Fig 2: Proposed model: GCs impaired the wound healing functions of EPCs via the impairment of CXCR4 regulated pathways. GCs downregulated the expression of CXCR4 under both normoxic and hypoxic conditions, thereby reducing the migration and wound healing ability of EPCs. Under normoxic condition, GCs impaired the expression of COX2/mPEGS1, thereby suppressing the PGE2/EP4/AKT pathway, which downregulated the CXCR4 expression. Under normoxic conditions, in addition to the PEG2 pathway, GCs independently impaired the HIF2a pathway, consequently decreasing the expression of CXCR4.

Fig 3: Analysis of the extracellular expression levels of CXCR4, c-met and CCR2 in fibroblasts, HUVECs and UC-MSC using flow cytometry. The cells were detached using 0.25% trypsin and analyzed using PerCP/Cy5.5-labeled anti-human CCR2, PE-labeled anti-human CXCR4 and fluorescein isothio-cyanate-labeled anti-human c-met antibodies. Red lines represent antibody isotype controls, and black lines represent the expression of the indicated markers. CXCR4, C-X-C chemokine receptor 4; CCR2, C-C chemokine receptor 2; HUVECs, human umbilical vein endothelial cells; UC-MSCs, umbilical cord mesenchymal stem cells.

Fig 4: GCs downregulated CXCR4 via the impaired PGE2/EP4/AKT signaling in EPCs. (A) The relative mRNA expression of EP4 in untreated, GC-treated EPCs, and GC-treated EPCs in the presence of PGE2. (B) The relative protein expression of pAKT/AKT in untreated, GC-treated EPCs, and GC-treated EPCs in the presence of PGE2. (C) The inhibition of PI3K/AKT pathway in GC-treated EPCs in the presence of PGE2. LY294002 was used as a PI3K inhibitor. (D) The relative mRNA expression of CXCR4 in GC-treated EPCs in the presence of PGE2 and PI3K inhibitor. Dexamethasone (100 nM, 24-h treatment) was used as a GC. Untreated: untreated EPCs, 100 nM: GC-treated EPCs, 100 nM + PGE2: GC-treated EPCs in the presence of PGE2, 100 nM + PGE2 + LY: GC-treated EPCs in the presence of PGE2 and PI3K inhibitor. LY: LY294002. The data represent the mean ± SD. n = 3, **P < 0.01, *P < 0.05. The experiment was repeated triplicate. The charts showed the overall mean data from all experiments.

Fig 5: GCs impaired the wound healing ability of EPCs. (A) Wound healing ability of untreated and GC-treated EPCs in flap mouse model. (B) The necrotic area of the flap tissues injected with untreated and GC-treated EPCs. (C) Immunohistochemistry of CD45-, MAC-1- (at day 3 post-transplantation), and CD31-positive cells (at day 7 post-transplantation) in flap tissues injected with untreated and GC-treated EPCs observing under a microscope at 200× magnification. (D) Quantification of CD45-, MAC-1- (at day 3 post-transplantation), and CD31-positive cell numbers (at day 7 post-transplantation) in flap tissues injected with untreated and GC-treated EPCs observing under a microscope at 200× magnification. (E) PKH-positive EPCs in flap tissues injected with untreated and GC-treated EPCs observing under a microscope at 200× magnification. (F) Quantification of PKH-positive EPCs in flap tissues injected with untreated and GC-treated EPCs observing under a microscope at 200× magnification. From A–F, the data were calculated from five individual mice from one representative experiment. The data represent the mean ± SD. n = 5, ***P < 0.001, **P < 0.01, *P < 0.05. (G) Wound healing ability of untreated and CXCR4 antibody-treated EPCs in flap mouse model. AMD3100 (100 nM, 24-h treatment) was used as a CXCR4 antibody. The data were calculated from three individual mice from one representative experiment. The data represent the mean ± SD. n = 3, **P < 0.01. Dexamethasone (100 nM, 24-h treatment) was used as a GC. Untreated: untreated EPCs, 100 nM: GC-treated EPCs. The scale bar indicates 100 µm. The experiment was repeated triplicate.