Tetrahydrobiopterin-dependent preservation of nitric oxide–mediated endothelial function in diabetes by targeted transgenic GTP–cyclohydrolase I overexpression
Nicholas J. Alp, … , Kirk A. Rockett, Keith M. Channon
Nicholas J. Alp, … , Kirk A. Rockett, Keith M. Channon
Published September 1, 2003
Citation Information: J Clin Invest. 2003;112(5):725-735. https://doi.org/10.1172/JCI17786.
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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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Figure 2

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(a) Evaluation of transgenic and native GTPCH mRNA expression by RT-PCR....
(a) Evaluation of transgenic and native GTPCH mRNA expression by RT-PCR. The top panel shows human-specific primers to detect transgenic GTPCH mRNA. A PCR product of 181 bp (arrowhead) was detected in lung and liver, and aorta from GCH-Tg but not WT littermates, and only after the RT step (RT+). The middle panel shows mouse-specific primers used to detect native GTPCH mRNA. A PCR product of 181 bp (arrowhead) was detected in lung, liver, and aorta from both GCH-Tg and WT mice. (b) Quantification of GTPCH expression by real-time fluorescent RT-PCR. Shown are means and SEMs of three samples, each quantified in triplicate. Note that arbitrary units are independent among the three graphs. In the top panel, transgenic GTPCH mRNA expression was 10-fold higher in lung than in liver and aorta among GCH-Tg animals but was not detected in WT littermates (**P < 0.01 for comparisons). In the middle panel, native GTPCH mRNA expression in liver was approximately 10-fold higher than in lung and 100-fold higher than in aorta, but similar between GCH-Tg and WT mice for each organ. In the bottom panel, total GTPCH mRNA expression in the lung was approximately 20-fold higher in GCH-Tg than in WT mice (*P < 0.05); in the liver there was no difference in GCH-Tg as compared with WT mice; in the aorta, there was a 5-fold increase in total GTPCH mRNA expression in GCH-Tg as compared with WT mice (**P < 0.01). (c) Immunoblotting with a rabbit anti-human GTPCH polyclonal antibody to detect transgenic GTPCH protein identified a specific 35-kDa band (arrowhead) in lysates from lung, liver, and aorta from GCH-Tg but not WT animals.