The gut microbe-derived metabolite trimethylamine-N-oxide induces aortic valve fibrosis via PERK/ATF-4 and IRE-1/XBP-1s signaling in vitro and in vivo

Background and aims: The gut microbe -derived metabolite trimethylamine-N-oxide (TMAO) has been implicated in the development of cardiovascular fibrosis. Endoplasmic reticulum (ER) stress occurs after the dysfunction of ER and its structure. The three signals PERK/ATF-4, IRE-1 alpha/XBP-1s and ATF6 are activated upon ER stress. Recent reports have suggested that the activation of PERK/ATF-4 and IRE-1 alpha/XBP-1s signaling contributes to cardiovascular fibrosis. However, whether TMAO mediates aortic valve fibrosis by activating PERK/ATF-4 and IRE-1 alpha/XBP-1s signaling remains unclear. Methods: Human aortic valve interstitial cells (AVICs) were isolated from aortic valve leaflets. PERK IRE-1 alpha, ATF4, XBP-1s and CHOP expression, and production of collagen I and TGF-81 were analyzed following treatment with TMAO. The role of PERK/ATF-4 and IRE-1 alpha/XBP-1s signaling pathways in TMAO-induced fibrotic formation was determined using inhibitors and small interfering RNA. Results: Diseased valves produced greater levels of ATF-4, XBP-1, collagen I and TGF-81. Interestingly, diseased cells exhibited augmented PERK/ATF-4 and IRE-1 alpha/XBP-1s activation after TMAO stimulation. Inhibition and silencing of PERK/ATF-4 and IRE-1 alpha/XBP-1s each resulted in enhanced suppression of TMAO-induced fibrogenic activity in diseased cells. Mice treated with dietary choline supplementation had substantially increased TMAO levels and aortic valve fibrosis, which were reduced by 3,3-dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) treatment. Moreover, a high-choline and high -fat diet remodeled the gut microbiota in mice. Conclusions: TMAO promoted aortic valve fibrosis through activation of PERK/ATF-4 and IRE-1 alpha/XBP-1s signaling pathways in vitro and in vivo. Modulation of diet, gut microbiota, TMAO, PERK/ATF-4 and IRE1-alpha/XBP1s may be a promising approach to prevent aortic valve fibrosis.