Fig 1.
Establishment of human wound healing assay and testing of effects of T4 on keratinocyte migration during epidermal repair of experimentally wound human skin.
(a) A schematic diagram of human wound healing culture model assay. A schematic diagram (b) and example of a wound punch sample (c) and longitudinal section (d). Sections from day 0 and day 6 of culture (vehicle control) are shown (e-g). The regenerative phenomenon is highlighted in (f). Magnification of marked area in (f) is shown in (g).
Table 1.
Characteristics of patients included in this study.
Table 2.
Antibodies used for immunohistology.
Fig 2.
T4 promotes human skin re-epithelialisation.
(a-d) Haematoxylin and eosin histochemistry: overview of wounded human skin fragment. After 3 days, epithelial tongue areas (blacked dotted line area) (ET) and length (blue dotted line) were significantly greater after treatment with T4 compared to control alone. (e-h) Cryosections of control-, or T4-treated human skin were examined by Ki-67 (red arrow)/TUNEL (green arrow) double-labelling [19, 23, 24, 56, 57] (e, f). The percentage of positive cells was analyzed in the new ETs (see dotted line). When compared to the control group, more Ki-67 positive cells after 3 day culture with 1000 nM T4 treatment (and more TUNEL positive cells at day 3 in T4 1000 nM treated group were found, although the differences were not statistically significant (e-h). (i, j) CK6 expression was significantly upregulated by T4, especially in the 1000 nM treatment group. Green fluorescence staining represented cytokeratin 6 IR in the new wound ET (dotted line area).(k, l) MTCO1 expression was significantly up-regulated by T4. Brown staining represents MTCO1 IR in the new ET. Staining intensity was measured in a defined reference area (dotted line) and normalized to the control (100%), as for CK6 expression. Number of independent experiments: n = 3 subjects (i.e. 1–2 punches per patient, per treatment group and per time point and at least 8 photomicrographs were analysed per condition); data were pooled since the results trends in all three independent experiments were comparable). *p<0.05, **p<0.01, ***<0.001. Scale bars = 50μm.
Fig 3.
T4 stimulates angiogenesis in wounded human skin.
(a-c) To analyze angiogenesis, the number of CD31+ cells (red) and of CD31+ blood vessel cross-sections (lumina) (yellow arrow, c) per visual field were counted by immunofluorescence microscopy (at least 12 visual fields per skin fragment were evaluated). In addition, the intensity of CD31 IR was measured. Scale bars in a, b = 50μm, c = 200μm. (d) CD31 IR was significantly up-regulated by T4 at days 3 and 6. Immunoreactivity data was normalized to the control data as were (e, f) the number of CD31 +ve endothelial cell nuclei (CD31+/DAPI+ cells) and lumina per microscopic field. Number of independent experiments: n = 3 subjects (i.e. 1–2 punches per patient, per treatment group and per time point and at least 8 photomicrographs were analyzed per condition); data were pooled since the results trends in all three independent experiments were comparable). MVD: Microvessel density; ibFGF ab: inhibitory bFGF antibody.
Fig 4.
T4 up-regulates bFGF and FGF receptor 1 IR.
(a-c) bFGF expression was significantly up-regulated by T4 in the epidermis of new epithelial tongues (staining intensity was measured in reference area) at day 3 and day 6 in 100nM T4 condition. Red fluorescence staining represents bFGF IR in the new epithelial tongue and the control IR value was normalized as 100%. (d-f) T4 increases FGFR1 expression. Red fluorescence staining represents FGFR1 IR in the new epithelial tongue. Staining intensity was measured in the dotted line reference area and the control IR value was normalized as 100%. One-Way ANOVA by appropriate post hoc comparisons was used. Data represent the mean±SEM of 3 independent experiments and normalized to the control as 100%. 1–2 punches per patient, per treatment group and per time point and at least 8 photomicrographs were analyzed per condition. *P<0.05; **P<0.01; ***P<0.001. Scale bars = 50μm.
Fig 5.
(a-f) T4 mediates its effect at least partially via bFGF.
T4 exerts its effect partly through inducing bFGF at day 3. Area and length of the ET (a: length of new ET; b: area of new ET), intensity of the IR of CD31 (c), number of CD31+ cells (d), Number of CD31+ lumina (e), and intensity of IR of bFGF in new ET (f) were significantly increased in the T4-treated test group compared to controls. 1–2 punches per patient, per treatment group and per time point and at least 8 photomicrographs were analyzed per condition. These effects could be abrogated by co-treatment with inhibitory bFGF antibody [45].