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

Abstract 

Background

We previously reported that iron chelators inhibit TNFα-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 TNFα-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 TNFα-mediated induction of VCAM-1. DMOG inhibition of VCAM-1 was dose-dependent, targeted VCAM-1 gene transcription independent of NF-κB nuclear translocation, and blocked TNFα-mediated chromatin modifications of relevant elements of the VCAM-1 promoter. Combined gene silencing of both HIF-1α and HIF-2α 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 TNFα-mediated VCAM-1 expression. Inhibition is mediated independent of nuclear translocation of NF-κB, appears to target TNFα-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)/α-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.

Abbreviations: α-KG, α-ketoglutarate, CAM, cell adhesion molecule, DMOG, dimethyl oxallyl glycine, DP, 2,2′-dipyridyl, egr-1, early growth response protein 1, FIH1, factor inhibiting HIF, HDMEC, human dermal endothelial cells, HIF, hypoxia inducible factor(s), hnRNA, heterogenous nuclear RNA, IRF-1, interferon regulatory factor 1, PHD, prolyl hydroxylase, RT-PCR, reverse transcription real-time polymerase chain reaction, TGFα, transforming growth factor-α, VCAM, vascular cell adhesion molecule

Keywords: Endothelial, Cell adhesion, TNF, Hypoxia, VCAM-1