Hemolytic Rate and Nitric Oxide Bioavailability Modulate the Expression of Hypoxia-Inducible Factor-1α (HIF-1α) in Transgenic Sickle Mice

In sickle cell disease (SCD), tissue hypoxia (caused by vaso-occlusive events, abnormal red cell rheology and anemia), intense oxidative stress and persistent hemolysis are three major components that determine pathogenesis. In SCD, ischemic events are likely to activate hypoxia-inducible factor-1α (HIF-1α), a transcription factor that is involved in regulation of several genes including genes for vasoactive molecules. Although tissue ischemia is the primary instigator of HIF-1α activation, the induction, stabilization and degradation of HIF-1α also involves participation of signaling molecules, oxygen sensors (prolyl hydroxylases or PHDs) and redox reactions. Nitric oxide (NO) is a major signaling molecule affecting the activity of PHDs. We hypothesize that increased oxidative stress and reduced NO bioavailability caused by cell-free plasma heme will have a significant effect on HIF-1α expression under in vivo conditions. To this end, we have explored the effect of hemolysis and NO bioavailability on HIF-1α expression:

• in transgenic-knockout sickle (BERK) mice expressing varying levels of anti-sickling fetal hemoglobin (HbF), and

• in transgenic sickle (NY1DD) mice subjected to arginine treatment and hypoxia.

We used BERK mice expressing different levels of HbF: BERK (HbF <1.0), BERKγM (~20% HbF) and BERKγH (~40% HbF). BERK mice show severe pathology, tissue hypoxia, significant hemolysis, and accumulation of HIF-1α under ambient conditions as determined in the cremaster tissue. Also, BERK mice showed maximal hemolytic rate (measured as plasma heme), and introduction of γ-transgene to elevate HbF levels to 20 and 40% caused significant reductions in plasma heme values as reported (Kaul et al. JCI, 2004). Increasing HbF expression and reducing hemolysis in BERK mice resulted in corresponding increases in NO metabolites (NOx) levels (P<0.05, multiple comparisons by ANOVA). Notably, in BERK mice, HIF-1α expression decreased by almost 50% with increased NOx levels. The present in vivo finding is in contrast with the reported NO-provoked HIF-1α accumulation in vitro under normoxic conditions. Next, we evaluated the effect of NO bioavailability on HIF-1α accumulation under hypoxic conditions. To this end, we treated in C57BL and transgenic sickle (NY1DD) mice with arginine (5% arginine in mouse chow). NY1DD mice show mild pathology but develop severe phenotype under hypoxic conditions. Both groups of mice were treated for 15 days with arginine followed by hypoxia (8% O₂) starting day 12 of arginine treatment. Compared with normoxic controls, hypoxia caused marked accumulation of HIF-1α in the cremaster tissue. Importantly, arginine markedly reduced hypoxia-induced HIF-1α activation in NY1DD mice to the control level, indicating that under hypoxic conditions increased NO bioavailability (arginine treatment) is associated with degradation of HIF-1α. Interestingly, arginine-treated NY1DD mice show increased NOx levels, and reduced expression of heme oxygenase-1 (HO-1), a marker of hemolysis, by almost 40%. Taken together, these results provide the first in vivo demonstration that the hemolytic rate and NO bioavailability have a major influence on HIF-1α expression in the mouse models of SCD.