Efectos moleculares de nuevos agentes promotores de la migración celular y la reepitelización de heridasmembrana amniótica y ácido oleanólico

Abstract

The cicatrisation of wounds is a complex process which implies different stages whose objective is to restore the integrity of the damaged tissue. If the normal process of healing is impaired, non-healing wounds can develop. Better described as chronic wounds, these are lesions that are unable to progress through the normal healing stages and thus they cannot to epithelize. The accumulated evidence shows the ability of some agents to promote the reversion of this situation and facilitate the epithelialization of these wounds. Amongst them it seems to stand out the abilities of a perinatal tissue, the amniotic membrane (AM), and the ones of a plant extract, the oleanolic acid (OA). The main objective of this research is to contribute to the knowledge of those molecular mechanisms through which the AM and the OA can promote the reepithelialization of skin wounds. For that purpose, several experiments have been directed to evaluate the effects of these agents on the functional properties of epithelial cells, migration and proliferation, as well as its effects on crucial molecular routes for these processes. In the execution of this strategy different types of epithelial cells have been employed, including mink lung epithelial cells (Mv1Lu), human breast epithelial cells MDA-MB-231 and the spontaneously immortalised human keratinocytes HaCaT cell line. To consider the effect of AM and OA on cell migration, artificial wound closure studies were executed in vitro in the presence of AM or OA, along with inhibitors of specific routes. This has allowed to understand how both AM and OA stimulate cell migration through the activation of the MAP Kinase pathway, including ERK and JNK. Also, it was proved that the migration promoted by the AM was affected by inhibiting TGF-ß signalling, whereas the migration promoted by the OA was affected by the inhibition of EGF receptor. Regarding the effects on proliferation, in the case of AM, as proposed by other authors, we confirmed that it promotes cell division and proliferation. In the case of OA, on the other hand, it seems to exert certain negative influence on the proliferation in the short term without affecting the cell cycle. Together with the indicated research, genetic expression studies were performed as well, aiming to define the transcriptomic effects of AM and OA on genes involved in cell proliferation and migration. The obtained results showed that both OA and AM are capable of altering the expression profile of relevant genes involved in cell migration, such as SNAI2, c-JUN and PAI-1, as well as genes involved in the regulation of the cell cycle, like CDKNIA. Furthermore, in order to obtain a complete vision, microscopy studies were carried out through inmunofluorescense techniques, allowing to precisely characterise the effects that the exposure to AM or OA has on the cell architecture and in relation to cell migration. At the migrating edge of artificial wound closure experiments, it was observed how the AM absolutely abates the nuclear signature of the SMADs. Moreover, it was proved how the treatment with AM was capable of promoting the remodelling of the focal adhesions of MV1LU and HaCaT cells, inducing the expression of paxillin, while also increasing the dynamism of the focal complexes in the migrating cells. This dynamism was related to an increase of the activation state of the focal adhesion kinase (FAK) on the migrating edge of HaCaT cells. Regarding the treatment with OA, its ability to promote the internalization of the EGF receptors was proved, the same as it occurs with the exposure the native ligand. These data make us propose the concurrence of diverse molecular routes in the migration promoted either by AM or OA. So, the stimulation of the cell migration caused by AM would be contributed by a mitigating effect on the signalling dependent on TGF-ß through the route of the SMADs, an effect observed both in Mv1Lu cells and as in HaCaT cells. Moreover, the AM triggers the recruitment of the MAP kinase pathway, involving the EGF receptor and promoting the activation of MEK1 jointly with the phosphorylation of c-Jun, a phenomenon that is crucial for cell migration at the edge and a correct wound closure. Regarding the OA, the effects of this agent also seem to involve the activation of the EGF receptor, also implying the activation of the MPA kinase pathway and specially JNK. This is coherent with the development of an expression profile indicative for the acquisition of tissue remodelling abilities, similar to a status of epithelial-mesenchymal transition, which is proved by changes in the cyto-architecture and the increase of migration. This specific knowledge disclosing the way of acting of these agents may open opportunities for future innovations in the management of chronic wounds.