Activation Of Epidermis Manipulate Skin Scar Formation In Response To Reduced Hydration In Wounds

Part I Expression of Proinflammatory Genes in Epidermal Cells Is Regualated by Reduced HydrationObjective:To investigate hydration status altering epidermal gene expression patterns in rabbit partial-thickness incisional wounds and try to provide an opportunity to elucidate the mechanisms in scar formation.Methods:Treatment of injured skin on rabbit ears with a semi-occlusive dressing preserves the hydration of the skin and then the scar formation in different hydration conditions was evaluated. Using microarray studies analyzed epidermal gene expression patterns in different conditions and classified the different genes according to their functions. Among them the genes related with inflammation (IL-11β, IL-8, TNF-α, COX-2) and S100A8, S100A9were focused and further verified in human ex vivo skin culture (HESC) and stratified keratinocytes culture (SKC). Hierarchical analysis of genes was performed by RNA interference (RNAi).Results:Using microarray studies on injured epidermis, we showed that global genes expression patterns in highly occluded versus non-occluded wounds were distinct. Decreased levels of hydration resulted in an increased expression of proinflammatory genes (IL-1β, IL-8, TNF-α, COX-2) and S100A8, S100A9in HESC and SKC. Hierarchical analysis of genes using RNA interference showed that both TNF-a and IL-lb regulate the expression of IL-8through independent pathways in response to reduced hydration. TNF-a/IL-8pathway was found to be mediated by COX-2and IL-8in turn controls the production of matrix metalloproteinase-9(MMP-9) in keratinocytes.Conclusion:Hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing. Part II S100A8and S100A9Expressed in Epidermal Cells Mediate Activation of Skin Fibroblasts When Reduced HydrationObjective:Microarray studies in rabbit partial-thickness incisional wounds showed that reduced hydration on epidermis upregulated the expression of S100A8and S100A9in the epidermal cells. Then their effect on the skin fibroblasts and role in dermal fibrosis were studied.Methods:Expression of S100A8and S100A9was compared between hypertrophic scar/keloid and normal skin by immunofluorescence (IFC) analyses. The characteristic of HaCaT cells was studied when knockdown of S100A8or S100A9by lentivirus mediated RNAi, then analyzed the effect of S100A8or S100A9from epidermal cells on cocultured fibroblasts by activation marker, α-SMA and Procollagen-I. The interaction of S100A8or S100A9and other elevated inflammatory factors (IL-1β, IL-8, TNF-a, COX-2) was investigated by q-PCR.Results:IFC showed that S100A8or S100A9were highly expressed in the epidermis of human hypertrophic scar and keloid tissues compared with normal skin. S100A8or S100A9genes in HaCaT cells can be silenced successfully by lentivirus mediated RNAi and the relative expression of S100A8was21%and S100A9was22%in gene knockdown cells. Knockdown of S100A8or S100A9resulted in a decrease of proliferation and migration of keratinocytes. Fibroblasts were activated when cocultured with HaCaT cells, but this activation effect would be alleviated when S100A8or S100A9gene was knockdown in HaCaT cells. Hierarchical analysis of genes by RNAi showed that S100A8and S100A9were down-stream genes of Cox-2, TNF-a, and IL-8, which were also upregulated in keratinocytes by reduced hydration in wounds.Conclusion:Our results indicate that S100A8and S100A9are increased in epidermal when reduced hydration, which play a critical role in scar formation. S100A8and S100A9are novel targets in preventing scarring and optimizing wound healing. Part IIIS100A8and S100A9Activate Skin Fibroblasts by TLR4and RAGEObjective:In order to study the effect of recombined human S100A8and S100A9on human skin fibroblasts and identify the receptor pathway involved in this effect.Methods:The pET-15b expression vector was designed from directly cloning of S100A8and S100A9PCR products. Then this expression vector was transformed into E.coli (BL21) and induced to expression proteins by IPTG. The products were identified by SDS-PAGE and purified by QIA expressionistTM. After Endotoxin removing and assay, the recombined proteins were used to treat human fibroblasts harvested from foreskin tissue (0.2ng/μl、 lng/μl、5ng/μl). Activation of treated fibroblasts was evaluated by the expression of a-SMA and procollagen-I in fibroblasts. After pretreated by special inhibitor for TLR-4(TAK-242) or RAGE (FPS-ZM1) the fibroblasts were stimulated by recombined S100A8and S100A9to illustrate which receptor was involved in this activation process.Results:By pET-15b expression vector in BL21we harvested highly qualified S100A8and S100A9proteins after purified by QIA expressionistTM. The expressed proteins were located at lOkDa and13kDa in SDS-PAGE and declared the expressed proteins were human S100A8and S100A9separately. Treatment of recombinant S100A8and S100A9proteins showed increased expression of a-SMA and procollagen-I in dermal fibroblasts. Pharmacological inhibition studies show that either blocking TLR4(TAK-242) or RAGE (FPS-ZM1) in fibroblasts alleviated the activation effect of S100A8and S100A9on dermal fibroblasts.Conclusion:Human S100A8and S100A9can activate dermal fibroblasts and this effect was carried out through both TLR4and RAGE in fibroblasts.