Increased Endothelial Tetrahydrobiopterin Synthesis by Targeted Transgenic GTP-Cyclohydrolase I Overexpression Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice

Abstract

Objective-Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). Methods and Results-Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. Conclusions-These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosisand is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression. Key Words: nitric oxide synthase Ⅲ endothelium Ⅲ atherosclerosis Ⅲ hypercholesterolemia N itric oxide (NO) produced in the endothelium by endothelial nitric oxide synthase (eNOS) is a key mediator of vascular homeostasis. NO bioavailability is reduced early in vascular disease states such as hypercholesterolemia and atherosclerosis because of reduced NO synthesis and increased NO consumption by reactive oxygen species. 1 A critical determinant of eNOS activity is the availability of the NOS cofactor tetrahydrobiopterin (BH4). When BH4 levels are inadequate, the enzymatic reduction of molecular oxygen by eNOS is no longer coupled to L-arginine oxidation, resulting in generation of superoxide rather than NO, thus contributing to vascular oxidative stress and endothelial dysfunction. BH4 bioavailability in the vasculature appears to be regulated at the level of biosynthesis by the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH) and by oxidative degradation of BH4 to dihydrobiopterin (BH2) that is inactive for eNOS cofactor function.Several pharmacologic studies suggest a possible role for BH4 availability in regulating NO-mediated endothelial function. Acute administration of BH4 improves some features of endothelial dysfunction in smokers, 2 and in patients with type See page 397II diabetes, 3 hypercholesterolemia, 4,5 or coronary atherosclerosis. 6 In hypercholesterolemic ApoE-knockout (ApoE-KO) mice, endothelium-dependent vascular relaxations are impaired, NO synthesis is reduced, and vascular superoxide production is increased. 7,8 However, endothelial dysfunction in ApoE-KO mice can be reduced by incubation of vessels in the BH4 p...

Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes

“…in 7). Reduced BH 4 levels have been reported in diabetic eNOS uncoupling (7,31,35), and restoration of BH 4 levels can "recouple" eNOS and enhance its regular enzymatic activity (7,40). In the present study we demonstrate for the first time a critical role for intracellular BH 4 levels for coupling and uncoupling of eNOS in EPCs.…”

“…In general, increased eNOS expression is considered to be beneficial. However, under certain pathophysiological conditions, upregulation of eNOS expression is associated with reduced endothelium-dependent vasodilatation (31)(32)(33)(34) explained by the so-called "eNOS uncoupling" (rev. in 7,33,34).…”

“…in 7,33,34). Under this condition eNOS itself can be a source of O 2 Ϫ , instead of NO (7,(31)(32)(33)(34). eNOS uncoupling was shown in a variety of experimental and clinical vascular disease states, especially in diabetes (6,7,33,35).…”

“…Diabetesassociated eNOS uncoupling has been described in the heart, vessels, and the kidney (4,34 -39), resulting in decreased NO bioavailibility, increased superoxide production, and disrupted eNOS dimer formation within the vascular wall, while eNOS mRNA and/or protein levels are maintained or even increased (36 -38). A reduction of the essential eNOS cofactor BH 4 has been described to be mechanistically involved in eNOS uncoupling (7,31,35,37,40).…”

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling

“…Kawashima and co-workers (23) reported that overexpression of eNOS in an apoE-KO background paradoxically resulted in increased vascular superoxide production, at least in part from uncoupled eNOS, which was prevented when supplementary BH4 was administered, or when these mice were further crossed with endothelium-targeted GTPCH transgenic mice (24). Although these data describe BH4 deficiency as a result of oxidation to BH2, evidence from this study and the observation that DHFR levels are diminished in mouse models of diabetes may suggest that insufficient recycling of BH2 to BH4 by DHFR is at least in part responsible, allowing the accumulation of BH2 and eNOS uncoupling to occur.…”