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Nitric oxide (NO), produced by endothelial NO synthase (NOS) is a key regulator of vascular tone and blood pressure. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial NOS. When BH4 levels become limiting, eNOS produces superoxide anion rather than NO and in turn further reduces NO bioavailability, resulting in endothelial dysfunction. Endothelial cell BH4 has been hypothesized to be critical in maintaining vascular function, but to date in vivo models of BH4 deficiency have achieved only partial global depletion of BH4 synthesis. We have investigated the specific role of endothelial cell BH4 using a novel line of conditional knockout mice.Mice homozygous for a floxed GCH1 (GCH1fl/fl) allele (encoding for GTPCH protein an essential enzyme in BH4 biosynthesis) were crossed with Tie2cre transgenic mice to produce GCH1fl/flTie2cre line, where the GCH1 gene is knocked out specific in endothelial cells. We have shown that the GCH1 gene is completely excised in isolated endothelial cells, with an accompanying lack of BH4 production in aortas from GCH1fl/flTie2cre mice. To determine the effects of endothelial cell BH4 deficiency on NO producing activity, we measured NO bioactivity by three complementary methods, each indicating deficient, or absent endothelial NO bioactivity in GCH1fl/flTie2cre mice compared with wild-type littermates. Superoxide production assessed by dihydroethidium HPLC was significantly 4-fold greater in primary endothelial cells from GCH1fl/flTie2cre mice compared with wild-type controls, which inhibited by L-NAME.Vasomotor studies demonstrated that GCH1fl/flTie2cre aortas and mesenteric arteries had significantly enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. The NOS-derived vasodilator in GCH1fl/flTie2cre aortas was identified as H2O2 using the scavenger PEG-catalase, which inhibited vasodilatation only in GCH1fl/flTie2cre aortas. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin abolished NOS-derived H2O2 production and restored normal endothelial function in GCH1fl/flTie2cre aortas. GCH1fl/flTie2cre mice had higher systemic blood pressure than wild-type littermates (96.4±0.8 mmHg vs 103.4±1.3 mmHg; P<0.01), which was normalised by L-NAME. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure, and identify endothelial cell BH4 as a pivotal regulator of NO vs. H2O2 as alternative eNOS-derived endothelial derived relaxing factors. [ABSTRACT FROM AUTHOR]

Purpose: Diabetes can impact on cardiovascular health by causing a distinct condition termed "diabetic cardiomyopathy". Its characteristic left ventricular (LV) diastolic dysfunction has been associated with interstitial fibrosis, reduced NO availability, and abnormal calcium handling. However, the early triggers and the underlying cellular mechanisms remain unknown. Here, we investigate changes in vascular and myocardial reactive oxygen species (ROS) and NO availability in a murine model of type 1 diabetes, and evaluate potential beneficial effects of inducing a myocardial-specific increase in the NOS cofactor tetrahydrobiopterin (BH4) on the development of LV dysfunction. Methods: Diabetes was induced in male mice by daily intraperitoneal streptozotocin (STZ) injection (43mg/kg, 5 consecutive days). To augment myocardial BH4 and increase NOS activity, transgenic mice were generated with myocardial-specific overexpression of the rate-limiting enzyme for BH4 synthesis, GTP cyclohydrolase 1 (mGCH1 Tg). Vascular function in isolated aortas was evaluated by isometric tension studies (myograph), NOS activity and biopterins by HPLC, and superoxide production by lucigenin-enhanced chemiluminescence. High-resolution echocardiography was used to assess LV function. Results: After 12 weeks of diabetes, WT and mGCH1 Tg mice showed impaired aortic endothelium-dependent vasodilatation, in association with increased superoxide production and reduced BH4 bioavailability (n=6-10 per group). By contrast, diabetic LV homogenates showed no increase in superoxide generation or reduced BH4:BH2 ratio and no reduction in NOS activity (n=9-12 per group). Nevertheless, in vivo echocardiography revealed significant LV diastolic dysfunction in WT diabetic mice, which was prevented in mGCH1 Tg mice (E'/A' diabetic vs control: 1.52±0.08 vs 1.53±0.08 in mGCH1 Tg; 0.89±0.07 vs 1.35±0.06 in WT, n=9 per group, P<0.01 for the interaction between genotype and diabetes). In line with these results, isolated LV myocytes from WT diabetic mice displayed prolonged relaxation, which was prevented in diabetic mGCH1 Tg (t50 relaxation, diabetic vs control: 105.3±2.8 vs 95.6±2.4 in WT and 77.3±3 vs 73.3±2 in mGCH1 Tg; n=25 cells from 2-5 hearts per group, P<0.05 for the interaction between genotype and diabetes). Conclusions: Impaired LV diastolic function in diabetic mice can be prevented by myocardial GCH1 overexpression in the absence of NOS dysfunction or increased oxidative stress, suggesting that GCH1/BH4 protect the diabetic myocardium by mechanisms other than redressing the local nitroso-redox balance. [ABSTRACT FROM AUTHOR]

