Comparative reactivity of the myeloperoxidase-derived oxidants HOCl and HOSCN with low-density lipoprotein (LDL): Implications for foam cell formation in atherosclerosis

Abstract

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo, and has implications for the progression of atherosclerosis.

Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

“…In the experiments with macrophage cell lysates, in general a more pronounced decrease in enzyme activity was seen when the LDL was pre-treated with HOCl or HOSCN for 30 min rather than 24 h. An exception to this was seen in lysates exposed to HOSCN-LDL, where similar loss in cathepsin B activity was observed at each incubation time. Overall, this difference is attributed to the formation of reactive species on the LDL, including N -chloramines in the case of HOCl [23], which are known to target intracellular thiol-containing enzymes [41, 42]. These data are consistent with a previous study showing that HOCl-modified LDL is capable of reducing isolated cathepsin B enzyme activity by a pathway involving N -chloramines formed from Lys residues present in the apoB100 protein [20].…”

“…LDL (1.019 < d < 1.06 g/ml) were isolated as previously described [23, 31]. The protein concentration of isolated LDL was assessed using the bicinchoninic acid (BCA) assay.…”

“…A correlation between serum levels of thiocyanate (SCN - ), the precursor to HOSCN, with higher macrophage foam cell populations [21] and fatty streak formation [22] in smokers supports a role for this oxidant in disease pathology. However, macrophages exposed to HOSCN-modified LDL accumulate cholesterol to a lesser extent compared to HOCl-modified LDL [19, 23], and human MPO transgenic atherosclerosis-prone mice supplemented with SCN - show a reduced extent of lesion formation [24]. …”

“…The effect of HOSCN-modified LDL on the activity of the lysosomal cathepsin enzymes (B, L and D) and LAL was compared to LDL exposed to HOCl and cyanate (OCN - ), which is a decomposition product of HOSCN that has also been implicated in atherogenesis [25]. This is important because HOCl, HOSCN and OCN - have different fingerprints of LDL modification and hence biological reactivity [23], which differ from that seen on exposure of LDL to Cu 2+ [26, 27]. …”

MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression

“…LDL was isolated from fresh plasma of healthy human volunteers by sequential density gradient ultracentrifugation as described previously [16]. Written informed consents were obtained from all volunteers prior to sampling.…”