The Role Of Glycine/oxalate Metabolism In Atherosclerosis

BackgroundAtherosclerosis,characterized by lipids accumulation in the artery wall,is the underlying cause of most ischemic cardiovascular diseases（CVD）.In recent years,with the development and wide application of metabolomics,more and more metabolites besides lipids have been related to the development of atherosclerosis and CVD,among which amino acids are an important category.The inverse correlations between circulating glycine levels and CVD have been reported by several prospective studies and meta-analyses.However,current researches focus on association analysis.The roles and mechanisms of glycine metabolism in atherosclerosis and CVD remain to be explored.Objectives1.To clarify the changes of glycine metabolism in atherosclerosis.2.To explore the role and possible mechanisms of glycine/oxalate metabolism in atherosclerosis.3.To explore the feasibility of glycine/oxalate metabolism as a therapeutic target for CVD.Methods1.The study enrolled patients with coronary artery stenosis（CAS）and age/gender-matched subjects without CAS.Liquid chromatography tandem-mass spectrometry（LC-MS/MS）was used to detect glycine and its related metabolites in the circulation of the two groups of subjects.2.Establish mouse atherosclerosis models with Apoe^-/-mouse and high-fat Western diet（WD）feeding.Knocking out Agxt which encoded for the key enzyme alanine glyoxylate aminotransferase in glycine/oxalate metabolism was used to induce glycine/oxalate metabolism dysregulation in mice.Supplementing hydroxy-L-proline into a normal Western diet（WD+HLP）was applied to induce oxalate overload.Construct an adeno-associated virus（AAV）vector overexpressing AGXT protein to correct glycine/oxalate metabolism dysregulation.Bone marrow-derived macrophages（BMDM）from mice were used as the in vitro model.3.Analyze the degree of atherosclerotic lesions with both En face oil red O staining of the whole aorta and H&E staining,oil red O staining,immunohistochemistry,and immunofluorescence of aortic sinus sections.RNAseq,q PCR,western blotting,immunofluorescence,enzyme-linked immunosorbent assay,seahorse analysis,Mito SOX staining,etc.,were applied to explore the mechanisms of glycine/oxalate metabolism dysregulation in atherosclerosis.Results1.After age and gender matching,the ratios between glycine and its related metabolites serine,threonine,and oxalate in patients with CAS were significantly lower than those in subjects without CAS.Atherosclerotic mice had significantly decreased glycine to oxalate ratio with downregulations of the key enzyme AGXT in livers.2.Compared with male Agxt^+/+Apoe^-/-mice,male Agxt^-/-Apoe^-/-mice had significantly lower plasma glycine to oxalate ratios,higher total cholesterol levels,and exacerbated atherosclerosis.However,female Agxt^-/-Apoe^-/-and Agxt^+/+Apoe^-/-mice showed comparable plasma glycine to oxalate ratios,total cholesterol levels,and atherosclerotic lesions.Moreover,Apoe^-/-mice receiving WD+HLP exhibited dysregulated glycine/oxalate metabolism,higher plasma total cholesterol levels,and severer atherosclerosis compared to those fed WD.3.Male Agxt^-/-Apoe^-/-mice showed significantly increased systemic and local inflammation,and enhanced oxidative stress in comparison to male Agxt^+/+Apoe^-/-mice.Female Agxt^-/-Apoe^-/-and Agxt^+/+Apoe^-/-mice had no significant differences in inflammation and oxidative stress indexes.Compared to WD mice,Apoe^-/-mice in the WD+HLP group exhibited enhanced inflammation and oxidative stress.4.Oxalate stimulation impaired mitochondria respiration in BMDMs,and significantly increased superoxide levels in mitochondria.Mitochondria dysfunction stimulates inflammation and CCL5 release from BMDMs.5.Overexpression of AGXT protein via AAV in Apoe^-/-mice significantly increase plasma glycine to oxalate ratios,attenuated inflammation and oxidative stress,and alleviated atherosclerosis.Conclusions1.Glycine/oxalate metabolism dysregulation was identified in both patients with CAS and atherosclerotic mice.2.Glycine/oxalate metabolism dysregulation induced by Agxt knocking out or HLP supplementation enhanced inflammation and oxidative stress,and exacerbated atherosclerosis in mice.3.Correction of glycine/oxalate metabolism dysregulation via AGXT overexpression attenuated atherosclerosis in mice.Figures 58,Tables 7,References 108...