerosis in PON2-def/apoE(-/-) mice on a Western diet. Our data suggest that macrophage PON2 modulates mechanisms that link ER stress, mitochondrial dysfunction and the development of atherosclerosis.

PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis

We report the engineering and characterization of paraoxonase‐3 knockout mice (Pon3KO). The mice were generally healthy but exhibited quantitative alterations in bile acid metabolism and a 37% increased body weight compared to the wild‐type mice on a high fat diet. PON3 was enriched in the mitochondria‐associated membrane fraction of hepatocytes. PON3 deficiency resulted in impaired mitochondrial respiration, increased mitochondrial superoxide levels, and increased hepatic expression of inflammatory genes. PON3 deficiency did not influence atherosclerosis development on an apolipoprotein E null hyperlipidemic background, but it did lead to a significant 60% increase in atherosclerotic lesion size in Pon3KO mice on the C57BL/6J background when fed a cholate‐cholesterol diet. On the diet, the Pon3KO had significantly increased plasma intermediate‐density lipoprotein/LDL cholesterol and bile acid levels. They also exhibited significantly elevated levels of hepatotoxicity markers in circulation, a 58% increase in gallstone weight, a 40% increase in hepatic cholesterol level, and increased mortality. Furthermore, Pon3KO mice exhibited decreased hepatic bile acid synthesis and decreased bile acid levels in the small intestine compared with wild‐type mice. Our study suggests a role for PON3 in the metabolism of lipid and bile acid as well as protection against atherosclerosis, gallstone disease, and obesity.—Shih, D. M., Yu, J. M., Vergnes, L., Dali‐Youcef, N., Champion, M. D., Devarajan, A., Zhang, P., Castellani, L. W., Brindley, D. N., Jamey, C. Auwerx, J., Reddy, S. T., Ford, D. A., Reue, K., Lusis, A. J. PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis. FASEB J. 29, 1185‐1197 (2015). www.fasebj.org