Angiopoietin-Like 4 Accelerates Skin Wound Re-Epithelialization By Promoting Epidermal Stem Cell Proliferation And Migration In Mice

Epidermal stem cells(EpSCs)residing in the epidermis of skin tissue have the ability to self-renew,proliferate and differentiate,and are key cells involved in the maintenance of skin homeostasis and the repair of epidermis after injury.However,the mechanisms underlying EpSC self-renewal and proliferation are not fully understood.Angiopoietin-like 4(ANGPTL4)is a secretory protein.It has been reported that knockout of Angptl4 in mice impaired keratinocyte migration and differentiation,and delayed re-epithelialization during skin wound healing.We hypothesize that ANGPTL4 may contribute to re-epithelialization and healing of skin wounds by regulating the proliferation and migration of epidermal stem cells.ObjectiveTo investigate the involvement of ANGPTL4 in re-epithelialization of skin wound and explore the underlying mechanisms by using Angptl4 gene knockout(Angptl4-/-)mice and cultured EpSCs.MethodsFull-thickness wounds were induced on the back of mice by using a skin biopsy punch.Wound re-epithelialization was examined by H&E staining.The expression of ANGPTL4,cell proliferation marker(PCNA)and EpSC markers(β1 integrin,α6 integrin)in skin tissues were detected by immunohistochemical staining.EpSCs were isolated from mouse skin and cultured.Flow cytometry analyses were performed to detect the expression of EpSC markers and cell cycle distribution of EpSCs.EpSC proliferation was detected by MTT assay and BrdU incorporation assay.The expression of cyclins was detected by RT-PCR and Western blot.The migration of EpSCs was detected by cell scratch assay.ResultsImmunohistochemical staining showed that the expression of ANGPTL4 was significantly increased in the basal layer cells of the epidermis around the wound during skin wound healing.Knockout of Angptl4 in mice delayed skin wound healing.H&E staining showed that ANGPTL4 deficiency in mice resulted in impaired skin wound re-epithelialization,including smaller thickness,length and area of the regenerated epidermis in Angptl4-/-mice than in WT mice.Immunohistochemical staining for PCNA,β1 integrin,and α6 integrin-expressing cells in regenerated epidermis of skin wounds revealed that the number and proliferation of EpSCs in the basal layers of the epidermis was significantly decreased in Angptl4-/-mice.In vitro studies showed that ANGPTL4 deletion in EpSCs significantly reduced cell proliferation,increased cell population in G1-phase cells,reduced cells in G2-phase,and down-regulated the expression of cyclins D1 and A2.Rescue the expression of ANGPTL4 in ANGPTL4 deficient EpSCs by transfection the cells with ANGPTL4 expression plasmid resulted in a decrease of G1-phase cells,an increase of S-phase cells,and elevation of cyclins D1 and A2 expression.In addition,ANGPTL4 deficiency inhibited the migration of EpSCs,which could also be reversed by ANGPTL4 overexpression.ConclusionThe expression of ANGPTL4 is elevated after skin injury.ANGPTL4 promotes EpSCs proliferation through upregulating cyclins D1 and A2 expression and accelerating cell cycle transition from G1 to S phase.ANGPTL4 promotes skin wound re-epithelialization by stimulating EpSC proliferation and migration.ANGPTL4 is a novel molecule involved in EpSC activation and re-epithelialization during skin wound healing.