Decreased low-density lipoprotein receptor-related protein 1 expression in pro-inflammatory monocytes is associated with subclinical atherosclerosis

Abstract

Subclinical atherosclerosis (SCA) occurs in asymptomatic individuals. Blood peripheral monocytes are involved in the development of atherosclerosis. Circulating monocytes acquire pro-inflammatory profiles, and they are involved in the early stages of atherosclerosis development. Low-density lipoprotein Receptor-related Protein 1 (LRP1) is expressed in monocytes, mainly in classical and intermediate subsets. Although LRP1 is highly expressed in macrophages and vascular smooth muscle cells (VSMCs) in atherosclerotic plaque formation, its expression in circulating monocytes has not been studied in SCA. The aim of this study was to characterize the LRP1 expression level in circulating monocytes of individuals with SCA and compared with individuals with low (LR) and intermediate (IR) risk of cardiovascular diseases, both without evidence of atherosclerotic lesions in carotid and coronary arteries. LRP1 and additional markers (CD11b, CD11c, and CD36) at cell surface of monocytes were analyzed by flow cytometry assays, whereas LRP1 and pro-inflammatory factors gene expressions were measured in isolated monocytes by quantitative RT-PCRs. Both LRP1 protein and LRP1 mRNA were significantly reduced in monocytes in SCA and IR respect to LR. Conversely, CD36, CD11b, and CD11c monocytic markers showed no significant changes between the different study groups. Finally, increased gene expressions of TNF-α and IL-1β were detected in monocytes of SCA, which were associated with decreased LRP1 expression at the cell surface in total monocytes. In summary, we propose that the decreased LRP1 expression at cell surface in total monocytes with pro-inflammatory profile is associated with the development of atherosclerosis in asymptomatic individuals.

Keywords: cardiovascular; cytokines; inflammation; lipids; lipoproteins; receptors.

FIGURE 1

LRP1 gene expression is also decreased in individuals with SCA and IR. (A) The levels of LRP1 specific transcripts were measured by quantitative RT-PCR (qPCR) in FACS-isolated total monocytes as was described in “Materials and Methods” section and relatively expressed at GAPDH levels from individuals with low risk (LR) (green symbols; n = 16), intermediate risk (IR) (blue symbols; n = 16) and subclinical atherosclerosis (SCA) (red symbols; n = 16) (Study II). (B) The LRP1 expression level at cell surface was measured by flow cytometry assays in total monocytes from peripheral blood extracted of individuals with low risk (LR) (green symbols; n = 16), intermediate risk (IR) of CVD (blue symbols; n = 16), and subclinical atherosclerosis (SCA) (red symbols; n = 16). In (A,B), results are shown as box plot representing the median value and interquartile range (IQR) of arbitrary units for qPCR and MIF (mean intensity of fluorescence), respectively. Maximal and minimal values for each study group are indicated (bar lines). Parameters were log-transformed to achieve normal distribution and to apply statistical parametric analysis, but in these table the original data were used. (C) Correlation analysis between LRP1 protein and LRP1 mRNA in monocytes of individuals with LR (green symbols), IR (blue symbols) and SCA (red symbols). p-values are shown and differences were considered statistically significant when p < 0.05.

FIGURE 2

Gene expression of pro-inflammatory factors is increased in circulating monocytes in SCA. The levels of specific transcripts of TNF-α (A), IL-1β (B), CCL2 (C), and CCR2 (D) were measured by quantitative RT-PCR (qPCR) in FACS-isolated total monocytes and relatively expressed at GAPDH levels from individuals with low risk (LR) (green symbols; n = 16), intermediate risk (IR) (blue symbols; n = 16) and subclinical atherosclerosis (SCA) (red symbols; n = 16) (Study II). Results are shown as box plot representing the median value and interquartile range (IQR) of arbitrary units for qPCR. Maximal and minimal values for each study group are indicated (bar lines). Parameters were log-transformed to achieve normal distribution and to apply statistical parametric analysis, but in these table the original data were used. p-values are shown and differences were considered statistically significant when p < 0.05.