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A. M. Guo, P. A. Edwards, J. C. Falck, R. J. Roman, A. G. Scicli; 20-HETE Is a Nonhypoxic Regulator of HIF in Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2652.
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We studied whether 20-HETE also leads to the activation of HIF-1α in EC.Methods and
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.
These results suggest that 20-HETE may be a novel non-hypoxic regulator of HIF-1α, and thus HIF-dependent genes in EC.
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