Hypoxia and hypoxia mimetics inhibit TNF-dependent VCAM1 induction in the 5A32 endothelial cell line via a hypoxia inducible factor dependent mechanism

Background: We previously reported that iron chelators inhibit TNF alpha-mediated induction of VCAM-1 in human dermal microvascular endothelial cells. We hypothesized that iron chelators mediate inhibition of VCAM-1 via inhibition of iron-dependent enzymes such as those involved with oxygen sensing and that similar inhibition may be observed with agents which simulate hypoxia. Objective: We proposed to examine whether non-metal binding hypoxia mimetics inhibit TNF alpha-mediated VCAM-1 induction and define the mechanisms by which they mediate their effects on VCAM-1 expression. Methods: These studies were undertaken in vitro using immortalized dermal endothelial cells, Western blot analysis, ELISA, immunofluorescence microscopy, quantitative real-time PCR, and chromatin immunoprecipitation. Results: Hypoxia and the non-iron binding hypoxia mimetic dimethyl oxallyl glycine (DMOG) inhibited TNF alpha-mediated induction of VCAM-1. DMOG inhibition of VCAM-1 was dose-dependent, targeted VCAM-1 gene transcription independent of NF-kappa B nuclear translocation, and blocked TNF alpha-mediated chromatin modifications of relevant elements of the VCAM-1 promoter. Combined gene silencing of both HIF-1 alpha and HIF-2 alpha using siRNA led to a partial rescue of VCAM expression in hypoxia mimetic-treated cells. Conclusion: Iron chelators, non-metal binding hypoxia mimetics, and hypoxia all inhibit TNF alpha-mediated VCAM-1 expression. Inhibition is mediated independent of nuclear translocation of NF-kappa B, appears to target TNF alpha-mediated chromatin modifications, and is at least partially dependent upon HIF expression. The absence of complete VCAM-1 expression rescue with HIF silencing implies an important regulatory role for an Fe(II)/alpha-ketoglutarate dioxygenase distinct from the prolyl and asparagyl hydroxylases that control HIF function. Identification of this dioxygenase may provide a valuable target for modulating inflammation in human tissues. Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.