Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1␣ is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1␣ stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1␣ stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1␣ hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1␣ is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1␣ is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1␣ is critical to reverse the pathological process.angiogenesis ͉ chronic complications ͉ hypoxia ͉ hyperglycemia ͉ chronic ulcers

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Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on hypoxia-inducible factor 1 (HIF-1), which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia-responsive element reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO-mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

The success of pancreatic β-cells transplantation to treat type 1 diabetes has been hindered by massive β-cell dysfunction and loss of β-cells that follows the procedure. Hypoxia-mediated cell death has been considered one of the main difficulties that must be overcome for transplantation to be regarded as a reliable therapy. Here we have investigated the mechanisms underlying β-cell death in response to hypoxia (1% O2). Our studies show that mouse insulinoma cell line 6 (Min6) cells undergo apoptosis with caspase-3 activation occurring as early as 2 h following exposure to hypoxia. Hypoxia induces endoplasmic reticulum stress in Min6 cells leading to activation of the three branches of the unfolded protein response pathway. In response to hypoxia the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) is upregulated. The important role of CHOP in the apoptotic process was highlighted by the rescue of Min6 cells from hypoxia-mediated apoptosis observed in CHOP-knockdown cells. Culturing isolated pancreatic mouse islets at normoxia showed intracellular hypoxia with accumulation of hypoxia-inducible factor-1α and upregulation of CHOP, the latter one occurring as early as 4 h after isolation. Finally, we observed that pancreatic islets of type 2 db/db diabetic mice were more hypoxic than their counterpart in normoglycemic animals. This finding indicates that hypoxia-mediated apoptosis may occur in type 2 diabetes.

Objective. Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)-blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti-TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF-B ligand (RANKL) in synovial tissue.Methods. The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by doubleblind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one-way analysis of variance, with Tukey's post hoc test.Results. Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF-primed osteoblasts and endothelial cells.Conclusion. Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti-TNF therapy.Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with skeletal complications, such as focal bone erosions at the site of inflammation and systemic osteopenia, that lead to joint-related disability. The formation and activation of osteoclasts at the cartilagepannus junction appear to be essential for RA-associated bone loss. These processes are driven by the interaction between receptor activator of NF-B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG) (1).A link between bone biology and the immune system is consistent with the finding that several cytokines known to mediate immune functions are also implicated in bone resorption. For instance, tumor necrosis factor (TNF) is one of the more potent osteoclastogenic cytokines produced in the setting of inflammation and is able to induce RANKL expression on the surface of stromal/osteoblast cells (2). The importance