De Novo Leukotriene B-4 Production From High-Density Lipoprotein (HDL) in Recurrent Cardiovascular Disease Patients Deteriorates Anti-Inflammatory Properties of HDL

Introduction: We recently reported that serum myeloperoxidase/paraoxonase 1 (MPO/PON1) ratio could be used as a useful marker for high density lipoprotein (HDL) function and elevated MPO/PON1 ratio correlated to recurrent cardiovascular diseases. We hypothesized that HDL from high MPO/PON1 ratio cases (high MPO/PON1 HDL) might have specific bioactive molecules which contributed dysfunctional HDL and atherosclerotic diseases. Methods and Results: HDL was purified by ultracentrifuge from cases with various MPO/PON1 ratios. Comprehensive liquid chromatography/mass spectrometry (LC/MS) based lipid mediator analysis revealed that high MPO/PON1 HDL produced potent pro-inflammatory lipid mediator, leukotriene B4 (LTB 4 ). We also found that high MPO/PON1 HDL carried higher MPO, lower PON1, as well as abundant LTB 4 producing enzymes, 5-lipoxygenase (5-LO) and LTA 4 hydrolase. To investigate biological impact of HDL on activated macrophages, lipid mediator productions from activated macrophages were analyzed with HDL treatment. Low MPO/PON1 HDL treatment attenuated pro-inflammatory lipid mediator production from macrophages, especially LTB 4 . We found that low MPO/PON1 HDL enhanced proteasome-mediated degradation of 5-LO in activated macrophages. However, high MPO/PON1 HDL failed to reduce 5-LO in macrophages. Consequently, activated macrophages maintained pro-inflammatory properties with high MPO/PON1 HDL treatment. As the mechanisms, low MPO/PON1 HDL, but not high MPO/PON1 HDL, was transferred into macrophages via clathrin-mediated endocytosis. HDL endocytosis seems to be a critical step for their anti-inflammatory properties, as endocytosis inhibitor Pitstop2 abolished 5-LO degradation in macrophages by HDL. nM range of LTB 4 completely attenuated HDL endocytosis into macrophages, indicating that LTB 4 produced from high MPO/PON1 HDL might play critical roles in dysfunctional, inflammatory HDL characteristics. Conclusions: Recurrent cardiovascular disease patients have elevated MPO/PON1 ratio in serum as well as in HDL. High MPO/PON1 HDL produces pro-inflammatory LTB 4 , which attenuates endocytotic HDL uptake into macrophages, fails to reduce proinflammatory lipid mediator production from activated macrophages.