Tetrahydrobiopterin deficiency exaggerates intimal hyperplasia after vascular injury

Abstract

Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 (n ϭ 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 Ϯ 0.3 vs. 4.5 Ϯ 0.4 pmol/min/mg protein, P Ͻ 0.01), and higher aortic superoxide content (5.2 Ϯ 2.0 ϫ 10 5 cpm vs. 1.6 Ϯ 0.7 ϫ 10 5 cpm, P Ͻ 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 Ϯ 0.3 to 4.1 Ϯ 0.4 pmol⅐min Ϫ1 ⅐mg protein Ϫ1, P Ͻ 0.05), and attenuated superoxide production (from 5.2 Ϯ 2.0 ϫ 10 5 cpm to 0.8 Ϯ 0.7 ϫ 10 5 cpm, P Ͻ 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 Ϯ 0.5 vs. wild-type 0.7 Ϯ 0.1, P Ͻ 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 Ϯ 0.1 vs. hph-1, P Ͻ 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis. restenosis; superoxide; nitric oxide synthase NEOINTIMAL FORMATION AND RESTENOSIS after vascular injury have been ascribed to a number of factors, including endothelial dysfunction, increased reactive oxygen species production, and accelerated smooth muscle cell migration and proliferation (5, 23). Nitric oxide (NO) produced by endothelium plays an important role in attenuating smooth muscle migration and proliferation, and decreasing neointimal hyperplasia by numerous mechanisms (25, 28). Tetrahydrobiopterin (BH4) is an absolute cofactor required for nitric oxide synthase (NOS) and maintains NOS as a NO vs. superoxide-producing enzyme (18). BH4 exerts this action through serving as an electron donor for the hydroxylation of L-arginine. Suboptimal concentrations of BH4, as observed in states of cardiovascular disease, led to an uncoupling of NOS with diminished NO and exaggerated superoxide anion production. NOS may become a source of oxygen-de...

Aliskiren Improves Nitric Oxide Bioavailability and Limits Atherosclerosis

“…The authors suggest that aliskiren may restore eNOS uncoupling via augmenting tetrahydrobiopterin (BH 4 ) levels, an essential cofactor for eNOS. 7 This multidomain enzyme contains 2 functional regions: a flavin-containing reductase domain and a heme-containing oxygenase domain, which has binding sites for the triad of heme, L-arginine, and BH 4 . Between these 2 domains, there is a regulatory calmodulin-binding sequence.…”

Tetrahydrobiopterin Inhibits PDGF-stimulated Migration and Proliferation in Rat Aortic Smooth Muscle Cells via the Nitric Oxide Synthase-independent Pathway

“…Previous investigations reported that BH4 stimulated proliferation of various cells, including vascular endothelial and pheochromocytoma (PC12) cells [18,24]. Moreover, BH4 deficiency is known to promote neointimal formation and cell proliferation in injured vascular walls [8]. It was reported that CCR2-stimulated kidney cell migration was inhibited in plasma or aorta homogenates in mice with elevated BH4 levels due to GTPCH overexpression [9], implying the possibility that BH4 may affect VMSC migration.…”

“…The salvage pathway can also generate BH4 from its oxidized forms via sepiapterin and SR [6]. The role of BH4 associated with NOS activity is generally relevant to the cardiovascular system in pathological and physiological conditions, including hypertension, cardiac hypertrophy, and vascular remodeling [7][8][9]. Recently, we found that deficiency in BH4 regeneration caused by diminished DHPR expression is involved in vascular disorders in hypertensive rats [10].…”

“…BH4 treatment has been reported to be pharmacologically beneficial for hypertension and atherosclerosis [17]. Moreover, some investigators have shown that BH4 can regulate cell proliferation in various cells, including PC12 and endothelial cells [8,18,19], and migration in kidney cells [9]. It is known that BH4 induces NO production by promoting NOS activity [3] and NO exerts inhibitory effects on migration and proliferation [20,21].…”

Hypertension Management and End Organ Protection: Focus on Aliskiren

“…Aliskiren may restore eNOS uncoupling via augmenting tetrahydrobiopterin (BH 4 ) levels, an essential cofactor for eNOS. 29 This multidomain enzyme contains 2 functional regions: a fl avin-containing reductase domains and a heme-containing oxygenase domain, which has binding sites for the triad of heme, L-arginine, and BH 4 . Between these 2 domains, there is a regulatory calmodulin-binding sequence.…”