Local arginine supplementation results in sustained wound nitric oxide production and reductions in vascular endothelial growth factor expression and granulation tissue formation.
OBJECTIVE: The goal of this work was to test the functional role of L-arginine in promotion of nitric oxide (NO) production and the vigorous granulation tissue formation characteristic of this wound model. BACKGROUND: Therapeutic use of supplemental arginine has been proposed as a safe and efficacious method to produce NO from nitric oxide synthase (NOS) and to produce proline and polyamines from arginase to improve wound healing. Although NO appears to be necessary to promote wound healing, the preferential metabolism of arginine to NO via NOS 2 may be detrimental if maintained beyond the initial days of healing. METHODS: A ventral hernia, surgically created in the abdominal wall of 12 swine, was repaired with silicone sheeting and skin closure. Osmotic infusion pumps, inserted in remote subcutaneous pockets, continuously delivered saline (n = 6) or L-arginine (n = 6) into the wound environment. Granulation tissue thickness was determined by ultrasonography. Fluid was aspirated serially from the wound compartment for measurements of nitrite/nitrate (NOx), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and amino acid concentrations. On day 14, the animals were sacrificed and the abdominal wall was harvested for immunohistochemical and molecular analysis. RESULTS: In animals receiving saline, a nearly linear four-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, quantitative ultrasound analysis detected significant reductions in L-arginine infused granulation tissue thickness compared with controls between days 4 and 14 (P < 0.05). Wound vessel count and luminal vascular surface area estimates derived from image analysis of histological sections were two- to three-fold lower in the L-arginine animals compared with controls (P < 0.05). Significant and sustained increases in wound fluid NOx levels were noted in L-arginine animals compared to saline controls (230 microM versus 75 microM at day 14, P < 0.05). Conversely, late VEGF levels (days 11 to 14) were reduced in the L-arginine animals compared to controls (7500 pg/ml versus 10,000 pg/ml at day 11, P < 0.05; 7250 pg/ml versus 11,101 pg/ml at day 14, P < 0.05). Arginine concentrations remained two- to four-fold greater in L-arginine treated animals compared with controls over the entire time course (P < 0.05). There were no significant differences in concentrations of ornithine, citrulline, or proline noted between groups over the 14-day period. Finally, TGF-beta1 levels were unaffected by L-arginine treatment. CONCLUSION: Although NO appears to be necessary for granulation tissue formation, early supplemental arginine may disturb the reciprocal regulation of NOS 2 and arginase, leading to the preferential metabolism of arginine to excess NO rather than ornithine, with consequent reductions in angiogenesis and granulation tissue formation.