We demonstrate a critical role for endothelial cell Gch1/BH₄ biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH₄ leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH₄ biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Abnormal uteroplacental remodeling leads to placental hypoperfusion, causing fetal growth restriction and pregnancy-related hypertension, which are associated with endothelial dysfunction and markers of reduced vascular NO bioavailability and oxidative stress. Tetrahydrobiopterin (BH4) is a redox cofactor for eNOS (endothelial NO synthase) with a required role in NO generation. Using mice models and human samples, we investigated the physiological requirement for endothelial cell BH4 in uteroplacental vascular adaptation and blood pressure regulation to pregnancy. In pregnant mice, selective maternal endothelial BH4 deficiency resulting from targeted deletion of Gch1 caused progressive hypertension during pregnancy and fetal growth restriction. Maternal endothelial cell Gch1 deletion caused defective functional and structural remodeling in uterine arteries and in spiral arteries, leading to placental insufficiency. Using primary endothelial cells isolated from either normal or hypertensive pregnancies, we found that hypertensive pregnancies are associated with reduced endothelial cell BH4 levels, impaired eNOS activity, and reduced endothelial cell proliferation, mediated by reduced GTPCH (GTP cyclohydrolase 1) protein. In rescue experiments, high blood pressure and fetal growth restriction in pregnant endothelial cell Gch1 deficient mice was not rescued by oral BH4 supplementation, due to systemic oxidation of BH4 to dihydrobiopterin. However, the fully reduced folate, 5-methyltetrahydrofolate prevented BH4 oxidation, reduced blood pressure to normal levels, and normalized fetal growth. We identify a critical requirement for maternal endothelial cell BH4 biosynthesis in uteroplacental vascular remodeling in pregnancy. Restoration of endothelial cell BH4 with reduced folates identifies a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension such as preeclampsia.

Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation. Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined. Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days. Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin. We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.

Nitric oxide (NO) has fundamental roles in numerous physiological and pathophysiological processes. In macrophages, NO produced by inducible nitric oxide synthase (iNOS) modulates metabolic changes that are essential to macrophage activation and plasticity, driving the characteristic metabolic switch from oxidative phosphorylation to glycolysis ^1,2 . Itaconate, derived from the TCA cycle by decarboxylation of cis -aconitate by IRG1 (also referred to as CAD, ACOD1), is one of the most upregulated metabolites during the inflammatory response ³ . Itaconate regulates macrophage polarization by electrophilically modifying cysteines of key enzymes that control inflammatory states (such as ATF3, Jak1, IFNβ), participate in glycolysis (for example, GAPDH, LDHA) and limit oxidative stress through structural competitive inhibition of succinate dehydrogenase ^4–9 . We recently reported that macrophages that are deficient in iNOS, and subsequent NO generation, produce strikingly higher levels of intracellular itaconate (up to ~15-fold) compared to wild-type cells when stimulated with inflammatory cytokines ^1,2,10 . Here we show that iNOS inhibits IRG1 activity and itaconate levels through a conformation-dependent protein–protein interaction rather than through the production of NO. Using a variety of biochemical and computational approaches, we show that a direct interaction between iNOS and IRG1 occurs within mitochondria, in mouse and human cells, and that it depends on binding of the cofactor BH4 to iNOS but does not require its capability to produce NO. Our findings reveal a non-canonical cellular function for iNOS that places it at the centre of a signalling hub, linking redox signalling and metabolism to modulation of the inflammatory response in macrophages.

BACKGROUND: Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. METHODS: We analyzed the maternal blood pressure in pregnant wild-type ( Gch1 ^fl/fl ) and Gch1 ^fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. RESULTS: In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. CONCLUSIONS: Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4.