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. 2014 Nov 1;37(11):1825-32.
doi: 10.5665/sleep.4180.

Altered in vitro endothelial repair and monocyte migration in obstructive sleep apnea: implication of VEGF and CRP

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Altered in vitro endothelial repair and monocyte migration in obstructive sleep apnea: implication of VEGF and CRP

Anne Briançon-Marjollet et al. Sleep. .

Abstract

Study objectives: Although obstructive sleep apnea (OSA) causes cardiovascular morbidities through atherosclerosis induced by inflammation and endothelial dysfunction, OSA patients exhibit elevated plasma vascular endothelial growth factor (VEGF), which may represent an adaptive response to intermittent hypoxia. The aims of this study were to investigate whether in vitro endothelial wound healing and monocyte migration are affected by patient serum, and to determine the implication of circulating factors (VEGF and C-reactive protein).

Patients: Serum was collected from healthy controls (HC), "healthy" OSA, and metabolic syndrome (MS) patients with or without OSA.

Measurements and results: Along with the presence of OSA and/or MS, both VEGF and hsCRP were significantly elevated in patient serum. Their specific role was tested with blocking antibodies on primary endothelial cells for wound healing assay and on human monocytes for migration assay. Endothelial wound healing was reduced with OSA compared to HC serum, and even more significantly using MS+OSA patient serum. Altered wound healing with OSA serum was unmasked when blocking VEGF and restored when blocking CRP. Monocyte migration was activated with OSA serum, and further enhanced by MS+OSA patient serum. Blocking CRP in serum inhibited this migration.

Conclusions: Serum from OSA patient alters in vitro endothelial cell repair function and activates monocyte migration; this is further aggravated with the presence of metabolic syndrome. These effects are partly driven by VEGF and CRP, suggesting an unfavorable balance between the pro healing (VEGF) and pro injury (CRP) factors that may promote vascular injury in OSA with and without metabolic syndrome.

Keywords: VEGF; endothelial repair; hsCRP; metabolic syndrome; obstructive sleep apnea.

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Figures

Figure 1
Figure 1
Serum VEGF and high sensitivity-CRP are elevated in OSA and MS patients. (A) Serum VEGF is elevated in 31 OSA patients and 13 MS patients compared to 16 non-OSA healthy controls, but not in 21 MS+OSA patients. * P < 0.05 vs controls, # P < 0.05 vs MS group, Two-way ANOVA. (B) Positive Spearman correlation between serum VEGF and time spent under 90% O2 saturation during sleep in OSA patients (P = 0.046) but not in healthy controls. (C) Positive Spearman correlation between serum VEGF and hsCRP in OSA patients and healthy controls (n = 47 subjects, P = 0.023). (D) Serum hsCRP is elevated in 31 OSA patients and 13 control MS patients compared to 16 non-OSA healthy controls, but not in 21 MS+OSA patients. * P < 0.05, Two-way ANOVA. (E) Positive Spearman correlation between hsCRP and severity of OSA measured by apnea-hypopnea index (AHI), n = 47 subjects, P = 0.015. (F) Negative Spearman correlation between hsCRP and minimal O2 saturation during sleep, both in healthy controls and OSA patients, n = 47 subjects, P = 0.003. In graphs B, C, E, and F, gray circles represent healthy controls (n = 16) and closed circles represent OSA patients (n = 31), both without MS.
Figure 2
Figure 2
Recombinant VEGF and CRP regulate endothelial repair in vitro. (A) Pictures in phase contrast microscopy (x10 magnification) showing examples of wounds at the beginning of experiment and after 7.5 h of healing by HMVEC (Bar = 50 μm). (B) VEGF (500 pg/mL) added in EBM medium activates wound healing compared to control EBM condition, and addition of anti-VEGF blocking antibody (0.5 μg/mL) or CRP (2 μg/mL) block this effect, whereas CRP alone has no significant effect. Addition of anti-CD32a (10 μg/mL) antibody reverses CRP action. One-Way ANOVA, ** P < 0.01 compared to EBM control, ## P < 0.01 compared to VEGF, $ P < 0.05 compared to VEGF+CRP. N = at least 5 independent experiments, error bars represent SEM.
Figure 3
Figure 3
Serum VEGF activates endothelial repair in vitro. (A) Pictures in phase contrast microscopy (x10 magnification) showing examples of wounds after 7.5 h of healing by endothelial cells, in presence of pooled Healthy Controls serum or pooled OSA patients serum in EBM medium. Bar = 50 μm. (B) Serum from OSA patients diminish endothelial healing compared to healthy controls serum, and anti-VEGF blocking antibody (0.5 μg/mL) but not control antibody added in OSA patient serum inhibits endothelial healing. Two-Way ANOVA, * P < 0.001 compared to control, # P < 0.05 compared to OSA without antibody or control antibody. N = at least 6 independent experiments, error bars represent SEM.
Figure 4
Figure 4
CRP in OSA patient serum inhibits endothelial repair in vitro. (A) Recombinant CRP (2 μg/mL) added in pooled serum from healthy controls inhibits endothelial healing (Mann-Whitney rank sum test, P = 0.002). (B) Blocking anti-CD32a antibody added in pooled serum from control or OSA patients induces an increase in endothelial healing by 8% and 22%, respectively. Two-Way ANOVA, * P < 0.05 vs healthy controls, # P < 0.05 vs OSA patients without antibody. (C) MS combined to OSA reduces by 18% endothelial wound healing, expressed in percent of control subjects, whereas in non-MS subjects, this decrease is only 5%. Two-way ANOVA, * P < 0.05 vs healthy controls, # P < 0.01 vs MS group. N = at least 4 independent experiments, error bars represent SEM.
Figure 5
Figure 5
CRP in OSA patient serum activates THP-1 monocyte migration in vitro. (A) Recombinant CRP (2 μg/mL) added in RPMI medium with 20% FBS activates THP1 migration through Transwells under MCP1 gradient (5 nM in lower chamber). Anti-CD32a blocking antibody (10 μg/mL) but not control antibody significantly blocks this effect. One-Way ANOVA, * P < 0.05 compared to RPMI control, # P < 0.05 compared to CRP. (B) Serum from moderate and severe but not from mild OSA patient activates monocyte migration compared to serum from healthy controls. Anti-CD32a blocking antibody (10 μg/mL) added in OSA patients serum inhibits monocyte migration. Two-Way ANOVA, * P < 0.05 compared to control without antibody, $ P < 0.05 compared to mild OSA patient, # P < 0.005 compared to respective condition without antibody. (C) Recombinant CRP (2 μg/mL) added in serum of healthy controls induces an increase in monocyte migration. Mann-Whitney test, P = 0,002. (D) OSA, MS and combination of OSA+MS induce monocyte migration, expressed in percent of control. Two-Way ANOVA, * P < 0.05 compared to healthy controls, # P < 0.05 compared to OSA only group, $ P < 0.05 compared to MS only group. N = at least 4 independent experiments, error bars represent SEM.

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