To investigate the expression of pigment epithelium-derived factor (PEDF) in wound healing and its effect and mechanism on human dermal microvascular endothelial cells (hDMEC).

The expression of PEDF during wound healing was determined by bioinformatics and gene chip analysis in NCBI GEO database; 3 SD rats were selected to make wound models, and skin tissue samples were collected for real-time fluorescence quantitative PCR and western blotting to verify the expression changes of PEDF in the process of wound healing. The cell scratch test was conducted to explore the effect of PEDF on the migration ability of hDMEC, the cell counting kit (CCK)-8 test was conduccted to explore the effect of PEDF on the proliferation of hDMEC, and the tube formation experiment was conducted to explore the effect of PEDF on the angiogenesis of hDMEC. STRING was used to predicte the PEDF-protein interaction relationship, and finally passed western blotting was used to detect the relationship between PEDF and hDMEC Wnt/β-catenin pathway. Data were compared with one-way ANOVA and LSD-t test.

(1) Bioinformatics analysis of gene chip GSE28914 by R showed that PEDF expression in human normal skin tissue was -0.04±0.03, and PEDF increased transiently during the coagulation phase (immediately after trauma) (0.84±0.12), the difference was statistically significant (Fold change= 0.89, P=0.002), decreased rapidly on the third day after trauma(-0.60±0.09), the difference was statistically significant (Fold change= -0.55, P=0.010), and finally returned to normal skin level(0.03±0.06), the difference was statistically significant (Fold change=-0.07, P=0.602). (2) The expression of PEDF in skin tissue of SD rats was higher than that in wound tissue on the third day after trauma. The relative expression level of PEDF RNA in skin of SD rats was 1.15±0.13, and that in wound tissue of SD rats on the third day after trauma was 0.45±0.04, the difference was statistically significant (t= 9.21, P< 0.001). The relative expression level of PEDF protein in skin tissue of SD rats was 1.32±0.15, and the expression level of PDEF protein in wound tissue of SD rats was 0.83±0.08 on the third day after trauma, the difference was statistically significant (t= 5.11, P< 0.001). (3) PEDF inhibited the migration of hDMEC. The migration rates of endothelial cells in siPEDF group, rPEDF group and control group were (92.01±3.04)%, (19.51±3.93)%, (57.03±1.065)%, respectively, and the difference was statistically significant (F=459.30, P< 0.001). The migration rate of siPEDF group was significantly higher than that of control group, the difference was statistically significant (t=25.29, P< 0.001), and the rPEDF group was significantly lower than that of control group, the difference was statistically significant (t=15.98, P< 0.001). The migration rate of endothelial cells in siPEDF group was two times that in control group, and that in rPEDF group was 1/3 of that in control group. (4) PEDF inhibited the proliferation of hDMEC. The proliferation rates of endothelial cells in siPEDF group, rPEDF group and control group were (442.60±58.90)%, (248.90±52.19)%, (333.80±47.70)%, respectively. There was statistically significant difference among the three groups (F= 16.69, P< 0.001). The proliferation rate of the siPEDF group was significantly higher than that of the control group, and the difference was statistically significant (t= 3.21, P= 0.013). The proliferation rate of the rPEDF group was significantly lower than that of the control group, and the difference was statistically significant (t= 2.69, P= 0.028). (5) PEDF inhibited hDMEC tube forming. The number of endothelial tube forming nodes in siPEDF group, rPEDF group and control group were (38.00±4.58), (11.33±3.51), (23.67±6.66) respectively, and the difference was statistically significant (F= 20.64, P= 0.002). The number of endothelial tube forming nodes in siPEDF group was significantly higher than that of control group, the difference was statistically significant (t= 3.07, P= 0.037). The number of nodes in rPEDF group was significantly less than that in control group, the difference was statistically significant (t= 2.84, P= 0.047); the tube length of siPEDF group, rPEDF group and control group were (1 518.00±250.90), (365.8±91.52) and (925.50±193.00) ppi, respectively, and the difference was statistically significant (F= 27.53, P< 0.001). The tube length of siPEDF group was significantly longer than that of control group, and the difference was statistically significant (t= 3.24, P= 0.032). The tube length of rPEDF group was significantly shorter than that of control group, the difference was statistically significant (t= 4.54, P= 0.011). (6) PEDF interacts with key proteins in the classical Wnt/β-catenin signaling pathway. STRING were used to predict the protein-protein interaction relationship and founded that there were co-acting proteins between PEDF and Wnt/β-catenin signaling pathway. (7) PEDF inhibited Wnt1 and β-catenin expression level. The relative expression levels of Wnt1 protein in hDMEC of siPEDF group, rPEDF group and control group were 0.84±0.04, 0.12±0.05 and 0.66±0.08, respectively. The differences among the three groups was statistically significant (F=114.80, P< 0.001). The protein expression level of Wnt1 in siPEDF group was significantly higher than that of control group, the difference was statistically significant (t=4.95, P= 0.036). The protein expression level of Wnt1 in rPEDF group was significantly lower than that of control group, the difference was statistically significant (t=13.94, P=0.005). The relative expression levels of β-catenin protein in siPEDF group, rPEDF group and control group were 0.77±0.05, 0.50±0.07 and 0.63±0.06, respectively, the differences between the three groups was statistically significant(F= 15.04, P= 0.005). The protein expression level of β-catenin in siPEDF group was significantly higher than that of the control group, and the difference was statistically significant (t=6.51, P=0.023). The protein expression level of β-catenin rPEDF group was significantly lower than that in control group, the difference was statistically significant (t=4.56, P=0.045).

The expression of PEDF changes dynamically within the wound healing process; PEDF might inhibit the ability of migration, proliferation, and tube formation in hDMEC by regulating Wnt/β-catenin pathway, and ultimately affect wound healing.