Bach1 Inhibitors Reduce Stasis in Townes Sickle Cell Mouse Model

In sickle cell disease (SCD) excessive release of Hb and heme into the vasculature can activate innate immune toll-like receptor 4 (TLR4) signaling in the vasculature leading to inflammation and vaso-occlusion. Heme-mediated TLR4 signaling can be interrupted by inducing heme oxygenase 1 (HO-1) to degrade heme or ferritin heavy chain (FTH1) to sequester iron. Globin synthesis, HO-1 expression, heme/iron homeostasis, and antioxidant levels are regulated in part by antioxidant response genes, which is a set of hundreds of genes that possess antioxidant response elements (ARE) in their promoter regions. Bach1 complexes bind to ARE sites and dominantly repress Nrf2 transcriptional activity. This transcriptional repression can be inactivated by heme binding to Bach1. We have discovered small molecule BACH1 inhibitors that increase HO-1 and FTH1 mRNA in HepG2 cells. Additionally, they restore glutathione levels and inhibit VCAM-1 expression in human primary aortic endothelial cells upon hemin or TNFα challenge. To investigate if Bach1 inhibitors could have an effect on vaso-occlusion in vivo, microvascular stasis was examined in Townes-SS mice with implanted dorsal skin-fold chambers. Townes-SS mice were orally gavaged with vehicle or Bach1 inhibitors at different doses once daily for 8 days. Microvascular stasis was determined in 20-25 preselected flowing subcutaneous venules in response to intravenous infusion of hemin (3.2 μmol/kg). Stasis was significantly inhibited by Bach1 inhibitors in a dose responsive manner. After stasis measurement, blood was collected and the F-cells were stained using the Kleihauer-Betke Test. F-cell % was significantly increased by Bach1 inhibitor treatment at all doses tested.