Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Bi-modal dose-dependent cardiac response to tetrahydrobiopterin in pressure-overload induced hypertrophy and heart failure.

The exogenous administration of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase (NOS), has been shown to reduce left ventricular hypertrophy, fibrosis, and cardiac dysfunction in mice with pre-established heart disease induced by pressure-overload. In this setting, BH4 re-coupled endothelial NOS (eNOS), with subsequent reduction of NOS-dependent oxidative stress and reversal of maladaptive remodeling. However, recent studies suggest the effective BH4 dosing may be narrower than previously thought, potentially due to its oxidation upon oral consumption. Accordingly, we assessed the dose response of daily oral synthetic sapropterin dihydrochloride (6-R-l-erythro-5,6,7,8-tetrahydrobiopterin, 6R-BH4) on pre-established pressure-overload cardiac disease. Mice (n=64) were administered 0-400mg/kg/d BH4 by ingesting small pre-made pellets (consumed over 15-30 min). In a dose range of 36-200mg/kg/d, 6R-BH4 suppressed cardiac chamber remodeling, hypertrophy, fibrosis, and oxidative stress with pressure-overload. However, at both lower and higher doses, BH4 had less or no ameliorative effects. The effective doses correlated with a higher myocardial BH4/BH2 ratio. However, BH2 rose linearly with dose, and at the 400mg/kg/d, this lowered the BH4/BH2 ratio back toward control. These results expose a potential limitation for the clinical use of BH4, as variability of cellular redox and perhaps heart disease could produce a variable therapeutic window among individuals. This article is part of a special issue entitled ''Key Signaling Molecules in Hypertrophy and Heart Failure.''

Depletion of LAG-3+ T Cells Translated to Pharmacology and Improvement in Psoriasis Disease Activity: A Phase I Randomized Study of mAb GSK2831781.

Activated T cells drive a range of immune-mediated inflammatory diseases. LAG-3 is transiently expressed on recently activated CD4+ and CD8+ T cells. We describe the engineering and first-in-human clinical study (NCT02195349) of GSK2831781 (an afucosylated humanized IgG1 monoclonal antibody enhanced with high affinity for Fc receptors and LAG-3 and antibody-dependent cellular cytotoxicity capabilities), which depletes LAG-3 expressing cells. GSK2831781 was tested in a phase I/Ib, double-blind, placebo-controlled clinical study, which randomized 40 healthy participants (part A) and 27 patients with psoriasis (part B) to single doses of GSK2831781 (up to 0.15 and 5 mg/kg, respectively) or placebo. Adverse events were generally balanced across groups, with no safety or tolerability concern identified. LAG-3+ cell depletion in peripheral blood was observed at doses ≥ 0.15 mg/kg and was dose-dependent. In biopsies of psoriasis plaques, a reduction in mean group LAG-3+ and CD3+ T-cell counts was observed following treatment. Downregulation of proinflammatory genes (IL-17A, IL-17F, IFNγ, and S100A12) and upregulation of the epithelial barrier integrity gene, CDHR1, was observed with the 5 mg/kg dose of GSK2831781. Psoriasis disease activity improved up to day 43 at all GSK2831781 doses (0.5, 1.5, and 5 mg/kg) compared with placebo. Depletion of LAG-3-expressing activated T cells is a novel approach, and this first clinical study shows that GSK2831781 is pharmacologically active and provides encouraging early evidence of clinical effects in psoriasis, which warrants further investigation in T-cell-mediated inflammatory diseases.

Tetrahydrobiopterin supplementation reduces atherosclerosis and vascular inflammation in apolipoprotein E-knockout mice.

BH4 (tetrahydrobiopterin) supplementation improves endothelial function in models of vascular disease by maintaining eNOS (endothelial nitric oxide synthase) coupling and NO (nitric oxide) bioavailability. However, the cellular mechanisms through which enhanced endothelial function leads to reduced atherosclerosis remain unclear. We have used a pharmaceutical BH4 formulation to investigate the effects of BH4 supplementation on atherosclerosis progression in ApoE-KO (apolipoprotein E-knockout) mice. Single oral dose pharmacokinetic studies revealed rapid BH4 uptake into plasma and organs. Plasma BH4 levels returned to baseline by 8 h after oral dosing, but remained markedly increased in aorta at 24 h. Daily oral BH4 supplementation in ApoE-KO mice from 8 weeks of age, for a period of 8 or 12 weeks, had no effect on plasma lipids or haemodynamic parameters, but significantly reduced aortic root atherosclerosis compared with placebo-treated animals. BH4 supplementation significantly reduced VCAM-1 (vascular cell adhesion molecule 1) mRNA levels in aortic endothelial cells, markedly reduced the infiltration of T-cells, macrophages and monocytes into plaques, and reduced T-cell infiltration in the adjacent adventitia, but importantly had no effect on circulating leucocytes. GCH (GTP cyclohydrolase I)-transgenic mice, with a specific increase in endothelial BH4 levels, exhibited a similar reduction in vascular immune cell infiltration compared with BH4-deficient controls, suggesting that BH4 reduces vascular inflammation via endothelial cell signalling. In conclusion, BH4 supplementation reduces vascular immune cell infiltration in atherosclerosis and may therefore be a rational therapeutic approach to reduce the progression of atherosclerosis.

Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease.

NO produced by eNOS (endothelial nitric oxide synthase) is a key mediator of vascular homoeostasis. NO bioavailability is reduced early in vascular disease states, such as hypercholesterolaemia, diabetes and hypertension, and throughout the progression of atherosclerosis. This is a result of both reduced NO synthesis and increased NO consumption by reactive oxygen species. eNOS enzymatic activity appears to be determined by the availability of its cofactor BH4 (tetrahydrobiopterin). When BH4 levels are adequate, eNOS produces NO; when BH4 levels are limiting, eNOS becomes enzymatically uncoupled and generates superoxide, contributing to vascular oxidative stress and endothelial dysfunction. BH4 bioavailability is determined by a balance of enzymatic de novo synthesis and recycling, versus oxidative degradation in dysfunctional endothelium. Augmenting vascular BH4 levels by pharmacological supplementation, by enhancing the rate of de novo biosynthesis or by measures to reduce BH4 oxidation have been shown in experimental studies to enhance NO bioavailability. Thus BH4 represents a potential therapeutic target for preserving eNOS function in vascular disease.