Hypoxia-Inducible Factor (HIF-1alpha) Has a Crucial Role in Microvascular Regulation in Transgenic/Knockout Sickle Mouse Models.

In sickle cell anemia (SCA), abnormal red cell rheology, hemolytic anemia and chronic hypoxia will impact endothelial function and result in compromised microvascular regulation. In SCA, chronic hypoxia and hypoxic events are likely to activate hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional factor that is involved in regulation of several genes including genes for vascular endothelial growth factor (VEGF) and vasoactive molecules (e.g., inducible NOS, neuronal NOS, heme oxygenase [HO-1], endothelin). However, the status and role of this critical factor remains unexplored in SCD. We hypothesize that in SCA, chronic hypoxia caused by intravascular sickling (hemoglobin S polymerization), low hematocrit and vaso-occlusive events will induce HIF-1alpha, which will lead to activation of vasoregulatory genes and affect vascular tone mechanisms. To this end, we have explored the expression of HIF-1alpha:

• under normoxic conditions in transgenic-knockout sickle BERK mice (express exclusively human alpha- and beta S-globins; severe pathology) and

• in transgenic sickle (NY1DD) mice (mild pathology) subjected to prolonged hypoxia.

In BERK mice, low hematocrit coupled with intravascular sickling will contribute to chronic hypoxia. We selected the cremaster muscle preparation used for our microcirculatory studies in BERK mice that have shown in vivo sickling, red cell adhesion and frequent episodes of transient ischemic events (Kaul et al. JCI, 2004). Western blotting of cytoplasmic and nuclear extracts of rapidly excised cremaster muscle from normoxic C57BL and BERK mice was carried out using mouse-reactive anti-HIF-1alpha antibodies. HIF-1alpha positive bands were detected at 90 kDa. Densitometric analysis confirmed average increases of 1.6-and 2.3-fold, respectively, in cytoplasmic and nuclear extracts of BERK mice (P<0.05 and 0.01 vs. C57BL), suggesting significant translocation of HIF-1 dimer to the nucleus. These findings show that HIF-1alpha is stabilized in BERK mice under normoxic conditions. The stabilization is probably facilitated by associated 6-fold increase in prostaglandin E2, increased COX-2 expression and higher cytokine (e.g., TNF-alpha) levels, which have been implicated previously in HIF1-alpha stabilization in other conditions. Secondly, we subjected transgenic NY1DD sickle mice to long-term hypoxia (4 days) using 8% oxygen in the breathing mixture, which has been shown to induce sickling and dense cell formation (Fabry et al. PNAS, 1992). In NY1DD mice, hypoxia caused 2.3-fold increase in HIF-1alpha expression as compared to normoxic controls (P<0.03). This response was exaggerated as compared with hypoxic C57BL mice (P< 0.05). The greater expression of HIF-1alpha in NY1DD mice was accompanied by 5.6-fold increase in VEGF expression and induction of inducible NOS. Importantly, the induction of HIF in NY1DD mice was accompanied by significant diameter and volumetric flow increases in A2 and A3 arterioles as compared with normoxic values (each P<0.01) approaching those seen in BERK mice. Our results predict that induction/stabilization of HIF-1alpha will have a crucial effect on genes for vasoactive molecules, and thereby on microcirculatory and hemodynamic parameters in SCA.