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GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats

CYNTHIA J. MEININGER^*,1, SHIJIE CAI, JANET L. PARKER^*, KEITH M. CHANNON, KATHERINE A. KELLY^*, ELIZABETH J. BECKER^*, M. KATHY WOOD, LAURA A. WADE^* and GUOYAO WU^*,

^* Cardiovascular Research Institute, Department of Medical Physiology, Texas A&M University System Health Science Center, Temple, Texas, USA;
Department Of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK;
Department of Biology, University of Mary Hardin-Baylor, Belton, Texas, USA; and
Department of Animal Science, Texas A&M University, College Station, Texas, USA

¹ Correspondence: CVRI/Dept. of Medical Physiology, Texas A&M Univ. System H.S.C., 702 SW H. K. Dodgen Loop, MRB 110G, Temple, TX 76504, USA. Email: cjm{at}tamu.edu

SPECIFIC AIMS

We addressed the hypothesis that decreased expression of GTP cyclohydrolase I (GTPCH) in endothelial cells from diabetic rats is responsible for decreased tetrahydrobiopterin (BH4) levels in these cells and subsequent impairment of their ability to synthesize nitric oxide (NO). The specific aims were to 1) utilize adenoviral GTPCH gene transfer to reverse BH4 deficiency and repair the ability of endothelial cells from type I and type II diabetic rats to produce NO; and 2) demonstrate an improvement in NO-mediated vascular reactivity in isolated vessels from type I (BBD) and type II (ZDF) diabetic rats after GTPCH gene transfer.

PRINCIPAL FINDINGS

1. GTPCH gene transfer increases BH4 levels and NO synthesis in endothelial cells from diabetic BB (BBd) rats
Cultured BBd endothelial cells expressed only 0.17 ± 0.02 pmol of BH4 per million cells. After AdGTPCH infection, BH4 concentrations increased >400-fold (73.37±14.42). This increased BH4 biosynthesis was the result of GTPCH gene transfer, as transduction with control virus (AdGFP) did not significantly increase BH4 levels (0.26±0.04, P>0.05). Furthermore, blocking GTPCH activity with 10 mM 2,4-diamino-6-hydroxypyrimidine (DAHP) prevented the increase in BH4 levels brought about by the GTPCH gene transfer (0.18 pmol/million cells with DAHP vs. 0.17 pmol/million cells without DAHP). The rise in BH4 brought about by AdGTPCH infection was sufficient to increase NO synthesis by the same cells (18.74±5.52 nmol/24 h/million cells compared with 0.77±0.07 in control cells). NO synthesis was not significantly increased in AdGFP-infected cells (2.77±0.61, P>0.05). Production of NO could not be detected if the cells were pretreated with DAHP before infection with AdGTPCH.

2. GTPCH gene transfer increases NO-dependent dilation in isolated BBd vascular rings
We used GTPCH gene transfer to increase BH4 and NO synthesis in isolated aortic rings from BBd rats. Vascular rings incubated with AdGTPCH showed significantly increased vasodilatory responses to acetylcholine (Fig. 1 ). The vasodilation was due entirely to NO synthesis as acetylcholine-induced relaxation did not occur when rings from the same vessel were pretreated with N^G-monomethyl-L-arginine (L-NMMA; 100 µM), an arginine analog that blocks NO synthesis.

View larger version (18K): [in this window] [in a new window]   Figure 1. Acetylcholine-induced relaxation of BBd aortic rings exposed to medium only (control) or medium containing AdGTPCH vector (GTPCH). Other rings were transduced in the same manner but were pretreated with L-NMMA for 30 min prior to addition of acetylcholine (control-NMMA and GTPCH-NMMA). Data are mean ± SE (number of vessel rings indicated in parentheses). *Statistically significant difference (P<0.005) in the response between GTPCH-infected rings and control rings at that dose of acetylcholine.

The maximal vasodilatory response of AdGTPCH-treated BBd rings (10¹⁰ virus particles/mL) to acetylcholine (10 µM) was significantly higher than the response of sham-treated (control) rings (64% vs. 37%, P<0.005). Rings infected with AdGFP control virus showed no significant increase in relaxation compared with sham-treated control rings (P>0.05), indicating that transfer of the GTPCH gene was responsible for increased vasorelaxation.

