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In recent years, the reconstruction of human skin by tissue engineering represents a clinical challenge and has offered a therapeutic alternative. Avascular engineered skin equivalents have been available for several years and used to treat wounds due to burns, nonhealing ulcers, and surgical excisions. They are constituted by different types of cultured cells included in a three-dimensional structure that permits cellular proliferation to create tissue substitutes. The major drawback of these artificial skin substitutes is their lack of blood supply, since the endurance and cell proliferation of the substitute depend on an adequate oxygen and nutrient supply and on toxin removal. These functions are served by the vascular system. We have produced a new model of endothelialized skin substitute that promotes the formation of capillary-like structures by seeding human umbilical vein endothelial cells (HUVECs) with dermal fibroblasts and human adipose-derived mesenchymal stem cells (hADMSCs) in a fibrin matrix. Dermal fibroblasts and hADMSCs produce extracellular matrix that stimulates cellular growth and proliferation. hADMSCs secrete significant quantities of angiogenic and antiapoptotic factors (vascular endothelial growth factor and hepatocyte growth factor), which induce in vitro differentiation of these cells into endothelial cells promoting angiogenesis and participating in tissue repair and skin regeneration processes. We obtained the artificial skin substitute with similar structure to native skin, including dermis and epidermis. We demonstrated that endothelial cells (CD31 and von Willebrand factor positive) proliferated and organized themselves into capillary-like structures within the fibrin matrix. The epidermis showed a complete epithelization by squamous cells (AE1/AE3 cytokeratin positive) with intracytoplasmic keratohyalin granules, hyperkeratosis, and parakeratosis. We have established a novel artificial skin substitute that facilitates the formation of capillary-like structures that may provide a novel therapeutic approach to different skin defects and prove to be a useful tool for regenerative medicine.

Brain derived neurotrophic factor (BDNF) increases the levels of somatostatin (SS) and its mRNA. To test the hypothesis that the regulation of SS synthesis by BDNF occurs at the transcriptional level and requires specific promoter sequences, cerebrocortical and PC12trkB neurons were transiently transfected with different constructs of the SS promoter fused to the luciferase and CAT reporter genes. We demonstrated that BDNF triggered the transcription of the SS gene through the CRE sequence located in the SS promoter. BDNF and SS are genes regulated by K(+)-induced neuronal activity. Using BDNF blocking antibodies, we investigated whether K(+)-induced BDNF was required for K(+)-dependent SS mRNA induction. We found that K(+)-induced SS mRNA was partially prevented when BDNF was blocked. This finding indicated that BDNF mediated the induction of SS mRNA by K(+) depolarization. To identify the mechanisms by which BDNF activates SS gene transcription we first elucidated the signaling pathways activated by BDNF in cerebrocortical cells. We confirmed that BDNF activates the MAPK/ERKs and PI3K/Akt pathways. Both signaling pathways are, in turn, implicated in the activation of CREB by BDNF. In addition we observed that the PKA inhibitors, H89 and Rp-cAMPS decreased BDNF-induced CREB activation. These findings suggested that BDNF-induced CREB activation was also mediated by the cAMP/PKA pathway. We next elucidated the mechanism by which BDNF induces SS mRNA. We observed that H89, PD0998059, KN62 and LY294002 diminished BDNF-induced SS mRNA suggesting that BDNF-induced SS mRNA is mediated by the activation of cAMP/PKA, MAPK/ERKs, CaMK and PI3K pathways., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Ca(2+) influx through L-type voltage-gated Ca(2+) channels (L-VSCC) is required for K(+)-induced somatostatin (SS) mRNA. Increase in intracellular Ca(2+) concentration leads to the activation of cyclic AMP-responsive element binding protein (CREB), a key regulator of SS gene transcription. Several different protein kinases possess the capability of driving CREB upon membrane depolarization. We investigated which of the signalling pathways involved in CREB activation mediates SS gene induction in response to membrane depolarization in cerebrocortical cells exposed to 56 mM K(+). Activity dependent phosphorylation of CREB in Ser(133) was immunodetected. Activation of CREB was biphasic showing two peaks at 5 and 60 min. The selective inhibitors of extracellular signal related protein kinase/mitogen-activated protein kinase (ERK/MAPK) PD098059, cyclic-AMPdependent protein kinase (cAMP/PKA) H89 and RpcAMPS, and Ca(2+)/calmodulin-dependent protein kinases (CaMKs) pathways KN62 and KN93 were used to determine the signalling pathways involved in CREB activation. Here we show that the early activation of CREB was dependent on cAMP/PKA along with CaMKs pathways whereas the ERK/MAPK and CaMKs were implicated in the second peak. We observed that H89, RpcAMPS, KN62 and KN93 blocked K(+)-induced SS mRNA whereas PD098059 did not. These findings indicate that K(+)-induced SSmRNA is mediated by the activation of cAMP/PKA and CaMKs pathways, thus suggesting that the early activation of CREB is involved in the induction of SS by neuronal activity. We also demonstrated, using transient transfections of cerebrocortical cells, that K(+) induces the transcriptional regulation of the SS gene through the cAMP-responsive element (CRE) sequence located in the SS promoter.