Spatial and Temporal Expression of Parathyroid Hormone-Related Protein during Wound Healing  Eric A.G. Blomme, Charles C. Capen, Thomas J. Rosol  Journal of Investigative Dermatology  Volume 112, Issue 5, Pages 788-795 (May 1999) DOI: 10.1046/j.1523-1747.1999.00561.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histology of the three phases of the healing process. (a) Edge of a wound at day 1. Arrowhead points to the flap of keratinocytes migrating toward the center of the wound (on the left side of the photograph). Note the band of degenerate leukocytes and cellular debris underneath the fibrin clot. (b) Wound at day 10. Wounds were completely re-epithelialized and contained dense granulation tissue, but the newly formed epidermis was only weakly attached to the underlying basement membrane, as shown by the epidermal–dermal separation (arrowhead) induced during biopsy sampling. (c) Wound at day 28. There was still evidence of granulation tissue in the dermis (arrowhead) and mild epidermal hyperplasia with orthokeratotic hyperkeratosis. Scale bar: 400 μm (a), 1 mm (b, c). Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 α-Smooth muscle actin immunohistochemistry of granulation tissue fibroblasts. Note the intense immunoreactivity of most fibroblasts. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Griffonia simplicifolia Lectin I histochemistry of granulation tissue endothelial cells. Note the intense brown staining of endothelial cells forming neocapillaries (arrows). Scale bar: 50 μm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Histology of the wounds at day 12 after treatment. (a) Vehicle control. (b) PTHrP(1–36). (c) PTH(7–34). When compared with wounds treated with the vehicle, wounds treated daily by topical application of either PTHrP(1–36) or PTH(7–34) did not differ histologically. Scale bar: 1 mm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 PTHrP expression in normal guinea pig skin. (a) Immunohistochemistry. (b) In situ hybridization. (c) Negative control for in situ hybridization. The epidermis (e), hair follicles (f), sebaceous glands (s), and arrector pili muscles (a) were stained positive with both techniques. Arrowhead points to the nonspecific immunohistochemical staining of the granular layer. Scale bar: 10 μm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 PTHrP expression by keratinocytes involved in re-epithelialization. Immunohistochemistry (a) and in situ hybridization (b) of wound edges at day 4. Flaps of migrating keratinocytes demonstrated intense immunoreactivity for PTHrP and increased levels of PTHrP mRNA in comparison with adjacent normal skin. Myofibroblasts of the early granulation tissue also were mildly positive with both techniques (arrowheads). Scale bar: 200 μm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 PTHrP expression during the granulation tissue phase. Immunohistochemistry (a) and in situ hybridization (b) of wounds at day 13. The newly formed epidermis stained intensely for PTHrP protein and mRNA. The intensity of staining was more intense than in the adjacent normal skin. Myofibroblasts (arrowheads) also were mildly positive with in situ hybridization, and the intensity of staining was comparable with that of endothelial cells (arrow), a cell known to produce PTHrP. Scale bar, 400 μm (a), 100 μm (b). Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 PTHrP expression by infiltrating macrophages. Mononuclear inflammatory cells infiltrating early wounds were weak to moderately stained by immunohistochemistry (a) and in situ hybridization (b). These cells (arrows) were determined to be macrophages based on morphology and expression of lysozyme (c). Scale bar: 100 μm. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 PTHrP secretion by THP-1 cells detected via radioimmunoassay. PTHrP could not be detected in the 24 h conditioned medium of unactivated cells. Following activation with IL-1α or PMA, THP-1 cells secreted increased levels of PTHrP into conditioned medium. Data are expressed as mean ± SEM for triplicate samples. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Ethidium bromide-stained agarose gel of reverse transcriptase-PCR amplified PTHrP mRNA. PTHrP mRNA was detected in THP-1 cells treated with PBS (lane 1), IL-1α (lane 2), and PMA (lane 3), as evidenced by a 321 bp amplification product. The 229 bp GAPDH mRNA amplification product was detected in all three lanes. Lane 4 is the negative control, consisting of RNA-free water; lane L is the 123 bp DNA ladder. Journal of Investigative Dermatology 1999 112, 788-795DOI: (10.1046/j.1523-1747.1999.00561.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions