Abstract
Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays an essential role in vascular remodeling of collateral arteries and perfusion recovery in response to hindlimb ischemia. In ischemic conditions, decreased NO bioavailability was observed because of increased oxidative 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 oxidative stress, increase nitric oxide and improve blood flow in response to acute 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 unilateral hindlimb ischemia. Cosupplementation with BH4 + L-arginine resulted in greater eNOS expression, Ca²⁺-dependent NOS activity and NO concentration in gastrocnemius from the ischemic hindlimb, as well as greater recovery of foot perfusion and more collateral artery enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + L-arginine regimen did further increase these dependent variables, although only the increase in eNOS expression reached statistical significances. In addition, rats given all three supplements demonstrated significantly less Ca²⁺-independent activity, less nitrotyrosine accumulation, greater glutathione:glutathione disulfide (GSH:GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, cosupplementation with BH4 + L-arginine + vitamin C significantly increased vascular perfusion after hindlimb ischemia by increasing eNOS activity and reducing oxidative stress and tissue necrosis. Oral cosupplementation of L-arginine, BH4 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
The following study hypothesis was supported by our findings: dietary cosupplementation with tetrahydrobiopterin (BH4), L-arginine and vitamin C acts synergistically to decrease oxidative stress, increase nitric oxide and thereby improve perfusion and tissue recovery in response to acute hindlimb ischemia more than supplementation with a single supplement
Summary
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 oxidative 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 oxidative stress
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