Gut Microbial Associations To Serum Metabolites Linked To Atherosclerosis Severity And Major Adverse Cardiac Event Risk

Background:Recent studies have shown that gut microbiota alterations is associated with coronary artery disease(CAD)in different populations.Gut microbe-derived metabolites such as trimethylamine N-oxide(TMAO),has been proven to promote the onset of atherosclerosis in animal experiment and is a prognostic marker of adverse cardiovascular events.However,the characteristics of gut microbiome in different stages of CAD pathophysiology remain to be established by a systematic study.And few studies aim to perform longitude cohort study and put effort in dissecting prognostic value of gut microbial markers on cardiovascular disease outcomes.Methods:In the first exploration part of the baseline period,we conducted multi-omics analysis(16S rRNA gene sequencing of the V3-V4 region and non-targeted metabolomics)on 201 subjects.We found that both the composition of the intestinal microbiota and serum metabolites profiles changed significantly with CAD severity.In the second part,fecal mixtures from healthy controls and CAD patients were transplanted into germ free C57BL/6 J mice and fed on high-fat diet for 12 weeks.We aimed to evaluate the influence of CAD-related intestinal microbiota on bile acid metabolism and serum cholesterol levels,meanwhile evaluated the systematic inflammatory and intestinal immune response through transcriptome sequencing and flow cytometry.Subsequently,we continued to enroll in the expansion cohort of CAD patients.Metagenomic analysis was performed on the expanded cohort containing 319 subjects,and longitudinal follow-ups were launched.We built random survival forest model identifying microbial prognostic markers related to adverse cardiovascular outcomes.Fecal microbiota transplantation experiment was performed on ApoE-/-mice to explore the promotion of atherosclerosis and inflammation phenotypes of the disordered CAD-associated gut microbiota.Finally,single strain colonization procedures demonstrated the effect of prognostic-related bacteria on platelet function and thrombus phenotype.Results:In the first phase of the analysis including 201 subjects,we identified 29 metabolite modules that were either positively or negatively correlated with the CAD severity phenotype,as well as microbial co-abundance group(CAGs)that were characteristically enriched in different subgroups of CAD.Intestinal bacteria with significant changes such as Roseburia,Ruminococcaceae and Clostridium ? affect the development of atherosclerosis by regulating the metabolic activity of bile acids and aromatic compounds.The disease classifier constructed based on these characteristics of bacteria and metabolites could accurately distinguish and diagnosed different subgroups of CAD in another independent validation cohort.Through animal experiments in the second part,we found that the recipient mice colonized with CAD associated microbiota showed enhanced vascular stiffness compared to healthy controls.The CAD mice exhibited more enriched abundance of Clostridium symbiosum and Eggerthella,which modulated the secondary bile acids pool,leading to an increase in lithocholic acid and keto-derivatives and resulted in elevated circulatory cholesterol.In addition,we observed that CAD-microbiota promote the abnormal immune response of the intestinal lamina propria with Th17/Treg imbalance,and deteriorated the permeability of the intestinal barrier.In the metagenomic study of the third part,we conducted an in-depth analysis at the strain level to further prove that the diversity and composition of gut microbiota were closely related to the CAD severity.By fecal microbiota transplantation experiments,we proved that the long-term colonization of fecal bacteria from CAD patients exacerbated the formation of atherosclerotic plaque and plaque instability in the aorta.Compared with healthy controls,disordered CAD-related microorganisms produce higher levels of lipopolysaccharide,which act on the aortic TLR4-CD14 signaling pathway,causing the activation of vascular and systemic immune inflammatory responses.We observed an increase in the proportion of aortic neutrophils and activation of CD4+ T lymphocytes,as well as an immune imbalance in the spleen characterized by an increase of Th1/Th2 ratio.Importantly,we evaluated the microbial characteristics predicting major adverse cardiovascular events,and verified the prognostic-related bacterial marker Bacteroides thetaiotaomicron has the potential to promote thrombosis and stimulate platelet activation.Conclusions:Gut microbiota and relative metabolites were closely related to CAD severity.The disordered gut microbiota of CAD contributed to the occurrence and development of atherosclerosis.The key members of the bacterial and metabolic features may become biomarkers for predicting the cardiovascular disease outcomes.The current study provides ideas for understanding the interaction of host-intestinal microbiota in the pathogenesis of atherosclerosis and for the development of personalized secondary prevention in the future.