Chronic Wound Healing by Amniotic Membrane: TGF-β and EGF Signaling Modulation in Re-epithelialization

Abstract

The application of amniotic membrane (AM) on chronic wounds has proven very effective at resetting wound healing, particularly in re-epithelialization. Historically, several aspects of AM effect on wound healing have been evaluated using cell models. In keratinocytes, the presence of AM induces the activation of mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) pathways, together with the high expression of c-Jun, an important transcription factor for the progression of the re-epithelialization tongue. In general, the levels of transforming growth factor (TGF)-β present in a wound are critical for the process of wound healing; they are elevated during the inflammation phase and remain high in some chronic wounds. Interestingly, the presence of AM, through epidermal growth factor (EGF) signaling, produces a fine-tuning of the TGF-β signaling pathway that re-conducts the stalled process of wound healing. However, the complete suppression of TGF-β signaling has proven negative for the AM stimulation of migration, suggesting that a minimal amount of TGF-β signaling is required for proper wound healing. Regarding migration machinery, AM contributes to the dynamics of focal adhesions, producing a high turnover and thus speeding up remodeling. This is clear because proteins, such as Paxillin, are activated upon treatment with AM. On top of this, AM also produces changes in the expression of Paxillin. Although we have made great progress in understanding the effects of AM on chronic wound healing, a long way is still ahead of us to fully comprehend its effects.

Keywords: EGF signaling; TGF-β signaling; amniotic membrane; cell models; re-epithelialization; wound healing.

FIGURE 1

Progression of a wound. A full-thickness wound is depicted at day 1 post-injury; the skin epithelium with its cell layers indicates that the keratinocytes from the spinous layer are activated for migration, whereas keratinocytes from the basal layer proliferate to sustain cell numbers during migration. Hemostasis occurring on day 0 creates a fibrin clot with immune cells, such as macrophages. Later, an acute wound will proceed with healing with reduction of the fibrin clot, formation of granulation tissue, and re-epithelialization by creating a migrating tongue. However, in a chronic wound, granulation tissue is diminished, re-epithelialization is halted, and wound remains open after an extended time. Drawn cells in the dermis represent fibroblasts.

FIGURE 2

Chronic wounds. Intracellular pathways leading to migration and cell proliferation during AM-stimulated re-epithelialization. Proposed mechanisms of action: (A) AM, possibly by secreting EGF and low levels of TGF-β, stimulates JNK1 and MEK1-ERK1/2, which leads to active c-Jun protein. N-terminus-phosphorylated, P, c-Jun, together with nuclear Smad, enhances c-Jun expression. Additionally, AM activation of JNK1 increases remodeling of FS by phosphorylation of Paxillin (Pax). Finally, the stimulation of FAK by AM can also promote cell migration. P70 S6 kinase (p70 S6K). (B) An excess of TGF-β induces the expression of cell cycle inhibitor genes: CDKN1A and CDKN2B. AM induces an attenuation of the Smad phosphorylation mediated by MEK, which prevents proper expression of CDKN1A and CDKN2B and resumes cell cycle. (C) AM proposed mechanism of action on chronic wound re-epithelialization. The effect of AM on halted chronic wound can be summarized by the re-initiation of the migrating tongue that leads to re-epithelialization by the proposed mechanisms: (i) FS remodeling by Paxillin remodeling at the leading front, (ii) overexpression of c-Jun at the migrating tongue, and (iii) attenuation of TGF-β signaling at the rear of the migrating tongue.