Role Of Intestinal Microbiota Metabolite TMAO In Aortic Aneurysm Progression In Marfan Syndrome Mice

[Background]Marfan syndrome is an inherited connective tissue disorder caused by mutations in the gene encoding fibrillin 1.Connective tissue throughout the body provides mechanical strength and flexibility to cells,tissues and organs.Therefore,Marfan syndrome is a multisystem disease often characterized by abnormalities of the eye,lung,skeletal muscle,and cardiovascular system.Among them,aortic aneurysm and dissection rupture are fatal complications,and inhibition of aortic dilatation is the focus of treatment.At present,there is no effective drug treatment,and it is often intervened by aortic root replacement surgery.However,due to the relatively poor overall prognosis,mechanism-orientated drug development is of enormous significance.The gut also has a rich presence of connective tissue,but the gut in Marfan syndrome has been characterized histologically or functionally.Therefore,we explored the changes of intestinal structure,intestinal bacterial community composition and metabolic pathways in Marfan syndrome mice.Trimetlylamine oxide(TMAO),as a gut-derived microbiota metabolite,has been proved to be a potential risk factor for various cardiovascular diseases and other chronic diseases in clinical and animal studies.Liquid chromatography-tandem mass spectrometry detected elevated levels of TMAO in the plasma of mice with Marfan syndrome.However,it remains unclear whether it is a causative factor contributing to aortic aneurysm progression,or just a potential pathological marker.This study,hence,investigated the role of TMAO in aortic aneurysms in Marfan syndrome.Fluoroquinolones,one of the most commonly used antibiotics,are also used in patients with aortic aneurysms.A large body of clinical data suggest that oral fluoroquinolones may increase the risk of aortic aneurysm and dissection/rupture.The deleterious effects of fluoroquinolones were also observed in mouse models of sporadic aortic aneurysm and Marfan syndrome,but the exact etiology is unclear.Long-term use of antibiotics is known to affect gut microbiota composition and its metabolites,including TMAO,prompting us to explore whether TMAO mediates ciprofloxacin-aggravated aortic aneurysm in Marfan syndrome.[Methods]1.H&E and AB-PAS staining were used to observe the changes of intestinal structure and composition in Fbn1+/+ and Fbn1C1039G/+mice.2.LC-MS/MS to analyze the composition of gut microbiota in Fbn1+/+and Fbn1C1039G/+ mice.3.TMAO-targeted metabolomic analysis of plasma TMAO and its related metabolites in Fbn1+/+ and Fby1C1039G/+ mice.4.The 6-month-old Fbn1+/+ and Fbn1C1039G/+ mice were given ciprofloxacin or vehicle for 4 months;the diameter of the aorta was detected by echocardiography,and the structure of the aorta and colon was examined by histopathology.5.Fbn1+/+ and Fbn1C1039G/+ mice aged 2-3 months were given TMAO or vehicle for 4 weeks;the diameter of the aorta was detected by echocardiography,and the structure of the aorta and colon was examined by histopathology.6.Fbn1+/+ and Fbn1C1039G1/+ mice aged 2-3 months were fed with choline,L-carnitine or vehicle,or co-administered with DMB for 4 weeks;the diameter of the aorta was detected by echocardiography,and the structure of the aorta and colon was examined by histopathology.7.Fbn1+/+ and Fbn1C1039G/+ mice aged 2-3 months were given ciprofloxacin or vehicle,or in combination with DMB for 4 weeks.Echocardiography detects aortic diameter,and histopathology examines aorta and colon structure.8.Fbn1+/+ and Fbn1C1039G/+ mice aged 2-3 months were given DMB or vehicle for 8 weeks;the diameter of the aorta was detected by echocardiography,and the structure of the aorta and colon was examined by histopathology.[Results]1.Colonic crypts in Fbn1C1039G/+ mice were significantly shortened and accompanied by inflammatory infiltration,and the secretion of acidic mucin from goblet cells in individual crypt was reduced.2.The composition of intestinal flora in Fbn1C1039G/+ mice was changed,as evidenced by up-regulation of Firmicutes and down-regulation of Bacteroidetes.3.TMAO levels in the plasma of Fbn1C1039G/+ mice was increased,and the levels of TMA,choline,carnitine and betaine were not significantly changed.4.Drinking water-supplemented TMAO significantly increased the rate of aortic dilatation in Fbn1C1039G/+ mice,and aggravated aortic wall destruction(as evidenced by elastic fiber breakdown,higher structure score and thickened medial layer),and promoted collogen deposition.5.Dietary choline and L-carnitine significantly increased the rate of aortic dilation in Fbn1C1039G/+ mice,and aggravated aortic wall destruction and collagen deposition.Co-administration of DMB could reverse the effects of choline and L-carnitine-aggravated aortic aneurysm.6.DMB inhibited the rate of progressive aortic dilatation in Fbn1C1039G/+mice,and ameliorated aortic wall destruction and collagen deposition.No histological toxicity was observed during the course DMB treatment.7.Ciprofloxacin significantly increased the incidence of aortic aneurysm-induced dissection and aorta rupture in Fbn1C1039G/+ mice,and aggravated aortic wall destruction and collagen deposition.Concurrent use of DMB reduced mortality and mitigated ciprofloxacin-aggravated aortic aneurysm.[Conclusions]1.Marfan mice display abnormal colonic structure and altered composition of intestinal flora.2.The gut microbial metabolite TMAO promotes the progression of aortic aneurysms in Marfan syndrome mice.3.The gut microbial metabolite TMAO mediates ciprofloxacinaggravated aortic aneurysm in Marfan syndrome mice.