Gut microbiota mediate vascular dysfunction in a murine model of sleep apnoea: effect of probiotics

Abstract

Background: Obstructive sleep apnoea (OSA) is a chronic prevalent condition characterised by intermittent hypoxia (IH), and is associated with endothelial dysfunction and coronary artery disease (CAD). OSA can induce major changes in gut microbiome diversity and composition, which in turn may induce the emergence of OSA-associated morbidities. However, the causal effects of IH-induced gut microbiome changes on the vasculature remain unexplored. Our objective was to assess if vascular dysfunction induced by IH is mediated through gut microbiome changes.

Methods: Faecal microbiota transplantation (FMT) was conducted on C57BL/6J naïve mice for 6 weeks to receive either IH or room air (RA) faecal slurry with or without probiotics (VSL#3). In addition to 16S rRNA amplicon sequencing of their gut microbiome, FMT recipients underwent arterial blood pressure and coronary artery and aorta function testing, and their trimethylamine N-oxide (TMAO) and plasma acetate levels were determined. Finally, C57BL/6J mice were exposed to IH, IH treated with VSL#3 or RA for 6 weeks, and arterial blood pressure and coronary artery function assessed.

Results: Gut microbiome taxonomic profiles correctly segregated IH from RA in FMT mice and the normalising effect of probiotics emerged. Furthermore, IH-FMT mice exhibited increased arterial blood pressure and TMAO levels, and impairments in aortic and coronary artery function (p<0.05) that were abrogated by probiotic administration. Lastly, treatment with VSL#3 under IH conditions did not attenuate elevations in arterial blood pressure or CAD.

Conclusions: Gut microbiome alterations induced by chronic IH underlie, at least partially, the typical cardiovascular disturbances of sleep apnoea and can be mitigated by concurrent administration of probiotics.

Fig.1:. Alterations in fecal bacterial composition between IH and RA FMT with or without probiotic treatment.

Principal coordinate analysis plots ordinated using weighted UniFrac (A) and unweighted UniFrac (B) distances. Relative abundance of the five distinct ASVs found differentially abundant by ANCOM and ALDeX2 (C). FMT: fecal matter transplantation, IH: intermittent hypoxia, PRO: probiotic, RA: room air. Statistical analysis was done using two-way ANOVA followed by Tukey post-test •p<0.05 vs. IH-FMT, *p < 0.05 vs. IH-FMT-PRO, ^#p < 0.05 vs. RA-FMT, ^$p < 0.05 vs. RA-FMT-PRO. FMT: fecal matter transplantation, IH: intermittent hypoxia, PRO: probiotic, RA: room air, VEH: vehicle.

Fig. 2:. FMT from IH-exposed mice elevates BP in naïve mice, and such BP changes are prevented by VSL3 probiotic treatment.

Systolic blood pressure (SBP) (A), diastolic blood pressure (DBP) (B), mean blood pressure (MBP) (C). Values are displayed as mean ± S.D (n = 10) mice. Statistical analysis was done using two-way ANOVA followed by Tukey post-test *p < 0.05 vs. IH-FMT-PRO, ^#p < 0.05 vs. RA-FMT, ^$p < 0.05 vs. RA-FMT-PRO. FMT: fecal matter transplantation, IH: intermittent hypoxia, PRO: probiotic, RA: room air, VEH: vehicle

Fig. 3:. FMT from IH-exposed mice impairs coronary arteries function in naïve mice and VSL3 probiotic administration prevents such effects.

Cumulative concentration response curve of U46619 (A), acetylcholine (ACh) (B), and sodium nitroprusside (SNP) (C). Values are displayed as mean ± S.D (n = 9 −10) mice. Statistical analysis was done using two-way ANOVA followed by Tukey post-test *p < 0.05 vs. IH-FMT-PRO, ^#p < 0.05 vs. RA-FMT, ^$p < 0.05 vs. RA-FMT-PRO. FMT: fecal matter transplantation, IH: intermittent hypoxia, PRO: probiotic, RA: room air, VEH: vehicle

Fig. 4:. FMT from IH exposed mice increases TMAO plasma levels and decreases acetate plasma levels, and VSL3 probiotics restore TMAO but not acetate plasma concentrations.

Plasma TMAO levels (A), Plasma acetate concentrations (B). Values are displayed as mean ± S.D (n = 5) mice. Statistical analysis was done using two-way ANOVA followed by Tukey post-test *p < 0.05 vs. IH-FMT-PRO, ^#p < 0.05 vs. RA-FMT, ^$p < 0.05 vs. RA-FMT-PRO. FMT: fecal matter transplantation, IH: intermittent hypoxia, PRO: probiotic, RA: room air, TMAO: trimethylamine N-oxide, VEH: vehicle

Fig. 5:. VSL3 probiotic administration does not lower elevated BP or prevent coronary artery dysfunction in mice exposed to IH.

Mean arterial blood pressure (MBP) (A), Cumulative concentration response curve of U46619 (B), acetylcholine (ACh) (C), and sodium nitroprusside (SNP) (D). Values are displayed as mean ± S.D (n = 8–10) mice. Statistical analysis was done using two-way ANOVA followed by Tukey post-test *p < 0.05 vs. RA. IH: intermittent hypoxia, PRO: probiotic, RA: room air