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Background-The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease. Methods and Results-Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue. Conclusions-Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00423280.

1 Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. -methyladenosine3 0 ,5 0 -bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. 8 Taken together, these results point to the presence of functional P2Y 1 (ADP), P2Y 2 (ATP, UTP) and P2Y 6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.

Objectives This study sought to determine the effects of endogenous tetrahydrobiopterin (BH4) bioavailability on endothelial nitric oxide synthase (eNOS) coupling, nitric oxide (NO) bioavailability, and vascular superoxide production in patients with coronary artery disease (CAD). Background GTP-cyclohydrolase I, encoded by the GCH1 gene, is the rate-limiting enzyme in the biosynthesis of BH4, an eNOS cofactor important for maintaining enzymatic coupling. We examined the associations between haplotypes of the GCH1 gene, GCH1 expression and biopterin levels, and the effects on endothelial function and vascular superoxide production. Methods Blood samples and segments of internal mammary arteries and saphenous veins were obtained from patients with CAD undergoing coronary artery bypass grafting (n = 347). The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G

FR104 is a monovalent pegylated Fab' Ab, antagonist of CD28, under development for treatment of transplant rejection and autoimmune diseases. In contrast to CD80/86 antagonists (CTLA4-Ig), FR104 selectively blunts CD28 costimulation while sparing CTLA-4 and PD-L1 coinhibitory signals. In the present work, FR104 has been evaluated in a first-in-human study to evaluate the safety, pharmacokinetics, pharmacodynamics, and potency of i.v. administrations in healthy subjects. Sixty-four subjects were randomly assigned to four single ascending dose groups, two double dose groups and four single ascending dose groups challenged with keyhole limpet hemocyanin. Subjects were followed up over a maximum of 113 d. Overall, the pharmacokinetics of FR104 after a single and double infusions was approximately linear at doses ≥0.200 mg/kg. CD28 receptor occupancy by FR104 was saturated at the first sampling time point (0.5 h) at doses above 0.02 mg/kg and returned to 50% in a dose-dependent manner, by day 15 (0.020 mg/kg) to 85 (1.500 mg/kg). FR104 was well tolerated, with no evidence of cytokine-release syndrome and no impact on blood lymphocyte subsets. Inhibition of anti-keyhole limpet hemocyanin Ab response was dose-dependent in FR104 recipients and was already apparent at a dose of 0.02 mg/kg. Abs to FR104 were detected in 22/46 (48%) of FR104 recipients and only 1/46 (2.2%) was detected during drug exposure. In conclusion, selective blockade of CD28 with FR104 was safe and well tolerated at the doses tested. The observed immunosuppressive activity indicated that FR104 has potential to show clinical activity in the treatment of immune-mediated diseases.

In this model of MS, statin treatment reverses myocardial remodelling and improves ventricular relaxation through AMPK-mediated anti-fibrotic effects.

1 Based on pharmacological criteria, we previously suggested that in the mouse aorta, endotheliumdependent relaxation by nucleotides is mediated by P2Y 1 (adenosine diphosphate (ADP)), P2Y 2 (adenosine triphosphate (ATP)) and P2Y 6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y 2 , P2Y 6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y 2 -deficient mice to clarify the action of UTP. 2 Thoracic aorta segments (width 2 mm) of P2Y 2 -deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. 3 Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. 4 P2Y 2 -deficient mice showed impaired ATP-and adenosine 5 0 [g-thio] triphosphate (ATPgS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgS activate predominantly the P2Y 2 subtype. 5 The ATP/ATPgS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y 1 , as they were sensitive to 2 0 -deoxy-N 6 -methyladenosine 3 0 ,5 0 -bisphosphate (MRS2179), a P2Y 1 -selective antagonist. 6 In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y 6 (like) receptor subtype. 7 In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y 2 . But this P2Y 2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y 6 (like) receptor.

Background and purpose: Transgenesis of human paraoxonase 1 (PON1), a HDL-associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E-deficient (apoE À/À ) mice with advanced atherosclerosis. Experimental approach: ApoE À/À mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n ¼ 10) or control adenovirus (AdRR5, n ¼ 10). Non-transfected apoE À/À (n ¼ 9) and C57Bl/6J (WT, n ¼ 6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. Key results: PON1 gene transfer raised total PON1 serum activity 13-15 fold during the 3-week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque-free thoracic aorta rings from apoE À/À mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86 ± 2%), ATP (90±2%) or UTP (83±3%). In contrast, in plaque-bearing segments amplitude (55±7%, 68±8%, 52±8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE À/À SMCs released less intracellular calcium than WT upon sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. Conclusions and implications: These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca 2 þ homeostasis in segments with pre-existing atherosclerosis, independently of an effect on plaque size.