Depletion and Restoration of the Intravascular Nitrite (NO2-) Reserve Modulate Susceptibility to Liver Vaso-Occlusive Infarction in Sickle Cell Transgenic Mice.

Multiple studies link low bioavailability of nitric oxide (NO) and major complications of sickle cell disease, especially pulmonary hypertension and acute vaso-occlusive pain episodes. A gender dimorphism has been suggested in human studies, with lower levels of NO availability observed in males. These studies support efforts to develop NO-based therapeutics and the newly characterized small molecule nitrite represents an ideal candidate. Nitrite is a circulating anion that is reduced to NO during physiological and pathological hypoxia and mediates potent cellular cytoprotection at physiologic concentrations in the setting of ischemia and reperfusion injury. Supporting a role for nitrite in sickle cell pathogenesis, we found that nitrite levels are depleted in transgenic mice expressing exclusively sickle hemoglobin (sickle mice), with whole blood nitrite levels 50% lower compared to control mice, and are further reduced by 19% in male sickle mice compared with females. Thus, the murine model demonstrates a sexual dimorphism in nitrite levels, consistent with data on human NO bioavailability. We then examined hepatic vaso-occlusion by challenging sickle mice with 2 hours of moderate normobaric hypoxia (10% oxygen), restoring them to normoxia, and then 18 hours later drawing blood to assay plasma alanine aminotransferase (ALT) levels as a quantitative measure of tissue injury. Sickle mice showed a 4- fold rise in ALT with this hypoxia-reoxygenation (HR) challenge; wild-type controls or non-sickling colony control mice with the same HR challenge had no change in ALT. Sexual dimorphism was again evident such that female mice were less susceptible to HR challenge than males. Therapeutic nitrite supplementation, administered either as 2.4 nanomole/g intraperitoneal injection during HR or oral supplementation (estimated 4 micromole/g/day) for 7 days before HR challenge, abrogated the ALT rise in sickle mice. Pharmacokinetic data indicated that nitrite supplementation at these injected and oral doses raise the nitrite levels in blood and tissue, without affecting methemoglobin levels. These studies provide additional mechanistic evidence for a gender contribution to sickle cell endothelial dysfunction and vasculopathy, and suggest that reduced NO bioavailabity in SCD results in dysregulated nitrite homeostasis. Depleted nitrite reserves are associated with enhanced injury with hypoxia-induced vaso-occlusion and therapeutic restoration of nitrite levels, both by intraperitoneal delivery and in the diet, reduces this injury.