Background— Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. Methods and Results— Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64–0.96]; P =0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke ( P <0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the l -arginine:glycine amidinotransferase ( AGAT ) gene ( P <2.1×10 −8 ; n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT −/− ) and (2) a guanidinoacetate N -methyltransferase deletion (GAMT −/− ) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT −/− mice and increased in GAMT −/− mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT −/− mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT −/− mice was decreased compared with controls. Conclusions— Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Objectives The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous NOS inhibitors ADMA and L-NMMA. Methods and results We generated a global-DDAH1 gene deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation, and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several fold higher than in wild type mice, but growth and development of these DDAH1−/− mice was similar to their wild type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ~20 mmHg higher in the DDAH1−/− mice than in wild type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+ mice, DDAH1+/− mice and DDAH1−/− mice at anticipated ratios of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusions Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, while DDAH2 had no detectable role for degrading ADMA and L-NMMA.

BackgroundDisrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity.ObjectivesThis study evaluates the potential of TRAF-STOP treatment in atherosclerosis.MethodsThe effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe−/−) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages.ResultsTRAF-STOP treatment of young Apoe−/− mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe−/− mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair “classical” immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe−/− mice.ConclusionsTRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.