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

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process.MethodsPolyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin.ResultsProtein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin.ConclusionsWe provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

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

Purpose: Neoangiogenesis is essential for tumor development. Hypoxia-inducible factor (HIF), a transcriptional factor composed of two subunits (a and h), plays a key role in this process, activating proangiogenic factors such as vascular endothelial growth factor (VEGF). The HIF a subunits are critically regulated by oxygen and are also modulated by growth factors. Kaposi sarcoma (KS) is a highly vascular tumor that releases large amounts of VEGF and for which we have recently described an essential role for the insulin-like growth factor (IGF) system. We therefore investigated the expression of HIF a subunits in biopsies from KS tumors and their modulation by IGF-I in KSIMM, a KS cell line. Results: Both HIF-1a and HIF-2a were expressed in KS biopsies in all tumoral stages. HIF-1a immunopositivity increased through the tumor development with highest expression in the late nodular stages. In KSIMM cells, IGF-I induced accumulation of both HIF a subunits.The induction suggests a translation mechanism as documented by cycloheximide chase experiment coupled with constant RNA levels as evaluated by quantitative real-time PCR. IGF-I^induced HIF a accumulation was followed by an increase in HIF function as assessed both by reporter gene assay and by induction of endogenous target gene expression (VEGF-A). Specific blockade of IGF-I receptor with aIR3 antibody or with picropodophyllin, a specific IGF-IR tyrosine kinase inhibitor, diminishes the basal and IGF-I^dependent induction of both HIF a congeners. Conclusion: These novel findings show the coupling between the IGF and HIF signaling in KS and suggest a coordinated contribution by these pathways to the characteristic vascular phenotype of this tumor.

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.MethodsFibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.ResultsChallenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.ConclusionWe propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.