Connective tissue growth factor promoter activity in normal and wounded skin

Abstract

In skin, connective tissue growth factor (CTGF/CCN2) is induced during tissue repair. However, what the exact cell types are that express CTGF in normal and wounded skin remain controversial. In this report, we use transgenic knock-in mice in which the Pacific jellyfish Aequorea victoria enhanced green fluorescent protein (E-GFP) gene has been inserted between the endogenous CTGF promoter and gene. Unwounded (day 0) and wounded (days 3 and 7) skin was examined for GFP to detect cells in which the CTGF promoter was active, alpha-smooth muscle actin (alpha-SMA) to detect myofibroblasts, and NG2 expression to detect pericytes. In unwounded mice, CTGF expression was absent in epidermis and was present in a few cells in the dermis. Upon wounding, CTGF expression was induced in the dermis. Double immunolabeling revealed that CTGF-expressing cells also expressed alpha-SMA, indicating the CTGF was expressed in myofibroblasts. A subset (approximately 30%) of myofibroblasts were also NG2 positive, indicating that pericytes significantly contributed to the number of myofibroblasts in the wound. Pericytes also expressed CTGF. Collectively, these results indicate that CTGF expression in skin correlates with myofibroblast induction, and that CTGF-expressing pericytes are significant contributors to myofibroblast activity during cutaneous tissue repair.

Figure 1

Connective tissue growth factor (CTGF) promoter activity in skin pre- and post-wounding. Skin of CTGF/enhanced green fluorescent protein (E-GFP) hemizygous mice (pre-wounding, day 0, or post-wounding, day 3 or day 7) was fixed, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei or anti-GFP antibody to detect cells in which the CTGF promoter was active (lefthand panels, 10 × magnification of dermal tissue; righthand panels, 40 × magnification of dermal tissue). Semiquantitative analysis of GFP expression was performed as described in Methods. Note that CTGF promoter activity, as determined by GFP-positive cells, was absent in the epidermis (day 0); however, a small number of dermal cells were positive for GFP expression. Post-wounding, the CTGF promoter was active in granulation tissue. *, significant induction compared to day 0 unwounded skin. Representative data from four wounds from four separate animals per timepoint is shown.

Figure 2

The presence of α-smooth muscle actin (SMA) expression myofibroblast pre- and post-wounding. Skin of connective tissue growth factor/enhanced green fluorescent protein (CTGF/E-GFP) hemizygous mice (pre-wounding, day 0, or post-wounding, day 3 or day 7) was fixed, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei or anti-α-SMA antibody to detect myofibroblasts (lefthand panels, 10 × magnification of dermal tissue; righthand panels, 40 × magnification of dermal tissue). Quantitiation of myofibroblasts was performed as described in Methods. Note that α-SMA expression was absent in the epidermis (day 0); however, a small number of dermal cells were positive for α-SMA expression. Post-wounding, myofibroblasts appeared within granulation tissue. *, significant induction compared to day 0 unwounded skin. Representative data from four wounds from four separate animals per timepoint is shown.

Figure 3

The connective tissue growth factor (CTGF) promoter is active in myofibroblasts. Skin of CTGF/enhanced green fluorescent protein (E-GFP) hemizygous mice (post-wounding, day 3) was fixed, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei, anti-α-smooth muscle actin (SMA) antibody to detect myofibroblasts, and anti-GFP antibody to detect CTGF promoter activity (40 × magnification of dermal tissue). The percentage of fibroblasts within the wound that were α-SMA positive, GFP positive, and both GFP and α-SMA positive was calculated as described in Methods. Note that ~75% of the cells in the wound area were myofibroblasts that expressed GFP. Representative data from four wounds from four separate animals per timepoint is shown.

Figure 4

A subset of the myofibroblasts within wound tissue are pericytes. Skin of connective tissue growth factor/enhanced green fluorescent protein (CTGF/E-GFP) hemizygous mice post-wounding (a) day 3 and (b) day 7 was fixed, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei, anti-α-smooth muscle actin (SMA) antibody to detect myofibroblasts, and anti-NG2 antibody to detect pericytes (10 × magnification of dermal tissue). The center of the wound and wound edges were examined and the percentage of fibroblasts within the wound that were α-SMA positive, NG2 positive, and both NG2 and α-SMA positive was calculated as described in Methods. At day 3, note that ~20% of the cells in the wound area were both α-SMA and NG2 positive whereas ~40% of the cells at the wound edges were α-SMA and NG2 positive, indicating that pericytes are recruited to the wound from surrounding tissue. At day 7, note that essentially all cells in the wound area are myofibroblasts. Approximately 30% of the cells in the center of the wound are myofibroblasts of pericyte origin. In the wound edge, ~40% of the cells are myofibroblasts of pericyte origin. Representative data from four wounds from four separate animals per timepoint is shown.

Figure 5

The connective tissue growth factor (CTGF) promoter is active in pericytes. Skin of CTGF/enhanced green fluorescent protein (E-GFP) hemizygous mice post-wounding (a) day 3 and (b) day 7 was fixed, sectioned, and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei, anti-GFP antibody to detect cells in which the CTGF promoter is active, and anti-NG2 antibody to detect pericytes (10 × magnification of dermal tissue). The center of the wound and wound edges were examined and the percentage of fibroblasts within the wound that were GFP positive, NG2 positive, and both NG2 and GFP positive was calculated as described in Methods. At day 3 and 7, note that essentially all pericytes were GFP positive, and hence showed CTGF promoter activity. Representative data from four wounds from four separate animals per timepoint is shown.