EFFECTS OF NITRIC OXIDE INHALATION ON VASCULAR INJURY

Heart, Lung, And Blood Institute

Investigators

Abstract

Nitric oxide modulates the neointimal response to vascular injury, as shown in animal models using nitric oxide donor drugs and gene therapy with endothelial nitric oxide synthase. Conversely, mice that lack the gene encoding endothelial nitric oxide synthase have enhanced neointimal response to vascular injury. Recently, nitric oxide transport in blood has been described, with the proposal that nitric oxide binding to cysteine-93 of the beta chains of the hemoglobin molecule (S-nitrosohemoglobin) may be of particular importance in this transport mechanism from the lungs to the periphery. We proposed the hypothesis that nitric oxide inhalation following vascular injury may increase nitric oxide transport in blood and reduce neointima formation by inhibiting smooth muscle cell proliferation. To this end, 20 mice were studied in this experiment: 10 C57BL/6J wild-type mice and 10 p27 knock-out mice which lack the gene encoding the cyclin-dependent kinase inhibitor p27 and have an enhanced neointimal response to vascular injury. The femoral and saphenous arteries of each mouse were exposed via a longitudinal incision on the anterolateral surface of the left leg. A 0.010-inch flexible wire was introduced into the saphenous artery, advanced to the left femoral artery about 1.5 cm, and then retracted and re-advanced a total of 5 times to produce intimal injury. The right femoral artery was left intact and served as control. Five mice of each strain were placed in room air and 5 mice of each strain were placed in an airtight chamber where they inhaled 30 ppm nitric oxide and 20.9 % oxygen balanced with nitrogen. After 14 days in either environment, mice were euthanized and the left and right femoral arteries were harvested for morphometric analysis. Blood samples were obtained by a direct heart stick to measure methemoglobin and nitrate (reactant products of nitric oxide with oxyhemoglobin), S-nitrosohemoglobin (reaction product of nitric oxide with cysteine- 93 of the beta globin chain) and nitrosyl(FeII)hemoglobin (reactant product of nitric oxide with deoxyhemoglobin). We found that wild-type and p27 knock-out mice who received nitric oxide inhalation for 14 days had an approximately 10-fold increase in methemoglobin and nitrate over rom air animals, consistent with the reaction of nitric oxide with oxyhemoglobin in the pulmonary circulation. Nitrosyl(FeII)hemoglobin increased over 100-fold over room air values in wild-type and p27 knock-out mice after this priod of nitric oxide inhalation. However, levels of S-nitrosohemoglobin were unchanged with nitric oxide inhalation compared with animals in room air. Morphometric measurements of injured and uninjured arteries are in process to determine the effects of nitric oxide on the neointimal response. We conclude that nitric oxide inhalation for 2 weeks increases levels of nitrosyl(FeII)hemoglobin, which may represent an important mechanism of nitric oxide transport from the pulmonary circulation to the systemic vasculature. However, nitric oxide inhalation did not change levels of S-nitrosohemoglobin, levels of which were low and thus may not be an important transporter of nitric oxide to regional vascular beds.