Tetrahydrobiopterin, L-Arginine and Vitamin C Act Synergistically to Decrease Oxidant Stress and Increase Nitric Oxide That Increases Blood Flow Recovery after Hindlimb Ischemia in the Rat

Jinglian Yan,Louis M Messina,Guodong Tie

doi:10.2119/molmed.2011.00103.revised

Jinglian Yan, Louis M Messina + Show 1 more

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https://doi.org/10.2119/molmed.2011.00103.revised

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Abstract

Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays essential roles in neovascularization. During limb ischemia, decreased NO bioavailability occurs secondary to increased oxidant stress, decreased L-arginine and tetrahydrobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), L-arginine and vitamin C acts synergistically to decrease oxidant stress, increase NO and thereby increase blood flow recovery after hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or L-arginine (alone or in combination) or chow supplemented with BH4 + L-arginine + vitamin C for 1 wk before induction of hindlimb ischemia. In the is-chemic hindlimb, cosupplementation with BH4 + L-arginine resulted in greater eNOS and phospho-eNOS (P-eNOS) expression, Ca(2+)-dependent NOS activity and NO concentration in the ischemic calf region (gastrocnemius), as well as greater NO concentration in the region of collateral arteries (gracilis). Rats receiving cosupplementation of BH4 + L-arginine led to greater recovery of foot perfusion and greater collateral enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + L-arginine regimen further increased these dependent variables. In addition, rats given all three supplements showed significantly less Ca(2+)-independent activity, less nitrotyrosine accumulation, greater glutathione (GSH)-to-glutathione disulfide (GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, co-supplementation with BH4 + L-arginine + vitamin C significantly increased blood flow recovery after hindlimb ischemia by reducing oxidant stress, increasing NO bioavailability, enlarging collateral arteries and reducing muscle necrosis. Oral cosupplementation of BH4, L-arginine and vitamin C holds promise as a biological therapy to induce collateral artery enlargement.

Highlights

Endothelial dysfunction is a hallmark of peripheral artery disease (PAD) [1]
The combination of BH4 and L-arginine had an additive effect on endothelial nitric oxide synthase (eNOS) expression, and the addition of vitamin C provided an additional beneficial effect
Increased P-eNOS expression was only observed in rats fed BH4 + L-arginine and BH4 + L-arginine + vitamin C

Summary

Introduction

Endothelial dysfunction is a hallmark of peripheral artery disease (PAD) [1]. Central to the development of endothelial dysfunction, regardless of its cause, is a reduction in the bioavailability of nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). It is likely that all three mechanisms contribute to the endothelial dysfunction characteristic of PAD because increased oxidant stress is a common antecedent in the pathogenesis of this disease and can reduce NO bioavailability. BH4 maintains eNOS in its functional dimeric form; in the absence of BH4, eNOS becomes uncoupled so that the electron flux is diverted away from the L-arginine binding site and instead reduces molecular oxygen, generating O2– [6]. This circumstance initiates a vicious cycle, wherein eNOS catalytic activity produces O2–, not NO, worsening existent oxidant stress

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