Intermittent Hypoxia Enhances THP-1 Monocyte Adhesion and Chemotaxis and Promotes M1 Macrophage Polarization via RAGE

Abstract

Intermittent hypoxia (IH) that resulted from obstructive sleep apnea (OSA) has been found to be a risk factor of coronary artery disease. IH and the receptor for advanced glycation end products (RAGE) expression are known to activate monocyte/macrophage and associated with atherosclerosis development, while their effects on monocyte adhesion, chemotaxis to the endothelium, and macrophage polarization remain unknown. In the present study, RAGE in THP-1 monocytes was inhibited by shRNA lentiviral particles, followed by exposure to IH. Cell adhesion assay, transwell migration assay, and macrophage polarization assays were performed to study the effects of IH and RAGE. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that IH increased RAGE expression and activated NF-кB signalling in THP-1 monocytes. The results also revealed that IH enhanced the MCP-1-mediated THP-1 monocyte adhesion and chemotaxis and promoted macrophage polarization toward a proinflammatory phenotype, which was mediated by RAGE activity. Additionally, inhibition of chemokine receptor type 2 (CCR2) suppressed the IH-induced monocyte adhesion and chemotaxis. These results demonstrated a potential role of monocyte adhesion, chemotaxis, and macrophage polarization in the development cardiovascular diseases induced by IH and identified that RAGE could be a promising therapeutic target to prevent atherosclerosis in patients with OSA.

Figure 1

Intermittent hypoxia increased RAGE expression and activates the NF-кB pathway in THP-1 monocytes. (a and b) RAGE expression of THP-1 cells exposed to IH compared to cells in normoxia over a period of 48 h. (c) Representative images of immunofluorescence staining showing the nuclear translocation of NF-кB p65 (green) in THP-1 cells. Scale bars indicate 10 μm. (d–f) Phosphorylated NF-κB p65 and IkBα protein expression and quantification of THP-1 cells exposed to IH compared with cells cultured under normoxia over a period of 48 h. (g–i) Phosphorylated NF-κB p65 and RAGE protein expression and quantification of NF-κB siRNA treated cells compared to untreated cells exposed to normoxia or IH. Data were represented as mean + SEM. ∗ represents significant difference compared with normoxic group, ∗/^#p<.05, ∗∗p<.01, ∗∗∗p<.001.

Figure 2

RAGE knockdown inhibited chemotaxis and adhesion of THP-1 cells exposed to intermittent hypoxia. Knockdown of RAGE in THP-1 cells was verified via qRT-PCR (a) and western blotting (b and c). Representative images with quantification of the results for THP-1 cells (d) transwell migration assay and adhesion assay (e) in presence of MCP-1. Data were represented as mean + SEM. ∗ represents significant difference compared with untreated cells under normoxia condition; ∗^/#p<.05, ∗∗∗/^###p<.001. Scale bars indicate 100 μm.

Figure 3

Intermittent hypoxia promoted CCR2 expression via RAGE, which mediated chemotaxis and adhesion of THP-1 cells. Expression levels of CCR2 in THP-1 cells exposed to normoxia and intermittent hypoxia determined by qRT-PCR (a) and western blotting (b). The effects of CCR2 neutralizing antibody (10 μg/m) and CCR2 inhibitor (10 nM) on (c) the adhesion of THP-1 monocytes to HUVECs and the cell transwell migration (d). Data were represented as mean + SEM. ∗ represents significant difference compared with control group under normoxia condition; ∗p<.05, ∗∗/^##p<.001, ∗∗∗/^###p<.001.

Figure 4

Intermittent hypoxia polarizes macrophages to an M1 phenotype, which was mediated by RAGE. (a) Morphology of THP-1 macrophage cultured in normoxia or IH. F-actin cytoskeleton of the cells were labelled with phalloidin (red) and nuclei stained with DAPI (blue). Scale bar: 20 μm. (b) mRNA expression levels of M1 expression markers. (c) mRNA expression levels of M2 expression markers. ∗ represents significant difference compared with control group under normoxia condition; ∗p<.05, ^##p<.001, ∗∗∗/^###p<.001.