Hypoxia-Dependent HIF-1 Activation Impacts on Tissue Remodeling in Graves' Ophthalmopathy-Implications for Smoking.

CONTEXT: In Graves' ophthalmopathy (GO), inflammation with tissue expansion in a closed compartment like the bony orbit and smoking may cause tissue hypoxia. OBJECTIVES: In this study, we investigated whether hypoxia-inducible factor-1 (HIF-1) action impacts on tissue remodeling in GO with the aim to identify possible new therapeutic targets. DESIGN/SETTING/PARTICIPANTS: Orbital fibroblasts (OFs) were derived from GO patients and control (Ctrl) persons. We analyzed HIF-1α levels in response to hypoxia and cigarette smoke extract, as well as HIF-1-dependent vascular endothelial growth factor (VEGF) release and adipogenic differentiation, by using HIF-1α small interfering RNA, or HIF-1 inhibitor BAY 87-2243. MAIN OUTCOME MEASURES: Western blot, real-time PCR, ELISA, and immunohistochemistry were used to analyze HIF-1α, VEGF, CD31, and adiponectin. Adipogenic differentiation was measured with Nile red assay. RESULTS: Higher HIF-1α levels in OFs were correlated with the clinical activity score of GO patients. Cigarette smoke extract elevated HIF-1α levels. HIF-1-dependent VEGF secretion was enhanced in GO-OF compared to Ctrl-OF, and as an in vivo consequence, we found a higher vessel density in GO tissue than in Ctrl tissue. Hypoxia strongly stimulated HIF-1-dependent adipogenesis and adiponectin release of GO-OF and enhanced TSH receptor-mediated adipogenesis. CONCLUSIONS: Hypoxia impacts on tissue remodeling in GO by stimulating angiogenesis and adipogenesis through activation of HIF-1-dependent pathways in OFs. Our results offer a molecular mechanism for the detrimental influence of smoking on GO and an explanation as to why decompression can improve the outcome of patients. Drug-targeted inhibition of HIF-1/VEGF may provide a therapeutic option to control tissue expansion in GO.

Role of reactive oxygen species in the regulation of HIF-1 by prolyl hydroxylase 2 under mild hypoxia.

The function and survival of eukaryotic cells depends on a constant and sufficient oxygen supply. Cells recognize and respond to hypoxia by accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1ß subunit. Besides physiology, HIF-1 induction is involved in major pathological processes such as cardiovascular disease, inflammation and cancer, which are associated with the formation of reactive oxygen species (ROS). ROS have been reported to affect HIF-1 activity but the role for ROS in regulating HIF-1 has not been definitely settled. In order to shed light on the redox-regulation of HIF-1 by ROS, we studied the impact of exogenous ROS treatment (H(2)O(2)) on HIF-1α and HIF-1 regulatory protein prolyl hydroxylase 2 (PHD2) in the human osteosarcoma cell line U2OS. At early reaction periods, H(2)O(2) induced HIF-1α but at prolonged observation phases the opposite occurred. Herein, modulation of PHD activity appeared to be the key element, because knockdown and inhibition of the PHD2 prevented reduction of HIF-1α. However, H(2)O(2) treatment constantly suppressed HIF-1 transactivation at all time-points. Our data indicate a dual redox regulation of HIF-1α protein amount with a constant suppression of HIF-1 target gene expression by ROS.

Role of N-acetyl-N-nitroso-tryptophan as nitric oxide donor in the modulation of HIF-1-dependent signaling.

N-Acetyl-N-nitroso-tryptophan (NANT) is well known for its capacity to generate nitric oxide (NO)-releasing compounds. It is unknown, however, whether NANT can be successfully applied as a precursor of NO in a complex biological environment such as a cell culture system. NO donors can be useful to induce the transcription factor hypoxia-inducible factor 1 (HIF-1) that coordinates the protection of cells and tissues from the lack of oxygen, termed hypoxia. HIF-1 degradation is controlled by prolyl hydroxylase 2 (PHD2) which needs to be inhibited for HIF-1 accumulation. Here, the effects of NANT in inhibiting recombinant PHD2 and up-regulating of HIF-1 and HIF-1-mediated carboanhydrase-9 (CA9) mRNA expression were compared in living cells with the NO donors N-nitrosomelatonin (NOMela) and S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NANT was similar to NOMela being highly effective in inhibiting recombinant PHD2. NANT-mediated activation of HIF-1 in oxygenated cells was comparable to hypoxic activation of HIF-1 in all cases. In contrast, under hypoxia NANT was able to boost hypoxic cellular HIF-1 levels by further reducing the activity of cellular PHD2. The strong increase of HIF-dependent CA9 mRNA expression demonstrated that NANT-induced HIF-1 was transcriptionally active. Finally, the efficacy of NANT to increase both HIF-1 and CA9 mRNA did not depend on the absolute conformation of the tryptophan moiety. In conclusion, NANT appears to be an excellent NO donor for cells in culture and l-NANT should be useful for in vivo animal studies.