Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression
Nicholas J. Alp, Shafi Mussa, Jeffrey Khoo, Shijie Cai, Tomasz Guzik, Andrew Jefferson, Nicky Goh, Kirk A. Rockett, Keith M. Channon
Nicholas J. Alp, Shafi Mussa, Jeffrey Khoo, Shijie Cai, Tomasz Guzik, Andrew Jefferson, Nicky Goh, Kirk A. Rockett, Keith M. Channon
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Article Cardiology

Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression

Abstract

Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate–cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.

Authors

Nicholas J. Alp, Shafi Mussa, Jeffrey Khoo, Shijie Cai, Tomasz Guzik, Andrew Jefferson, Nicky Goh, Kirk A. Rockett, Keith M. Channon

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Oxidation of glutathione stores and superoxide generation in aorta from ...
Oxidation of glutathione stores and superoxide generation in aorta from diabetic GCH-Tg and WT mice. (a) The GSH/GSSG ratio was reduced in diabetic WT mice as compared with control WT mice (*P = 0.02, n = 3), but diabetes did not alter the GSH/GSSG ratio in GCH-Tg mice. (b) Lucigenin-enhanced chemiluminescence. Superoxide production in control WT aortas was similar to that in GCH-Tg aortas. Superoxide production was increased more than twofold in diabetic WT aortas as compared with control (*P < 0.05), but this increase was significantly smaller in diabetic GCH-Tg aortas (*P < 0.05 as compared with control and with diabetic WT, n = 4–6). RLU, relative light units. (c) DHE staining for whole-vessel superoxide production. Representative sections are shown (×10), with total red DHE fluorescence expressed in arbitrary units. Aortic DHE fluorescence was increased two- to threefold in both diabetic WT and GCH-Tg mice as compared with their respective controls (**P < 0.01, n = 3). Scale bar: 100 μm. (d) DHE staining for endothelial cell superoxide production. Representative sections are shown (×60), with specific endothelial cell red DHE fluorescence (arrowheads) expressed in arbitrary units. Endothelial DHE fluorescence was increased by more than 17-fold in diabetic WT aorta, whereas endothelial superoxide production in diabetic GCH-Tg aorta was increased only 3.5-fold as compared with control (**P < 0.01, n = 3). Elastic laminas exhibit green autofluorescence. Scale bar: 20 μm.