Tetrahydrobiopterin, L-arginine and vitamin C actsynergistically to decrease oxidative stress, increase nitricoxide and improve blood flow after induction of hindlimbischemia in the rat

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.

Figure 1

Effects of BH4, l-arginine (L-arg) and vitamin C (VitC) on eNOS and phosphorylated eNOS expression in the ischemic gastrocnemius muscle. Muscle was harvested 14 d after the induction of hindlimb ischemia, and supernatants from muscle homogenates were used in these assays. (A) Expression of eNOS was increased by all dietary additives, although the greatest increase was noted in rats that received BH4 + l-arginine + vitamin C. (B) Expression of p-eNOS was increased in rats fed BH4 + l-arginine or BH4 + l-arginine + vitamin C. Control rats were fed a standard diet, whereas the four treatment groups consumed diets supplemented with additives noted in the bar graph. Concentrations of these additives are noted in the text. Data are mean ± standard deviation (sd); n = 5–6. *p < 0.05 versus control; ^†p < 0.05 versus BH4 or l-arginine groups; ‡p < 0.05 versus BH4 + l-arginine group.

Figure 2

Effects of BH4, l-arginine (L-arg) and vitamin C (VitC) on NOS activity in the ischemic gastrocnemius muscle. Muscle was harvested 14 d after the induction of hindlimb ischemia, and supernatants from muscle homogenates were used in these assays. (A) Ca²⁺-dependent NOS activity was increased by all dietary additives, although the greatest increase was noted in mice receiving BH4 + l-arginine or BH4 + l-arginine + vitamin C. Data are mean ± sd; n = 5–6. *p < 0.05 versus control; ^†p < 0.05 versus BH4 or l-arginine groups; ‡p < 0.05 versus BH4 + l-arginine group. (B) NOx (nitrite + nitrate) was increased in mice receiving BH4 + l-arginine or BH4 + l-arginine + vitamin C. Data are mean ± sd; n = 5. *p < 0.05 versus control; ^†p < 0.05 versus BH4 or l-arginine groups. (C) Ca²⁺-independent NOS activity was less in mice receiving BH4 + l-arginine or BH4 + l-arginine + vitamin C than in control mice. Data are mean ± sd; n = 5–6. *p < 0.05 versus control. In all graphs, control rats were fed normal chow and underwent hindlimb ischemia, whereas the four treatment groups consumed diets supplemented with additives noted in the bar graph. Concentrations of these additives are noted in the text.

Figure 3

Effects of BH4, l-arginine (L-arg) and vitamin C (VitC) on oxidative stress in the ischemic gastrocnemius muscle. Muscle was harvested 14 d after the induction of hindlimb ischemia, and supernatants from muscle homogenates were used in these assays. (A) Ni-trotyrosine expression was decreased by all dietary additives, although this decrease was greatest in rats receiving BH4 + l-arginine + vitamin C. (B) The GSH:GSSG ratio was increased in rats receiving BH4 + l-arginine; the addition of vitamin C to this regimen caused an additional increase in this ratio. Data are mean ± sd; n = 5–6. *p < 0.05 versus control, BH4 or l-arginine groups; ^†p < 0.05 versus BH4 + l-arginine groups.

Figure 4

Effects of BH4, l-arginine and vitamin C on perfusion recovery after induction of hindlimb ischemia. (A) Laser Doppler perfusion imaging (LDPI) data, expressed as the ratio of blood flow from the ischemic to nonischemic hindlimbs, was determined before, immediately after and then serially over the ensuing 6 wks. Group identity is shown in the color key. Data are mean ± sd; n = 6. *p < 0.05 for BH4 + l-arginine or BH4 + l-arginine + vitamin C groups versus all other groups. (B) The angioscore, determined 42 d after the induction of hindlimb ischemia and calculated as described in the text, was greater in rats receiving BH4 + l-arginine or BH4 + l-arginine + vitamin C than in all other groups. Data are mean ± sd; n = 6. *p < 0.05 for BH4 + l-arginine or BH4 + l-arginine + vitamin C groups versus all other groups. (C) Representative angiogram from each study group.

Figure 5

The effects of BH4, l-arginine (L-arg) and vitamin C (VitC) on necrosis in the ischemic gastrocnemius muscle. (A) Gastrocnemius muscle was removed 7 d after induction of hindlimb ischemia. Transverse sections of this muscle were stained with nitroblue tetrazolium to determine the ratio of necrotic versus viable surface area. This ratio was less in rats receiving BH4 + l-arginine + vitamin C than in all other groups. Data are mean ± sd; n = 6. *p < 0.05 for BH4 + l-arginine + vitamin C groups versus all other groups. (B) Representative images from each study group. (C) Histological sections of gastrocnemius muscle taken from the ischemic hindlimb 7 d after the induction of hindlimb ischemia were stained with H&E. Representative sections are shown. Note the loss of myofibers and the intense cellular infiltrate in the control, BH4 and l-arginine groups. Best preservation of muscle architecture was consistently observed in rats receiving BH4 + l-arginine + vitamin C.

Figure 6

Schematic illustration of possible mechanism of three dietary combined regimen can increase NO bioavailability and decrease oxidative stress, accordingly increase blood flow recovery and reduce tissue necrosis. A^•, ascorbate; AH⁻, dehydroascorbate.