Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds

Abstract

BackgroundPost-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds.Methods and FindingsTo better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border.ConclusionsThe effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.

Amniotic membrane stimulates cell migration by modulating transforming growth factor‐β signalling

“…Moreover, its relevance is further demonstrated by the fact that AM co‐treatment with TGF‐ß provides increased migration both in MvLu1 and HaCaT cells, although treatment with just TGF‐ß reports mixed results. Interestingly, in line with observations about AM treatment inducing re‐epithelialization of chronic wounds (Alcaraz et al, ; Insausti, Alcaraz, et al, ), previous work in our laboratory has demonstrated that AM is able of attenuating the expression profile of relevant genes involved in cell cycle regulation under TGF‐ß control (Alcaraz et al, ), which resembles how attenuation of TGF‐ß signalling has also been demonstrated to be critical for some tumour cells to escape from cell cycle arrest (Nicolas & Hill, ). In that sense, exacerbated canonical TGF‐ß signalling has been linked to the promotion of skin fibrosis in an intricate process that involves fibroblast, keratinocyte and leucocyte interaction (Andrews, Marttala, Macarak, Rosenbloom, & Uitto, ; Ashcroft & Roberts, ).…”

“…AM was prepared as described in Alcaraz et al (). Briefly, term placenta from healthy donor mothers was obtained from uncomplicated caesarean section.…”

“…In the past, we have used AM as a biological dressing for infection control and, most importantly, to encourage re‐epithelialization in deep traumatic wounds (Insausti, Alcaraz, et al, ). Interestingly, when applied in vitro , AM induced the activation of extracellular signal‐regulated kinases 1/2 (ERK1/2) and c‐JUN N‐terminal kinases 1/2 (JNK1/2), with the overexpression of c‐Jun along the wound edge expression (Alcaraz et al, ; Insausti, Alcaraz, et al, ). Moreover, AM presence in epithelial cell cultures promoted the activation of R‐Smads and affected the expression profile of several TGF‐ß‐dependent genes (Alcaraz et al, ).…”

Mitogen-activated protein kinase phosphatase-3 (MKP-3) in the surgical wound is necessary for the resolution of postoperative pain in mice

“…An increase in p-ERK-1/2 and p-p38 expression as well as a functional association between the two have been observed in keratinocytes of psoriatic lesions in humans42–44 and in mouse keratinocytes under pruritogenic conditions 45. Furthermore, p-ERK expression is enhanced in human keratinocytes in in vitro models of acute and chronic wounds 46,47. The phosphorylation of ERK can also be induced by pro-algesic factors such as epidermal growth factor 47.…”

Antimicrobial peptide Epinecidin-1 promotes complete skin regeneration of methicillin-resistant Staphylococcus aureus-infected burn wounds in a swine model

“…The capacity of HaCaT cells to migrate, proliferate, and close a defined injury area into a confluent monolayer was evaluated by following a previously reported assay with some modifications [30]. Briefly, monolayer cells were scratched vertically with a P1000 pipette tip to create an artificial wound and washed twice with PBS to remove cellular debris.…”