GPR40 Agonism Modulates Inflammatory Reactions in Vascular Endothelial Cells

Abstract

Endothelial dysfunction is strongly linked with inflammatory responses, which can impact cardiovascular disease. Recently, G protein-coupled receptor 40 (GPR40) has been investigated as a modulator of metabolic stress; however, the function of GPR40 in vascular endothelial cells has not been reported. We analyzed whether treatment of GPR40-specific agonists modulated the inflammatory responses in human umbilical vein endothelial cells (HUVECs). Treatment with LY2922470, a GPR40 agonist, significantly reduced lipopolysaccharide (LPS)-mediated nuclear factor-kappa B (NF-κB) phosphorylation and movement into the nucleus from the cytosol. However, treatment with another GPR40 agonist, TAK875, did not inhibit LPS-induced NF-κB activation. LPS treatment induced expression of adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and attachment of THP-1 cells to HUVECs, which were all decreased by LY2922470 but not TAK875. Our results showed that ligand-dependent agonism of GPR40 is a promising therapeutic target for overcoming inflammatory reactions in the endothelium.

Keywords: Cell adhesion molecules; Human umbilical vein endothelial cells; Inflammation; Receptors, G-protein-coupled 40.

Fig. 1

G protein-coupled receptor 40 (GPR40)-specific agonist can modulate nuclear factor-kappa B (NF-κB) activation under lipopolysaccharide (LPS) treatment. Cells were pretreated with LY2922470 (25 or 50 μM), TAK875 (25 or 50 μM), or LY2922470 plus GW1100 (10 μM) for 24 hours and then stimulated with LPS (200 ng/mL) plus the indicated reagents for 0.5 hour. (A, B) Phosphorylated NF-κB level was determined by Western blot. (C, D) Intracellular location of NF-κB was visualized through immunofluorescence microscopy analysis (×200). White arrows indicate that NF-κB was translocated from cytosol into the nucleus (red fluorescence, NF-κB; blue fluorescence, nucleus). (E, F) Western blot shows the level of nuclear NF-κB. Lamin B1 was used as the loading control for nuclear protein. The mean±standard deviation was obtained from three separate experiments. NE, nuclear extract. ^aP<0.05 vs. vehicle group, ^bP<0.05 vs. LPS group, ^cP<0.05 vs. LPS plus LY2922470 group, as analyzed with analysis of variance (ANOVA) followed by an un-paired Student’s t-test.

Fig. 2

G protein-coupled receptor 40 (GPR40)-specific agonist can regulate adhesion molecule expression under lipopolysaccharide (LPS) treatment. Human umbilical vein endothelial cells (HUVECs) were pretreated with LY2922470 (25 or 50 μM), TAK875 (25 or 50 μM), or LY2922470 plus GW1100 (10 μM) for 24 hours and then stimulated with LPS (200 ng/mL) plus the indicated reagents for 6 hours. (A, B) Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) levels were determined by Western blot. (C, D) The stimulated HUVECs were co-cultured with green fluorescence-labeled THP-1 cells for 0.5 hour, and then THP-1 cells attached to HUVECs were analyzed using a fluorescence microscope (×200) and a spectrofluorometer. The mean±standard deviation was obtained from three separate experiments. ^aP<0.05 vs. vehicle group, ^bP<0.05 vs. LPS group, ^cP<0.05 vs. LPS plus LY2922470 group, as analyzed with analysis of variance (ANOVA) followed by an un-paired Student’s t-test.