Elevated atherosclerosis-related gene expression, monocyte activation and microparticle-release are related to increased lipoprotein-associated oxidative stress in familial hypercholesterolemia

Abstract

Objective: Animal and in vitro studies have suggested that hypercholesterolemia and increased oxidative stress predisposes to monocyte activation and enhanced accumulation of oxidized LDL cholesterol (oxLDL-C) through a CD36-dependent mechanism. The aim of this study was to investigate the hypothesis that elevated oxLDL-C induce proinflammatory monocytes and increased release of monocyte-derived microparticles (MMPs), as well as up-regulation of CD36, chemokine receptors and proinflammatory factors through CD36-dependent pathways and that this is associated with accelerated atherosclerosis in subjects with heterozygous familial hypercholesterolemia (FH), in particular in the presence of Achilles tendon xanthomas (ATX).

Approach and results: We studied thirty FH subjects with and without ATX and twenty-three healthy control subjects. Intima-media thickness (IMT) and Achilles tendon (AT) thickness were measured by ultrasonography. Monocyte classification and MMP analysis were performed by flow cytometry. Monocyte expression of genes involved in atherosclerosis was determined by quantitative PCR. IMT and oxLDL-C were increased in FH subjects, especially in the presence of ATX. In addition, FH subjects had elevated proportions of intermediate CD14++CD16+ monocytes and higher circulating MMP levels. Stepwise linear regression identified oxLDL-C, gender and intermediate monocytes as predictors of MMPs. Monocyte expression of pro-atherogenic and pro-inflammatory genes regulated by oxLDL-C-CD36 interaction was increased in FH, especially in ATX+ subjects. Monocyte chemokine receptor CX3CR1 was identified as an independent contributor to IMT.

Conclusions: Our data support that lipoprotein-associated oxidative stress is involved in accelerated atherosclerosis in FH, particularly in the presence of ATX, by inducing pro-inflammatory monocytes and increased release of MMPs along with elevated monocyte expression of oxLDL-C-induced atherosclerosis-related genes.

Fig 1. Flow cytometric analysis of monocyte subpopulations.

Representative example of monocyte subsets depicted as classical CD14++CD16- monocytes (blue color), intermediate CD14++CD16+ monocytes (red color), and nonclassical CD14+CD16++ monocytes (green color).

Fig 2. CD36 expression on monocyte subpopulations.

(A) Representative flow cytometric plot of CD36 expression on monocyte subpopulations. (B) Box plot analysis of CD36 surface expression levels on classical (blue color), intermediate (red color) and nonclassical (green color) monocytes in healthy controls and FH subjects. Median (horizontal dotted line), 25% and 75% quartiles (box), 10% and 90% quartiles (whiskers). Filled circles represent outliers. *p<0.010; **p<0.005; ***p<0.0001.

Fig 3. Real-time quantitative RT-PCR analysis on isolated monocytes.

Expression of pro-atherogenic and pro-inflammatory genes in FH and control subjects (A) and in FH subjects with and without ATX (B). Real-time quantitative RT-PCR was performed on total RNA obtained from freshly isolated monocytes. The amount of mRNA for the gene of interest was normalized relative to [beta]₂M mRNA, and the graphs were generated with the relative median values obtained after normalization. Mann-Whitney U test was used to compare the transcriptional expression of genes and statistically significant difference (p<0.05) is indicated by *.