3. GTPCH gene transfer does not increase NO synthesis in vascular smooth muscle cells
Because virus may enter either endothelial or smooth muscle cells in vessel rings, we investigated the effect of GTPCH gene transfer into cultured rat aortic smooth muscle cells. No BH4 could be detected by our assay in control smooth muscle cells (i.e., no virus exposure). After AdGTPCH infection, BH4 levels in smooth muscle cells increased to a level equivalent to that observed in cultured BBd endothelial cells exposed to the virus (69.70±3.90 pmol/million smooth muscle cells vs. 73.37±14.42 pmol/million endothelial cells). Transfer of GTPCH into smooth muscle cells did not lead to a significant increase in NO synthesis. NO production was 0.13 ± 0.01 nmol/24 h/million cells in control smooth muscle cells vs. 0.16 ± 0.01 nmol/24 h/million cells in AdGTPCH-treated smooth muscle cells. This is in contrast to BBd endothelial cells, which exhibited approximately a 25-fold increase in NO production after GTPCH gene transfer.

4. Coronary endothelial cells from ZDF rats exhibit a BH4 deficiency
To determine whether endothelial cells from ZDF rats exhibit a BH4 deficiency similar to endothelial cells from BBd rats, we isolated coronary endothelial cells from ZDF rats before onset of diabetes at 7 wk (cells collected at 6 wk of age), as well as 2 and 12 wk after the onset of diabetes (9 and 19 wk of age, respectively) and compared them to endothelial cells from age-matched lean control rats. There was no difference in BH4 levels between ZDF rats at 6 wk of age (0.82±0.02 pmol/10⁶ ZDF endothelial cells) and age-matched lean controls (0.85±0.03 pmol/10⁶ lean control cells). A significant decrease in BH4 levels was, however, evident within 2 wk of disease onset (0.64±0.02 pmol/10⁶ ZDF cells vs. 0.82±0.02 pmol/10⁶ lean control cells, P<0.05) and worsened after 12 wk of diabetes (0.42±0.02 vs. 0.80±0.04 pmol/10⁶ cells). Hyperglycemia, also evident after 2 wk of diabetes (24.4±0.69 mM in ZDF rats vs. 7.42±0.26 mM in age-matched lean controls), was maintained for the 12 wk of the experiment.

5. GTPCH gene transfer increased NO-dependent vasodilation in ZDF vessel segments
When aortic rings from ZDF rats were infected with AdGTPCH, vasodilatory responses to acetylcholine were also significantly (P<0.05) increased (Fig. 2 ). Maximal dilatory response increased from 44% in sham-treated ZDF rings (control) to 80% in AdGTPCH-treated rings, again indicating a beneficial effect of increased GTPCH expression. No response to acetylcholine was observed if vessels were pretreated with L-NMMA, indicating that the increase in the vasodilatory response to acetylcholine occurs via an increase in NO synthesis (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. Acetylcholine-induced relaxation of ZDF aortic rings exposed to medium only (control) or medium containing AdGTPCH vector (GTPCH). Data are mean ± SE (number of vessel rings indicated in parentheses). *Statistically significant difference (P<0.05) in the response between GTPCH-infected rings and control rings at that dose of acetylcholine.

CONCLUSIONS AND SIGNIFICANCE

Vascular disease is a major cause of morbidity and mortality associated with diabetes mellitus. Endothelial dysfunction underlies the vascular complications associated with this disease and represents a therapeutic target for prevention and treatment of vascular disease. We investigated the feasibility of modulating BH4 levels via gene transfer to reverse endothelial dysfunction accompanying types I and II diabetes.

We previously showed that BH4 levels are deficient in endothelial cells of BBd rats, leading to severely impaired NO synthesis. A consequence of this impaired NO production is decreased vascular reactivity. A BH4 deficiency is evident in freshly isolated endothelial cells from both BBd rats and rats made diabetic by treatment with streptozotocin, indicating that the deficiency is not an artifact due to culturing of cells. We show, for the first time, that BH4 levels are decreased in freshly isolated endothelial cells of the ZDF rat, a model of type II diabetes.

