Dietary antioxidants act as a first line of defense against lipids peroxidation in lipoproteins and in arterial cells, including macrophage foam cells. A second line of defense by antioxidants could be related to stimulation of paraoxonases (PONS). PON1 is associated in serum with high density lipoprotein (HDL). The activity of serum PON1 was shown to be inversely associated with the risk for atherosclerosis development and this phenomenon could be related to the ability of PON1 to protect lipids in LDL, HDL and arterial cells against oxidation, secondary to its hydrolytic action on specific oxidized lipids. PON1 was shown to play a major role in protecting against oxidative stress and its consequent cardiovascular diseases development. Dietary intervention with appropriate nutraceuticals, with most potent and diverse antioxidative properties most efficiently attenuate cardiovascular diseases progression. Major risk factors for atherosclerosis development include the quantity and quality of low density lipoprotein (LDL, "The bad cholesterol") and that of high density lipoprotein (HDL, "the good cholesterol"). High serum levels of LDL and law serum concentration of HDL are known risk factors for accelerated atherosclerosis. This is the result of LDL-induced increased cholesterol influx into the arterial wall, or of a decreased HDL-mediated cholesterol efflux from the arterial wall. The "quality" of both LDL and HDL is also an important determinant of their atherogenicity and upon oxidation of these lipoproteins, they become more atherogenic, i.e. oxidized LDL (Ox-LDL) is taken up by arterial cells at enhanced rate, and oxidized HDL (Ox-HDL) is less efficient than native HDL in promoting reverse cholesterol transport (RCT) from the atherosclerotic lesion to the liver for excretion (1). HDL is a unique anti-atherogenic lipoprotein which contain several enzymes including LCAT, PAF-AH and paraoxonase 1 (PON1), which is an esterase (lactonase). PON1 was shown to protect against lipid peroxidation in atherosclerotic lesion, in "oxidized macrophages" and in oxidized lipoproteins (Ox-LDL, Ox-HDL), a phenomena which could be attributed to PON1 hydrolytic activity on certain oxidized lipids (2, 3). Dietary antioxidants such as vitamin E, but mainly specific polyphenolic flavonoids found in pomegranate juice (PJ, tannins, anthocyannins) and in red wine (quercetin,resverotrol), significantly inhibited LDL oxidation and preserved POW activity. Dietary supplementation of these antioxidants to the atherosclerotic E-0 mice reduced serum oxidative stress by 30-60% and increased serum PON1 acitvity by 20-40% (4,5). Most important, the atherosclerotic lesion size was significantly reduced following PJ or red wine consumption in these mice, by up to 44%. Consumption of PJ for 2 weeks by healthy subjects resulted in a significant 20% increase in serum PON1 activity and their LDL and HDL were more resistant to oxidation. Most important, pomegranate juice consumption for one year by atherosclerotic carotid artery stenosis (CAS) patients resulted in a significant reduction, by up to 30%, in their common carotid intima-media thickness (IMT). These results were accompanied by an increase in the patients' serum PON1 activity by 83%, and by 60-90% decrease in the patients serum LDL basal oxidative state and LDL susceptibility to copper ion-induced oxidation (6). As PON1, unlike the above dietary antioxidants is not a free radical scavengers, we hypothesized that PON1 may act as a second line of defense against lipid peroxidation (the first line being the antioxidants) ( 7), acting as an hydrolase on preformed oxidized lipids (Figure 1). Indeed, PON1 was shown to decrease phospholipids hydroperoxides and cholesteryl linoleate hydroperoxides in oxidized LDL and in atherosclerotic lesions (8). We next questioned whether the hydrolytic action of PON1 on certain oxidized lipids could be operable also in vivo, and whether PON1 affects macrophage oxidative status, foam cell formation and atherosclerosis development. For this proposes, we used PON1-deficient mice, as well as PON1-transgenic mice (on the background of the atherosclerotic apo E deficient mice). On using macrophages from PON1(0)/E-0 mice, cellular lipid peroxides content and PMA-induced superoxide anion release were both substantially increased, in comparison to the control E-0 mice cells, by 80% and by 116% respectively. The increased superoxides release could be associated to activation of macrophage NADPH oxidase, as determined by a 130% increased P-47 (a cytosolic factor of the enzyme) translocation from the cytosol to the plasma membrane, where the active enzyme is formed (Figure 2). Most important, the atherosclerotic lesion size was increase by 42% in PON1(0)/E-0 vs. E-0 mice, and the lesion morphology indicated increased number of foam cells, which were enriched with crystalline cholesterol (9). Using macrophages from the PON1-transgenic mice, as well as PON1-transfected macrophages, an apposite pattern for macrophage foam cell formation was observed, in comparison to macrophages from the PON1 deficient mice (Figure 3). Macrophage peroxides and superoxide anion release from PON 1 Tg/E-0 vs. E-0 mice cells were decreased by 50% and 40% respectively, and this phenomena were associated with a 35% decrement in macrophage-mediated LDL oxidation, and a 30% increment in cellular glutathione (GSH) content (10). To find out whether POW active site is required for PON 1 protection against Ox-LDL accumulation, we used evolved PON1 that was mutated in the active site in the hisitidins 115 and 134 (11). Whereas the wild type PON1 decreased macrophage-mediated oxidation of LDL by 60%, the PON1 mutants did not significantly affect LDL oxidation. We next questioned the possible role of HDL-apolipoprotein A-I in PON1 protection against LDL oxidation. On using reconstituted HDL, composed of phosphotydil choline, cholesterol, Apo A-I and PON1, in comparison to phosphatydil choline, cholesterol and PON 1 (no apo A-I present), a 50% increased capability of PON1 to decrease LDL oxidation was noted, implicating the importance of apo A-I in POW activation (12). An in vivo support to the above observations was obtained from serum PON1 distribution analysis. In diabetic patient, unlike control healthy subjects PON1 was found to be dissociated from HDL to the lipoprotein deficient serum (LPDS) fraction, and in diabetic patients vs. control subjects, a 70% increase in HDL-free PON1 (LPDS-associated) was noted. Upon oxidation of HDL and LPDS with AAPH, the free radicals generator, a 35% decrement in HDL oxidation was noted when POW (5U/ml) was present in the oxidation system, whereas no significant effect of POW could be observed when added to LPDS under a similar oxidation conditions (13). These differences could be attributed to a stabilization of POW paraoxonase activity, when present in HDL, but not when present in LPDS. In conclusion, serum HDL-associated PON1 antiatherogenicity can be related to the reduction in OxLDL accumulation, a phenomenon that is associated with a direct hydrolytic effect of PON1 on Ox-LDIL, as well as to an indirect effect of PON1 on oxidized lipids in macrophages, leading to a less efficient cell-mediated oxidation of LDL. In diabetic patients, accelerated atherosclerosis development could be related to their serum PON1 pattern, i.e.: decreased POW levels and increased HDL-free PON1 concentration, two characteristics which lead to increased accumulation of atherogenic oxidized LDL, oxidized macrophages, and the consequent atherosclerotic lesion formation (Figure 4). Dietary intervention with appropriate nutraceuticals, with most potent and diverse antioxidative properties, is recommended to increase PON1, reduce oxidative stress and attenuate cardiovascular diseases progression (Figure 5). Copyright (c) 2006 S. Karger AG, Basel.
