Chronic sleep fragmentation induces endothelial dysfunction and structural vascular changes in mice

Abstract

Study objectives: Sleep fragmentation (SF) is a common occurrence and constitutes a major characteristic of obstructive sleep apnea (OSA). SF has been implicated in multiple OSA-related morbidities, but it is unclear whether SF underlies any of the cardiovascular morbidities of OSA. We hypothesized that long-term SF exposures may lead to endothelial dysfunction and altered vessel wall structure.

Methods and results: Adult male C57BL/6J mice were fed normal chow and exposed to daylight SF or control sleep (CTL) for 20 weeks. Telemetric blood pressure and endothelial function were assessed weekly using a modified laser-Doppler hyperemic test. Atherosclerotic plaques, elastic fiber disruption, lumen area, wall thickness, foam cells, and macrophage recruitment, as well as expression of senescence-associated markers were examined in excised aortas. Increased latencies to reach baseline perfusion levels during the post-occlusive period emerged in SF mice with increased systemic BP values starting at 8 weeks of SF and persisting thereafter. No obvious atherosclerotic plaques emerged, but marked elastic fiber disruption and fiber disorganization were apparent in SF-exposed mice, along with increases in the number of foam cells and macrophages in the aorta wall. Senescence markers showed reduced TERT and cyclin A and increased p16INK4a expression, with higher IL-6 plasma levels in SF-exposed mice.

Conclusions: Long-term sleep fragmentation induces vascular endothelial dysfunction and mild blood pressure increases. Sleep fragmentation also leads to morphologic vessel changes characterized by elastic fiber disruption and disorganization, increased recruitment of inflammatory cells, and altered expression of senescence markers, thereby supporting a role for sleep fragmentation in the cardiovascular morbidity of OSA.

Keywords: atherosclerosis; cell senescence; endothelial function; sleep apnea.

Figure 1

Food intake, endothelial function and blood pressure. (A) SF-induced hyperphagic behavior. C57BL/6J mice exposed to SF consumed more food on a daily basis over the 20-week period in comparison with CTL (n = 15/group). Data are expressed in grams of chow consumed per mouse per day. (B) Post-occlusive hyperemic response. Time to reach baseline perfusion levels during the post-occlusive period at time points 8, 9, 10, and 19 weeks of SF exposure (ratio between SF and CTL groups at each time point; SF vs. CTL – P < 0.03) (n = 15/group). (C) Representative 24-h blood pressure recordings in mmHg (upper tracing – systolic BP and lower tracings – diastolic BP at baseline (red lines) and following 20-week SF-exposures (black lines). Time 12-24 indicates daylight period. Bar graph indicates mean increases in systolic and diastolic BP in 3 mice following SF (P < 0.05 SF vs. baseline or CTL). (D) Post-occlusive hyperemic response. Representative example for the 3 phases of a laser Doppler post-occlusive hyperemic response test in mice exposed to SF and CTL conditions. AO, occlusion area; AH, hyperemia area; TL, time to latency; TR, time to recovery; TH1, time to half before hyperemia; TH2, time to half after hyperemia; TM, time to max; PF, peak flow; RF, rest flow; BZ, biological zero; PU, perfusion units.

Figure 2

Aortic structural changes associated with long-term SF. (A) Representative en face analysis of aortas from mice after 20-weeks SF exposures and CTL conditions (n = 5/group) using Sudan IV staining. (B) EVG staining for detection of elastic fiber disorganization and elastic fiber disruption on aorta arch from SF and CTL exposed mice. (C) Relative vessel luminal area in aortic arch and thoracic aorta after 20 weeks of SF exposures or CTL conditions (%). (D) Relative aortic wall thickness in aortic arch and thoracic aorta after 20 weeks SF exposures vs. CTL mice (%). (E) Number of fragmented elastic fibers per section after 20 weeks of SF and CTL exposures. * P < 0.01, SF vs. CTL.

Figure 3

Inflammatory cell recruitment and cell senescence markers in aorta following long-term SF. (A) SF and CTL root aortas were cryosectioned and stained with H&E for morphology and Oil Red O for foam cell detection. Consecutive cryosections were immunostained with monoclonal antibodies for detection of macrophages (F4/80) and senescence markers (TERT, p16INK4a, cyclin D1, cyclin A). Arrows indicate examples of labeled cells for each of the selected markers. (B) Plasma levels of SASPs KC/CXCL1 (pg/mL), (C) IL-6 (pg/mL) and (D) IGFBP3 (pg/mL) after 20 weeks of SF. * P < 0.01, SF vs. CTL.