Aortic rings from BBd rats exhibit decreased vasodilation in response to acetylcholine while maintaining the vascular smooth muscle vasodilatory response to NO donors, indicating endothelial but not smooth muscle dysfunction. We demonstrate that GTPCH gene transfer, in blood vessels from animals exhibiting a BH4 deficiency (both BBd and ZDF rats), causes a significant increase in the endothelium-dependent vasodilatory response to acetylcholine. This vascular relaxation, however, only occurred in the absence of L-NMMA, indicating that GTPCH was supporting NO production, presumably via an increase in BH4 concentration. GTPCH gene transfer in endothelial cells from the diabetic BB rat increased BH4 levels leading to an increase in NO production. This increase in NO synthesis is the basis for the GTPCH-induced reversal of impaired vessel reactivity in the diabetic animals.

GTPCH gene transfer restored arterial GTPCH activity and BH4 levels in carotid arteries, resulting in improved endothelium-dependent relaxation and basal NO release transfer in hypertensive deoxycorticosterone acetate (DOCA)-salt rats. These data provide support for the feasibility of using GTPCH gene transfer to correct endothelial dysfunction brought about by BH4 deficiency.

A transgenic mouse model in which human GTPCH overexpression was targeted to endothelial cells under the control of the Tie2 promoter exhibited increased BH4 levels in vascular tissues in vivo and increased NOS activity. When these transgenic mice were made diabetic by streptozotocin injection, they maintained sufficient BH4 levels to support normal endothelial vasodilatory responses to acetylcholine. This contrasted with the wild-type diabetic mice, which exhibited decreased BH4 levels and deficient NO-mediated endothelial function. Thus, a method for increasing BH4 concentrations in endothelial cells exhibiting impaired NO synthesis may be a means of alleviating endothelial dysfunction and increasing NO biosynthesis in diabetes.

While short-term treatment with exogenous BH4 has been shown to improve endothelial cell function and vessel reactivity, chronic in vivo pharmacological administration is not a practical solution. Exogenous BH4 is easily oxidized to dihydrobiopterin, which no longer functions as an eNOS cofactor and can actually compete with BH4 for eNOS binding. BH4 injected intravenously is rapidly cleared from the circulation by the liver and kidney, with uptake into skeletal muscle being relatively low. With a half-life of only 30 min, repeated injections are necessary to maintain muscle BH4 content at levels sufficient to support BH4-dependent enzyme activity.

We show that GTPCH gene transfer can significantly increase BH4 levels and NO production in coronary EC as well as increase NO-mediated dilation in vascular segments from type I and type II diabetic rats. While GTPCH gene transfer resulted in an equivalent increase in BH4 levels in vascular smooth muscle cells, there was no increase in NO synthesis, presumably due to lack of eNOS in smooth muscle cells. These data support our hypothesis that GTPCH gene transfer provides a means of increasing endogenous levels of BH4 for preservation of endothelial function in diabetes (Fig. 3 ).

View larger version (11K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized effects of GTPCH gene transfer. Increased expression of GTPCH in endothelial cells results in increased levels of BH4. This BH4 supports the eNOS-catalyzed synthesis of NO in the endothelial cell, improving endothelium-dependent vasodilation.

GTPCH gene transfer appears most beneficial in conditions where BH4 deficiency exists. GTPCH gene transfer restores BH4 levels necessary for basal NO production and normal vasoreactivity in hypertensive rats exhibiting a BH4 deficiency. In contrast, GTPCH gene transfer increased BH4 and GTPCH activity in carotid arteries in normal rabbits, yet produced no increase in endothelium-dependent relaxation in response to acetylcholine. It may be that increasing levels of BH4 beyond a supposedly "normal" level does not confer any additional benefit. Only a small increase in NO production occurs after GTPCH gene transfer in normal human dermal microvascular cells, despite a large increase in BH4 levels. These novel observations provide experimental support for overexpression of GTPCH as a therapeutic strategy for amelioration of the endothelial BH4 deficiency in diabetes and as a powerful and specific means of retarding or reversing progression of vascular disease in diabetic individuals. These studies lay the groundwork for gene therapy directed at GTPCH as a viable means of preventing and/or treating the vascular complications of diabetes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1702fje;

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