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., Competing Interests: None.

Background and Purpose: Pregnancy-associated vascular remodelling is essential for both maternal and fetal health. We have previously shown that maternal endothelial cell tetrahydrobiopterin (BH4) deficiency causes poor pregnancy outcomes. Here, we investigated the role and mechanisms of endothelial cell-mediated vasorelaxation function in these outcomes., Experimental Approach: The vascular reactivity of mouse aortas and uterine arteries from non-pregnant and pregnant endothelial cell-specific BH4 deficient mice (Gch1 fl/fl Tie2cre mice) was assessed by wire myography. Systolic blood pressure was assessed by tail cuff plethysmography., Key Results: In late pregnancy, systolic blood pressure was significantly higher (∼24 mmHg) in Gch1 fl/fl Tie2cre mice compared with wild-type littermates. This was accompanied by enhanced vasoconstriction and reduced endothelial-dependent vasodilation in both aorta and uterine arteries from pregnant Gch1 fl/fl Tie2cre mice. In uterine arteries loss of eNOS-derived vasodilators was partially compensated by upregulation of intermediate and large-conductance Ca 2+ -activated K + channels. In rescue experiments, oral BH4 supplementation alone did not rescue vascular dysfunction and pregnancy-induced hypertension in Gch1 fl/fl Tie2cre mice. However, combination with the fully reduced folate, 5-methyltetrahydrofolate (5-MTHF), restored endothelial cell vasodilator function and blood pressure., Conclusions and Implications: We identify a critical requirement for maternal endothelial cell Gch1/BH4 biosynthesis in endothelial cell vasodilator function in pregnancy. Targeting vascular Gch1 and BH4 biosynthesis with reduced folates may provide a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

