Increased C-C chemokine receptor 2 gene expression in monocytes of severe obstructive sleep apnea patients and under intermittent hypoxia

Abstract

Background: Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes.

Methods: Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes.

Results: Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia.

Conclusions: This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.

Figure 1. CCR2 mRNA expression significantly increased in monocytes of severe OSA patients.

(A) The monocytic CCR2 mRNA expression of 54 patients from four different groups was analyzed by RT/real-time PCR. Data were means and standard errors. (mean ± SE, *: P<0.05 vs. AHI ≤5, †: P<0.05 vs 5< AHI ≤15, ‡: P<0.05 vs 15< AHI ≤30) (B) Linear regression demonstrated the positive correlation between AHI and CCR2 mRNA expression levels in monocytes (p<0.01, r = 0.507). (C) Linear regression demonstrated the negative correlation between average oxygen saturation in patients and CCR2 mRNA expression levels in monocytes (p<0.05, r = 0.335). (D) Linear regression demonstrated the positive correlation between the monocytic CCR2 mRNA expression level and the time with SaO₂ <85% in OSA patients (p<0.05, r = 0.328).

Figure 2. Intermittent hypoxia enhanced CCR2 gene expression in monocytes.

THP-1 cells were treated with normoxia or intermittent hypoxia as described in methods. (A) RNA was isolated for the analysis of CCR2 gene expression by RT/real-time PCR. (B) Membrane proteins were prepared for western blot analysis. (C) Human peripheral monocytes were treated with the same conditions as in (A) and total RNA was isolated for the analysis of CCR2 gene expression by RT/real-time PCR. Data were present as means and standard errors from three independent experiments (mean ± SE, *: P<0.05 vs. Normoxia).

Figure 3. Up-regulation of monocytic CCR2 gene expression depended on the hypoxia level, but not TNF-α or CRP.

(A) THP-1 cells were treated with normoxia or intermittent hypoxia with different hypoxia levels (lowest O₂ set-point at 5% or 0.1%) as described in methods. RNA was isolated for the analysis of CCR2 mRNA expression by RT/real-time PCR. In the presence of TNF-α (B) or CRP (C), THP-1 cells were treated with intermittent hypoxia as described in methods. RNA was isolated for the analysis of CCR2 mRNA expression by RT/real-time PCR. (mean ± SE, *: P<0.05 vs. Normoxia, †: P<0.05 vs Normoxia, three independent experiments).

Figure 4. Intermittent hypoxia increased MCP-1-induced chemotaxis of monocytes.

THP-1 cells were pretreated with normoxia or intermittent hypoxia as described in methods and then processed for the MCP-1-mediated chemotaxis assay. (A) Representative photos for normoxia- and intermittent hypoxia-treated THP-1 cells that migrated toward lower chamber indicated by black arrow. (B) Statistical results from three independent experiments were shown. Data were means and standard errors. (mean ± SE, *: P<0.05 vs. Normoxia).

Figure 5. Intermittent hypoxia increased the MCP-1-enhanced adhesion of monocytes to vascular endothelial cells.

THP-1 cells pretreated with normoxia or intermittent hypoxia were activated by 20 ng/ml MCP-1 for another 24 hours, and then processed for cell adhesion assay. (A) Representative photos for THP-1 cells after cell adhesion assay. Adhered cells were indicated by black arrow. (Normoxia: without any treatment, Normoxia + MCP-1: with MCP-1 stimulation only, Intermittent hypoxia: with intermittent hypoxia pretreatment only, Intermittent hypoxia + MCP-1: with intermittent hypoxia pretreatment and MCP-1 stimulation) (B) Statistical results from three independent experiments were shown. Data were means and standard errors. (mean ± SE, *: P<0.05 vs. Normoxia, †: P<0.05 vs. Normoxia + MCP-1, ‡: P<0.05 vs Intermittent hypoxia).

Figure 6. Intermittent hypoxia induced the activation of p44/42 and p38 MAPK signal pathways in THP-1 cells.

THP-1 cells were treated with normoxia or intermittent hypoxia and cytosolic proteins were collected at 1, 3 and 6 hr for western blot analysis. The time-dependent activation of (A) p44/42 MAPK or (B) p38 MAPK through phosphorylation by intermittent hypoxia was investigated in THP-1 cells. Monocytes were pretreated with (C) PD98059 or (D) MSB202190 to inhibit p44/42 or p38 MAPK respectively for 1 hr and then the CCR2 mRNA expression was induced by intermittent hypoxia. (N: normoxia; IH: intermittent hypoxia) (mean ± SE, *: P<0.05 vs. Normoxia, †: P<0.05 vs Intermittent hypoxia + PD98059, ‡: P<0.05 vs Intermittent hypoxia + MSB202190).