Paraoxonase-1 (PON-1), an HDL-associated enzyme has been shown to possess antioxidant/anti-inflammatory properties and protect against atherogenesis. The aim of the study was to explore the association of PON-1, with lipids, antioxidant-vitamins and lifestyle in male subjects with and without angiographically documented coronary artery disease (CAD) and in healthy volunteers. PON-1 activity was measured spectrophotometrically using phenyl acetate as substrate. PON-1 and HDL-Cholesterol were significantly lower and lipid peroxides and triglycerides were higher in CAD patients than in normal coronary and control subjects. PON-1 activity showed positive association with HDL-C and inverse relation with lipid peroxides and no association with antioxidant vitamins in healthy subjects. Smoking habit, alcohol intake and diabetes mellitus did not seem to influence PON-1 activity. Serum levels of PON-1 and HDL-C showed inverse association with the presence of CAD but are not related to the severity of disease in terms of number of diseased vessels. Interventional means by diet/drugs to enhance PON-1 activity may contribute to attenuation of atherosclerosis.

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It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.

Although high-density lipoprotein is atheroprotective, it can become dysfunctional in chronic inflammatory conditions and increase cardiovascular risk. We previously demonstrated that HDL from subjects with documented coronary artery disease is dysfunctional and is pro-oxidant/proinflammatory in macrophages. Here we examined the influence of dysfunctional/proinflammatory HDL (piHDL) on lipid accumulation in human macrophages, in comparison to functional HDL (nHDL). Exposure of macrophages to piHDL, in contrast to nHDL, resulted in oxidative stress and marked uptake of lipids from piHDL, leading to the formation of foam cell phenotype as noted by oil red O staining with concomitant increase in total cellular cholesterol content. Using western blotting, we identified that piHDL profoundly upregulated the expression of scavenger receptor CD36 and suppressed the expression of ABCG1 and SRB1 in macrophages, thereby facilitating cholesterol influx capacity of macrophages. We then identified that CD36 did not act alone, indeed it was activated in macrophages along with ERK/MAPK, in response to piHDL, which in turn led to lipid accumulation as well as proinflammatory response via activation of NFkB and subsequent release of proinflammatory markers-TNF-ά and MMP-9. These effects were confirmed using pharmacological inhibitors for either CD36 or ERK/MAPK. Furthermore, piHDL treatment moderately activated PPAR-γ and Nrf2, the known regulators of CD36 in macrophages, suggesting that the two forms of HDL differentially regulate CD36 expression. Taken together, the results demonstrate that a novel CD36-ERK/MAPK-dependent mechanism is involved in macrophage lipid accumulation by piHDL, there by revealing the importance of functional deficiency in HDL and its potential link to atherogenesis.

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P-selectin (CD62p) exposure is an established marker for platelet activation. P-selectin exposure can trigger variety of thrombotic and inflammatory reactions. In patients with coronary artery disease (CAD), platelets are activated, and hence, there is increased P-selectin exposure. The role of P-selectin exposure in patients on treatment with statins and anti-platelets is conflicting. A case-control study was performed to determine P-selectin exposure in consecutively recruited 142 patients (age ≤ 55 years) with angiographically proven CAD on treatment and 92 asymptomatic controls. P-selectin exposure was determined by flow cytometry. Data on conventional risk factors were obtained along with estimation of levels of thrombotic [fibrinogen, lipoprotein (a), tissue plasminogen activator, plasminogen activator inhibitor-1, homocysteine and von Willebrand factor] and anti-thrombotic factors (antithrombin III). The P-selectin exposure was compared among patient groups who had different modes of presentation of CAD and categories of CAD disease severity. The patients were followed up for a period of 26 months. The results indicate that P-selectin exposure was significantly elevated in patients (mean ± SD 9.24 ± 11.81) compared to controls (mean ± SD 1.48 ± 2.85) with p < 0.0001. Similarly, conventional risk factors were significantly elevated in patients. P-selectin exposure showed significant negative correlation with antithrombin III levels. P-selectin exposure was higher in patients who presented with acute coronary syndromes than those who presented with effort angina. Cardiovascular event rate was 6 % on follow-up. The study establishes that thrombotic-inflammatory pathways enhancing P-selectin exposure unrelated to treatment might be activated in patients, while the event rate remained lowered, and hence, treatment strategies should be inclusive to control these factors.

Atherosclerosis can be considered as an inflammatory disease and oxidized low-density lipoprotein (oxLDL) is a critical factor in atherogenesis. Although high-density lipoprotein (HDL) is generally an antiatherogenic lipoprotein, this property can be compromised by functional impairment mainly due to oxidative modification. As such, understanding the proatherogenic properties exerted by oxidized-HDL (oxHDL) becomes more important. This study was focused on examining the role of oxHDL as a proatherogenic agent, using oxLDL as a positive control. The comparative toxicity of oxHDL and oxLDL having same range of malondialdehyde, to monocytes was evaluated. After treatment, markers for oxidative stress, inflammation, and cytotoxicity were quantitated. The results showed that like oxLDL, oxHDL induced significant oxidative stress, cytotoxicity, and release of TNF -alpha and MMP-9 in monocytes/macrophages, but was less potent than oxLDL in promoting these proatherogenic effects. Further, the effects of oxHDL for the enhanced formation of MMP-9 were found to be mediated by NADPH oxidase/ROS-JNK/ERK pathway, as one mechanism.

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