The cofactor tetrahydrobiopterin (BH 4 ) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH 4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH 4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH 4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1 fl/fl with Tie2cre mice, generating Gch1 fl/fl Tie2cre mice and littermate controls. GTP cyclohydrolase protein and BH 4 levels were reduced in heart tissues from Gch1 fl/fl Tie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH 4 . Deficiency in coronary endothelial cell BH 4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1 fl/fl Tie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH 4 led to mild cardiac hypertrophy in Gch1 fl/fl Tie2cre hearts. Endothelial cell BH 4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH 4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1 /BH 4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH 4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury. NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1 /BH 4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH 4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH 4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Aims: The non-coding locus at 6p24 located in Intron 3 of PHACTR1 has consistently been implicated as a risk allele in myocardial infarction and multiple other vascular diseases. Recent murine studies have identified a role for Phactr1 in the development of atherosclerosis. However, the role of PHACTR1 in vascular tone and in vivo vascular remodelling has yet to be established. The aim of this study was to investigate the role of PHACTR1 in vascular function., Methods and Results: Prospectively recruited coronary artery disease (CAD) patients undergoing bypass surgery and retrospectively recruited spontaneous coronary artery dissection (SCAD) patients and matched healthy volunteers were genotyped at the PHACTR1 rs9349379 locus. We observed a significant association between the PHACTR1 loci and changes in distensibility in both the ascending aorta (AA = 0.0053 ± 0.0004, AG = 0.0041 ± 0.003, GG = 0.0034 ± 0.0009, P < 0.05, n = 58, 54, and 7, respectively) and carotid artery (AA = 12.83 ± 0.51, AG = 11.14 ± 0.38, GG = 11.69 ± 0.66, P < 0.05, n = 70, 65, and 18, respectively). This association was not observed in the descending aorta or in SCAD patients. In contrast, the PHACTR1 locus was not associated with changes in endothelial cell function with no association between the rs9349379 locus and in vivo or ex vivo vascular function observed in CAD patients. This finding was confirmed in our murine model where the loss of Phactr1 on the pro-atherosclerosis ApoE-/- background did not alter ex vivo vascular function., Conclusion: In conclusion, we have shown a role for PHACTR1 in arterial compliance across multiple vascular beds. Our study suggests that PHACTR1 has a key structural role within the vasculature., Competing Interests: Conflict of interest: D.A. reports in kind support from Astra Zeneca Inc. for SCAD genetics research and research funding from Astra Zeneca for unrelated research. He has received educational funding from Abbott Vascular Inc. to support a clinical research fellow. He has conducted consultancy for General Electric Inc. to support general research funds. No other authors have any relationships to declare., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: Recent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans., Methods and Results: Study 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide (O2.-) sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with O2.- production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM., Conclusions: We demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

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Background: Obesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown., Objectives: The aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes., Methods: A screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints., Results: Because ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects., Conclusions: These results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183)., Competing Interests: Funding Support and Author Disclosures This study was supported by the Novo Nordisk Foundation Tripartite Immunometabolism Consortium Award (NNF15CC0018486), the British Heart Foundation (FS/16/15/32047, PG/13/56/30383, RG/17/10/32859 and RG/F/21/110040) and British Heart Foundation Chair award (CH/16/1/32013), British Heart Foundation Centre of Research Excellence award (RG/13/1/30181), the National Institute for Health Research Oxford Biomedical Research Centre, and the Swedish Heart Lung Foundation (HLF 20180290). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype., Objective: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes., Methods and Results: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca 2+ ]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31 phosphorous magnetic resonance spectroscopy), and myocardial function., Conclusions: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.

