Figure 5

Suppression of HGF production impairs EDK-mediated re-epithelialization of wounded HSEs. (a) Efficiency of shRNA knockdown of hepatocyte growth factor (shHGF) in EDK cells relative to a scrambled, shRNA control (shScram) as measured by ELISA. Secretion of hepatocyte growth factor (HGF; top panel) was reduced 95% relative to shScram (t-test: **P < 0.01), secretion of KGF (lower panel) was not affected by shHGF. Data are normalized to shScram and all results are expressed as the mean +/- standard deviation (SD) of three experiments and three technical replicates per experiment. (b) Representative morphology of wounded tissues constructed with EDK-shHGF and EDK-shScram cells 72 hours after wounding (black arrows demarcate the initial wound edges; white arrows demarcate the tip of epithelial tongues). Bars, 200 μm. (c) The degree of re-epithelialization was significantly lower in tissues containing EDK-shHGF cells as compared with EDK-shScram (t-test: **P < 0.01). (d) Relative levels of HGF in supernatants of wounded tissues 72 hours after wounding as measured by ELISA. Tissues containing EDK-shHGF cells produced significantly lower levels of HGF as compared with EDK-shScram (t-test: **P < 0.01). (e) Proliferation of basal keratinocytes in tissues containing EDK-shHGF and EDK-shScram cells was analyzed using immunoperoxidase staining with anti-BrdU antibody 72 hours after wounding (black arrows demarcate the initial wound edges; white arrows demarcate the BrdU-positive cells). (f) Quantification of BrdU-positive basal keratinocytes. Tissues containing EDK-shHGF demonstrated significantly lower percentage of proliferating basal keratinocytes compared with EDK-shScram (t-test: **P < 0.01). Bars, 100 μm. All results are presented as the mean +/- SD of three independent experiments and three technical replicates.