Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis

Abstract

Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe^-/-) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe^-/- mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe^-/- mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.

Keywords: AGXT; CCL5; amino acids; atherosclerosis; glycine; mitochondrial dysfunction; oxalate.

Figure 1.. Dysregulated Glycine and Oxalate Metabolism in Patients and Mice with Atherosclerosis

(A) Schematic representation of glycine metabolic pathways. (B-E) Targeted metabolomics assessing the ratios of glycine to (B) serine, (C) threonine, (D) alanine and (E) oxalate in serum from age- and sex-matched patients with or without sCAD (n=24). (F) En face analysis of atherosclerotic lesions in male Apoe^−/− mice fed a standard diet (SD) or Western diet (WD) for 12 weeks (n=5). (G-J) Targeted metabolomics assessing the ratios of glycine to (G) serine, (H) threonine, (I) alanine and (J) oxalate in plasma from male Apoe^−/− mice fed a SD or WD for 12 weeks (n=5). (K) Western blot analysis of AGXT protein abundance in livers from Apoe^−/− mice fed a SD or WD for 12 weeks (n=5). Mann-Whitney U test for B-E, G, H and K. Unpaired t test for F, I, J. Data are presented as mean ± SEM. All points and P values are shown.

Figure 2.. AGXT Deficiency Exacerbates Atherosclerosis in Male Apoe^−/− Mice

(A) Western blot analysis confirming the loss of AGXT in livers from male Agxt^−/−/Apoe^−/− mice (n=6). (B) Glycine to oxalate ratio in plasma from male Agxt^−/−/Apoe^−/− mice and their Agxt^+/+/Apoe^−/− littermates (n=9). (C-G) Male Agxt^−/−/Apoe^−/− (n=12) and Agxt^+/+/Apoe^−/− mice (n=10) were fed a WD for 12 weeks: (C) plasma total cholesterol (TC), (D) atherosclerosis in the aortic tree, (E) H&E staining, (F) Oil Red O (ORO) staining, and (G) Mac2 immunohistochemistry of the aortic sinus. (scale bar: 200 μm). Unpaired t test for B-C and E-G. Mann-Whitney U test for D. Data are presented as mean ± SEM. All points and P values are shown.

Figure 3.. Oxalate homeostasis is maintained and atherosclerosis is unaltered in female Agxt^−/−/Apoe^−/− mice

(A) Western blot analysis confirming the loss of AGXT in livers from female Agxt^−/−/Apoe^−/− mice (n=6). (B) Glycine to oxalate ratio in plasma from female Agxt^−/−/Apoe^−/− and Agxt^+/+/Apoe^−/− mice (n=10). (C-G) Female Agxt^−/−/Apoe^−/− and Agxt^+/+/Apoe^−/− mice were fed a WD for 12 weeks (n=10): (C) plasma total cholesterol (TC), (D) atherosclerosis in the aortic tree, (E) H&E staining, (F) Oil Red O staining, and (G) Mac2 immunohistochemistry of aortic sinus. (scale bar: 200 μm). Unpaired t test for B, D, E and G. Mann-Whitney U test for C and F. Data are presented as mean ± SEM. All points and P values are shown.

Figure 4.. Dysregulated Oxalate Metabolism Induces a Pro-inflammatory Response and CCL5 Release

(A-C) RNA-sequencing of livers collected from male Agxt^−/−/Apoe^−/− and Agxt^+/+/Apoe^−/− mice fed a WD for 12 weeks (n=5): (A) Volcano plot of DEGs (padj <0.05, log2 fold change >1) in male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (Blue: downregulated; Red: upregulated). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG)-based pathway analysis. The significance of the enrichment was determined by right-tailed Fisher’s exact test followed by Benjamini-Hochberg multiple testing adjustment. (C) Heatmap-based representation of 50 DEGs linking inflammation, fibrogenesis and the metabolism of lipids and sterols/steroids with atherosclerosis. (D) qPCR analyses using independent samples of DEGs regulating inflammatory responses in livers from male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=9). Gene expression levels were normalized to 18S. (E) CCL2 concentrations in plasma from male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=10). (F) CCL5 concentrations in plasma from male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=10). (G) qPCR analyses of genes regulating inflammatory responses in livers from female Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=9). Gene expression levels were normalized to 18S. (E) CCL2 concentrations in plasma from female Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=10). (F) CCL5 concentrations in plasma from female Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=10). Statistical differences in gene expression (D and G) were tested using unpaired t test or Mann-Whitney U test, depending on normality tests. *P<0.05, **P<0.01, ***P<0.001 vs. Agxt^+/+/Apoe^−/− mice. Unpaired t test for E and H. Mann-Whitney U test for F and I. Data are presented as mean ± SEM.