Aims: Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis., Methods and Results: Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad-/- mice were crossed to an ApoE-/- background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad-/-ApoE-/- mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad-/- mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt., Conclusion: The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Recent clinical trials have revealed that aggressive insulin treatment has a neutral effect on cardiovascular risk in patients with diabetes despite improved glycemic control, which may suggest confounding direct effects of insulin on the human vasculature. We studied 580 patients with coronary atherosclerosis undergoing coronary artery bypass surgery (CABG), finding that high endogenous insulin was associated with reduced nitric oxide (NO) bioavailability ex vivo in vessels obtained during surgery. Ex vivo experiments with human internal mammary arteries and saphenous veins obtained from 94 patients undergoing CABG revealed that both long-acting insulin analogs and human insulin triggered abnormal responses of post-insulin receptor substrate 1 downstream signaling ex vivo, independently of systemic insulin resistance status. These abnormal responses led to reduced NO bioavailability, activation of NADPH oxidases, and uncoupling of endothelial NO synthase. Treatment with an oral dipeptidyl peptidase 4 inhibitor (DPP4i) in vivo or DPP4i administered to vessels ex vivo restored physiological insulin signaling, reversed vascular insulin responses, reduced vascular oxidative stress, and improved endothelial function in humans. The detrimental effects of insulin on vascular redox state and endothelial function as well as the insulin-sensitizing effect of DPP4i were also validated in high-fat diet-fed ApoE -/- mice treated with DPP4i. High plasma DPP4 activity and high insulin were additively related with higher cardiac mortality in patients with coronary atherosclerosis undergoing CABG. These findings may explain the inability of aggressive insulin treatment to improve cardiovascular outcomes, raising the question whether vascular insulin sensitization with DPP4i should precede initiation of insulin treatment and continue as part of a long-term combination therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2-mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background and Purpose: The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and ROS signalling in vascular physiology. To determine the physiological requirement for de novo endothelial cell BH4 synthesis for the vasomotor function of resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation., Experimental Approach: The reactivity of mouse second-order mesenteric arteries was assessed by wire myography. High performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis., Key Results: Gch1 fl/fl Tie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1 fl/fl Tie2cre mice. Gch1 fl/fl Tie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was abolished by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1 fl/fl Tie2cre mice, compared with those from wild-type littermates. Loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H 2 O 2 and cyclooxygenase-derived vasodilator in Gch1 fl/fl Tie2cre mesenteric arteries., Conclusions and Implications: Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease., (© 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Overproduction of nitric oxide (NO) is thought to be a key mediator of the vascular dysfunction and severe hypotension in patients with endotoxaemia and septic shock. The contribution of NO produced directly in the vasculature by endothelial cells to the hypotension seen in these conditions, vs. the broader systemic increase in NO, is unclear. To determine the specific role of endothelium derived NO in lipopolysaccharide (LPS)-induced vascular dysfunction we administered LPS to mice deficient in endothelial cell tetrahydrobiopterin (BH4), the essential co-factor for NO production by NOS enzymes. Mice deficient in endothelial BH4 production, through loss of the essential biosynthesis enzyme Gch1 (Gch1(fl/fl)Tie2cre mice) received a 24hour challenge with LPS or saline control. In vivo LPS treatment increased vascular GTP cyclohydrolase and BH4 levels in aortas, lungs and hearts, but this increase was significantly attenuated in Gch1(fl/fl)Tie2cre mice, which were also partially protected from the LPS-induced hypotension. In isometric tension studies, in vivo LPS treatment reduced the vasoconstriction response and impaired endothelium-dependent and independent vasodilatations in mesenteric arteries from wild-type mice, but not in Gch1(fl/fl)Tie2cre mesenteric arteries. Ex vivo LPS treatment decreased vasoconstriction response to phenylephrine in aortic rings from wild-type and not in Gch1(fl/fl)Tie2cre mice, even in the context of significant eNOS and iNOS upregulation. These data provide direct evidence that endothelial cell NO has a significant contribution to LPS-induced vascular dysfunction and hypotension and may provide a novel therapeutic target for the treatment of systemic inflammation and patients with septic shock., (Copyright © 2015. Published by Elsevier Inc.)

Human genetic studies have revealed an association between GTP cyclohydrolase 1 polymorphisms, which decrease tetrahydrobiopterin (BH4) levels, and reduced pain in patients. We now show that excessive BH4 is produced in mice by both axotomized sensory neurons and macrophages infiltrating damaged nerves and inflamed tissue. Constitutive BH4 overproduction in sensory neurons increases pain sensitivity, whereas blocking BH4 production only in these cells reduces nerve injury-induced hypersensitivity without affecting nociceptive pain. To minimize risk of side effects, we targeted sepiapterin reductase (SPR), whose blockade allows minimal BH4 production through the BH4 salvage pathways. Using a structure-based design, we developed a potent SPR inhibitor and show that it reduces pain hypersensitivity effectively with a concomitant decrease in BH4 levels in target tissues, acting both on sensory neurons and macrophages, with no development of tolerance or adverse effects. Finally, we demonstrate that sepiapterin accumulation is a sensitive biomarker for SPR inhibition in vivo., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation., Background: 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., Methods: 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., Results: 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., Conclusion: 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.

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (•-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors., (© 2014 American Heart Association, Inc.)

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.