Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.

Methods for human skin gene therapy requires efficient and stable introduction of genes into skin cells. Transient cutaneous gene therapy is an attractive approach in the treatment of skin diseases. The ‘Achilles heel’ of adenoviral gene therapy is its immunogenicity and many aspects of adenovirus induced cutaneous immune reaction still remain unanswered, particularly the role of keratinocytes. Therefore, human keratinocytes were transfected with adenoviral DNA and cytokine expression was analyzed. Moreover, adenoviral transduction of full-skin was performed ex vivo and in vivo. We observed cytokine induction after cytoplasmatic internalization of adenoviral DNA into epidermal cells. Inhibition of AIM2, NALP3, DAI or mda5 downregulated the cytokine response. Transduction of immunocompetent mice led to a detectable transgene expression for 12 days. Re-application of the vector led to a decrease in intensity and duration of transgene expression limited to 4 days and an increased cytokine expression. In contrast, immunodeficient mice showed a reduced expression of cytokines after DNA internalization. AIM2, NALP3, DAI and mda5 are essential in the induction of an innate immune response towards adenoviral DNA. This immune reaction leads to a decrease in transduction efficiency of the vector after re-application and modulation of these receptor systems stabilizes transgene expression.

Background: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.