Figure 5.. Oxalate Overload Induces Mitochondrial Dysfunction and Overproduction of Superoxide Leading to CCL5 Release in Macrophages

(A) qPCR analyses of genes regulating redox homeostasis in livers from male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=9). Gene expression levels were normalized to 18S. (B) DHE fluorescence and Mac-2 immunofluorescence in the aortic sinuses of male Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (scale bar: 50 μm. n=9). (C) qPCR analyses of genes regulating redox homeostasis in livers from female Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (n=9). Gene expression levels were normalized to 18S. (D) DHE fluorescence and Mac-2 immunofluorescence in the aortic sinuses of female Agxt^−/−/Apoe^−/− vs. Agxt^+/+/Apoe^−/ mice (scale bar: 50 μm. n=10). (E) Oxygen consumption rate (OCR) measured in BMDM treated with 0.75 mM NaOX for 30 min and in control cells (CTL) using Seahorse extracellular flux analyzer (n=16). Oligomycin, FCCP and rotenone+antimycin A (R/A) were used at final concentrations of 1.5 μM, 1 μM and 0.5 μM, respectively. (F) Mitochondrial superoxide measured in BMDM treated with 0.75 mM NaOX for 30 min using the MitoSOX Red probe (n=12, scale bar: 50 μm). (G) Medium concentration of CCL5 in BMDM treated with 0.75 mM NaOX for 18 h in the absence or presence of mitoTEMPO (MT, 10 μM, n=12–20). Statistical differences in gene expression (A and C) were tested using unpaired t test or Mann-Whitney U test, depending on normality tests. *P<0.05, **P<0.01, ***P<0.001 vs. Agxt^+/+/Apoe^−/− mice. Unpaired t test for B, D and F. Mann-Whitney U test for E. Kruskal-Wallis test followed by Dunn’s post-hoc test for G. Data are presented as mean ± SEM. All points are shown.

Figure 6:. AAV-mediated Overexpression of AGXT Reduces Oxidative Stress and Inflammation

AAV-AGXT or AAV-GFP were injected into male Apoe^−/− mice and the mice were fed a WD for 12 weeks (n=8). (A) Western blot analysis confirming the overexpression of AGXT in livers from mice treated with AAV-AGXT. (B) Plasma glycine/oxalate ratio. (C-D) qPCR analyses of genes regulating (C) redox homeostasis and (D) inflammatory responses. Gene expression levels were normalized to 18S. (E-F) Plasma concentrations of (E) CCL2 and (F) CCL5. (G) DHE fluorescence and Mac-2 immunofluorescence in the aortic sinuses (scale bar: 50 μm). Data are presented as mean ± SEM. All points are shown. *P<0.05, **P<0.01, ***P<0.001 vs. AAV-GFP.

Figure 7:. AAV-mediated Overexpression of AGXT Reduces Atherosclerosis Independent of Circulating Cholesterol

AAV-AGXT or AAV-GFP were injected into male Apoe^−/− mice and the mice were fed a WD for 12 weeks (n=8). (A) Plasma total cholesterol (TC). (B) Atherosclerosis in the aortic tree. (C) H&E staining, (D) Oil Red O staining, (E) Mac2 immunohistochemistry of aortic sinus. (scale bar: 200 μm). (F) Proposed model of dysregulated oxalate metabolism as a driver and therapeutic target in atherosclerosis. Unpaired t test for A-E. Data are presented as mean ± SEM. All points and P values are shown.