Abstract
Purpose: :
We studied whether 20-HETE also leads to the activation of HIF-1α in EC.Methods and
Results: :
We used the stable 20-HETE analog WIT003, and found that there was a marked increase in HIF-1α expression 6 hr after the addition of 10 µM WIT003. VEGF increases 4 hr after WIT003 thus preceding changes in HIF-1α. These changes in HIF-1α were suppressed by a VEGF neutralizing antibody and by the VEGF Receptor2 kinase inhibitor SU5416, suggesting that the increase in HIF-1α induced by WIT003 are mediated by VEGF. Incubation with PEG-SOD (400 U/ml), apocynin (100 µM) or DPI (10 µM) inhibited the increases in HIF-1α expression, thus indicating that NADPH oxidase-dependent superoxide is involved in mediating the response. Furthermore, WIT003 induced an increased p47phox expression in EC prior to the activation of HIF-1α, as well as translocation to the cell membrane indicating the assembly and activation of the NADPH subunits. WIT003-induced HIF-1α is dependent on MAPK activation since incubation with the MEK1/ERK1/2 inhibitor U0126 completely abolishes the increases in both VEGF and HIF-1α. Since the erithropoietin receptor is downstream of HIF, we tested whether this protein was altered after treatment with WIT003. We found that EpoR doubled after WIT003 treatment.
Conclusions: :
These results suggest that 20-HETE may be a novel non-hypoxic regulator of HIF-1α, and thus HIF-dependent genes in EC.
Keywords: hypoxia • vascular endothelial growth factor • lipids