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 1

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(a) Schematic of the murine Tie2 promoter-enhancer/human GTPCH transgene...
(a) Schematic of the murine Tie2 promoter-enhancer/human GTPCH transgene. The murine Tie2 promoter and its intronic enhancer (10 kb) are depicted as gray bars, human GTPCH cDNA is depicted as a white bar, and SV40 poly A signal is depicted as a black bar. Oligonucleotide primers were used to screen genomic DNA for the presence of the transgene. Restriction endonuclease sites for SalI and XbaI are shown. GCH, GTPCH; pA, SV40 poly A; P1 and P2, oligonucleotide primers. (b) Genomic DNA analysis of potential founders. The top panel shows PCR reactions performed on DNA isolated from tail biopsies. The expected 150-bp product (filled arrowhead) was identified in founder mouse 16; linearized pTie2-GCH plasmid DNA was used as a positive control. The bottom panel shows Southern blot analysis performed to confirm the transgenic founders’ genotype and estimate transgene copy number. Founder mouse 16 showed a single hybridization fragment of 8 kb (open arrowhead), suggesting a single chromosomal integration site. The pTie2-GCH plasmid digested with XbaI was used as a positive control.