Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibodydriven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17-DMAG dose-dependently inhibited dermal-epidermal separation ex vivo in cryosections of human skin induced by co-incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17-DMAG dose-dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP AE LPS and EBAspecific immune complexes. In addition, coimmunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membranedegrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil-driven autoantibodyinduced tissue damage, thus being a relevant therapeutic target in patients with neutrophil-mediated autoimmune diseases such as inflammatory types of EBA.

Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic wounds.