Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages

Abstract

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.

Fig 1. mRNA and protein analysis of GPR55 in human macrophages and foam cells.

Human THP-1 macrophages (MΦ) were left untreated or were treated for 18 h with 100 μg/ml oxLDL to generate foam cells (FC). (A) mRNA content of GPR55 was analyzed by qRT-PCR. Data are shown as mean (±SD) of five independent experiments, each in duplicate. (B) Western blot analysis of GPR55, with the expected molecular mass of each protein shown on the left-hand side. Data are expressed in comparison with β-actin, and are the mean (± SD) of five independent experiments, each performed in duplicate. *p<0.05 versus MΦ. (C) Immunofluorescence of GPR55 was performed by confocal laser-scanning microscopy, and data are shown as pictures taken with a 63 objective (numerical aperture 1⁄4 1.4), and as densitometric analysis (mean ±SD) of at least six independent experiments. (D) GPR55 expression was assessed by flow cytometry upon staining cells with anti-GPR55 FITC either at cell surface or intracellularly upon cell permeabilization. Data are reported as mean fluorescence intensity (M.I.F.), and are representative of five independent experiments. *p<0.05 versus MΦ.

Fig 2. Regulation of lipid-droplets accumulation and cholesterol transporters in human macrophages and foam cells.

Human THP-1 macrophages (MΦ) were left untreated or were treated for 18 h with 100 μg/ ml oxLDL to generate foam cells (FC), that were then treated for further 24 h with selective GPR55 agonist O-1602, and were pretreated or not with the CBD antagonist. (A) Nile red-stained lipid droplets were analyzed by confocal laser-scanning microscopy, and data are shown as numbers of lipid-droplets per cell (mean ±SD) of at least six independent experiments. Representative images of MΦ and FC, as well as of FC treated with 10nM O-1602or 0.5 μM CBD are shown in the panel. *p<0.05versus MΦ; #p<0.05 versusFC+O-1602. (B) Effect of GPR55 activation on CD36 expression in human macrophages and foam cells. The latter cells were pretreated with the CBD antagonist, and then were treated with the O-1602 agonist. CD36 expression was assessed by flow cytometry upon staining cells with anti-CD36 FITC. Data are reported as mean fluorescence intensity, and are representative of four independent experiments. Western blot analysis of SR-BI (C), ABCA1 (D), ABCG1 (E), with the expected molecular mass of each protein shown on the left-hand side. Data are expressed in comparison with β-actin, and are the mean (±SD) of five independent experiments, each performed in duplicate. *p<0.05 versus MΦ; #p<0.05 versus FC+O-1602.

Fig 3. Effect of GPR55 activation on cytokines production and release.

THP-1 macrophages (MΦ) were left untreated or were treated for 18 h with 100 mg/ml oxLDL to generate foam cells (FC). FC were treated for 24h with O-1602, and pretreated or not with CDB. (A) mRNA content of IL-10, IL-12, TNF-α were analyzed by qRT-PCR. Data are shown as mean (±SD) of six independent experiments, each in duplicate. *p<0.05 versus FC; #p<0.05 versus FC+O-1602. Levels of intracellular cytokines production were assessed by flow cytometry (as detailed in Materials and Methods), upon intracellular staining with anti-TNF-α (B) and IL-10 (C) antibodies. Data are means (±SD) of four independent experiments. *p<0.05 versus FC; #p<0.05 versus FC+O-1602.

Fig 4. Effect of GPR55 activation on MMP-9 activity.

Human THP-1 macrophages (MΦ) were left untreated or were treated for 18 h with 100 μg/ml oxLDL to generate foam cells (FC). FC were treated for 24h with O-1602, and pretreated or not with CDB. After the treatments, surnatants were collected and gelatinase activity of MMP-9 was measured in conditioned media subjected to zymography. Gelatinase activity is expressed as area (mm²) exposed to MMP-9 activity. Data are shown as mean ± SD of five independent experiments. *p<0.05 versus FC; #p<0.05 versus FC+O-1602.

Fig 5. Effect of GPR55 activation on NFAT transcription factor members.

Human THP-1 macrophages (MΦ) were left untreated or were treated for 18 h with 100 μg/ml oxLDL to generate foam cells (FC). FC were treated for 24h with O-1602, and pre-treated or not with CDB. mRNA analysis of NFAT members was performed by qRT-PCR. Data are shown as mean ± SD of three independent experiments, each in duplicate. *p<0.05 versus FC; #p<0.05 versus FC+O-1602.