The inflammatory preatherosclerotic remodeling induced by intermittent hypoxia is attenuated by RANTES/CCL5 inhibition

Abstract

Rationale: The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients.

Objectives: To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA.

Methods: We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure.

Measurements and main results: IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia.

Conclusions: Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.

Figure 1. Intermittent hypoxia induces splenocyte activation

Splenocyte proliferation in response to concanavalin-A after 5 (A) and 14 (B) days of intermittent hypoxia (IH) or air (N); *p<0.05 vs N (n=6–12 per group). Splenocyte mRNA expression of RANTES/CCL5 (C), MIP-1α/CCL3 (D), MIP-1β/CCL4 (E) and MCP-1/CCL2 (F). Measurements were normalized to the eukaryotic 18S ribosomal-RNA (G) and expressed as fold induction of their baseline values; *p<0.05 vs N (n=6 per group). Splenocyte migration toward RANTES/CCL5 after 14 days of IH or air (H). Splenocytes were tested without stimulation or after 50 ng.ml−1 interferon-gamma (IFN-γ) stimulation; p<0.05 vs unstimulated* or stimulated† splenocytes from N-mice (n=4).

Figure 2. Intermittent hypoxia increases leukocyte-endothelium adhesive interactions

Leukocyte-microvessel interplay was assessed by leukocyte rolling and ICAM-1 expression in mesenteric vessels from mice exposed to intermittent hypoxia (IH) or air (N) for 14 days. (A) Representative photographs showing leukocyte rolling (arrow). (B) Rolling quantification (n=4–5 each). Representative immunoblotting (C) and quantitative analysis (D) of ICAM-1 expression (n=4 each).

Figure 3. Intermittent hypoxia induces structural aorta remodeling

Vascular remodeling was assessed in mice exposed to intermittent hypoxia (IH) or air (N) for 14 days. (A) Hematoxylin-eosin stainings (10x10 magnification and digitally magnified insets). (B) Histomorphometric analysis of intima-media thickness, n=7 each. (C) Verhoeff coloration showing thicker elastic fibers in IH-mice (10x40 magnification). Elastic fiber separation (D) and smooth-muscle cell (SMC) nuclei (E) in the media (n=7 each). (F) Alcian blue coloration showing mucoid depositions between the elastic fibers in IH-mice (arrows, 10x40 magnification).

Figure 4. Intermittent hypoxia induces aorta inflammation

Inflammation was assessed in mice exposed to intermittent hypoxia (IH) or air (N) for 14 days. (A) CD3 immunostaining with arrows showing T-cells (10x20 magnification). (B) Quantitative analysis of CD3-positive cell infiltration according to the various tunica of the aortic wall. Note that T-cells predominated in the adventitia-periadventitia tunica (n=9–10 per group). (C) Representative RANTES/CCL5 immunostaining (10x20 magnification). Immunoblottings and quantifications of nuclear NFkB-p50 (D) and cytosolic ICAM-1 (E) (n=4 each).

Figure 5. RANTES/CCL5 neutralization attenuates IH-induced aorta remodeling

Mice were exposed to intermittent hypoxia (IH) or air (N) for 14 days, and treated either with the anti-RANTES/CCL5 monoclonal antibody or with the control IgG throughout the exposure. (A) Intima-media thickness (n=7–8 each). (B) Cytosolic α-smooth muscle actin (α-SMA) expression with representative immunoblotting and quantitative analysis (n=5–7 each). (C) Nuclear NFkB-p50 expression with representative immunoblotting and quantitative analysis (n=4–7 each). (D) Quantification of T-cell infiltration in the aortic wall (n=7–8 each). (E) Representative RANTES/CCL5 immunostaining (10x20 magnification). (F) IFNγ mRNA expression normalized to ubiquitin (n=4–9 each). *p<0.05 vs N-IgG; †p<0.05 vs anti-RANTES treated IH-mice. (G) Regulation pathway of NFkB activation and subsequent leukocyte recruitment and activation (adapted from Ye et al, (44)).