Schwedhelm, Edzard

Abstract Background Atherosclerosis is a chronic inflammatory disease and the primary underlying cause of cardiovascular disease (CVD). Recently, intracellular metabolic pathways have been identified as master switches of immune cell function and thus seem promising targets for therapeutic interventions aimed at lowering inflammation and thereby atherogenesis. Decades of research have demonstrated the importance of the amino acid (AA) L-arginine (Arg) in CVD. Although most research has focused on endothelium-dependent effects of Arg by the nitric oxide synthase (NOS), Arg metabolism - mainly through its metabolizing enzyme arginase 1 (Arg1) - has also been shown to regulate immune cell responses independent of its endothelial functions. Despite these findings, the cell-specific contribution of Arg/Arg1 metabolism on atherogenesis is still largely unknown. Purpose Profiling the Arg metabolism in endothelial and immune cells to identify cell-specific changes during athero-progression. Methods To investigate Arg metabolism in atherogenesis, we fed apolipoprotein E deficient (Apoe-­/­-) mice a Western diet (WD) for 6 and 12 weeks. Chow diet-fed aged-matched Apoe-­/-­ mice were used as littermate controls and C57BL/6 mice as steady-state controls. Systemic and local Arg concentrations were measured by mass-spectrometry. Arg metabolism in organs was studied by qPCR. Arg1 expression in atherosclerotic lesions was examined by immunohistochemistry (IHC). To assess cell-specific expression of Arg-related enzymes, aortic leukocytes from WD-fed Apoe-­/-­ mice were subjected to CITE-seq analysis. The role of T cell Arg1 was determined by nor-NOHA inhibition in Jurkat cells. Results AA measurements showed that systemic Arg was reduced in early and advanced atherogenesis (p<0.02), and splenic Arg was reduced in advanced atherogenesis (p=0.006) compared with steady state. In advanced atherosclerosis, Arg1 was decreased in the liver but increased in the spleen and aorta, whereas Nos2 was increased in the liver and aorta compared with steady state. To identify the cell types driving these local changes, we performed IHC on atherosclerotic lesions. Arg1+ lesional macrophages accumulated in advanced compared to early lesions (p=0.03), whereas Arg1+ endothelial cells tended to decrease (p=0.2). Surprisingly, we also found an accumulation of Arg1+ CD4+ T cells in lesions and a further upward trend with athero-progression. CITE-seq confirmed a constitutive expression of Arg1 in lymphoid clusters. In vitro experiments with Jurkat cells showed that arginase inhibition suppressed T cell proliferation and promoted apoptosis and migration (all p<0.0001). Conclusion Our data indicate that Arg metabolism is strongly regulated during athero-progression and suggest a so far unknown role of T cell-intrinsic Arg/Arg1 metabolism in the disease. Murine atherosclerosis studies are now being performed to decipher the cell-specific contribution of Arg/Arg1 metabolism in atherosclerosis.

Background: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. Methods: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry–based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. Results: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3 + T cells in the atherosclerotic lesions suggested a T-cell–related effect of homoarginine supplementation, which was mainly attributed to CD4 + T cells. Macrophages, dendritic cells, and B cells were not affected. CD4 + T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. Conclusions: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.