Differing responses of human follicular and nonfollicular scalp cells in an in vitro wound healing assay: Effects of estrogen on vascular endothelial growth factor secretion

Abstract

Improved wound healing of hairy skin may involve mesenchymal hair follicle cells with stem cell potential and enhancement by estrogen therapy. How estrogen affects follicular dermal papilla (DP) and dermal sheath (DS) cells in wound healing is unknown. Therefore, a comparison of estradiol action on DP, DS, and corresponding interfollicular dermal fibroblasts (DF) in a scratchwound assay was performed using matching primary cultures established from female temporo-occipital scalp. All three cell types expressed mRNA transcripts and protein for estrogen receptors a (ERa) and b (ERb). DF ERa transcripts were half that of DP and one-third of DS cells, while DF ERb transcripts were two-thirds of DP and DS cells. In the scratch-wound assay all three cells types migrated at similar rates, but only the rate of DF was enhanced by estradiol. Mechanical wounding increased DNA synthesis rates of all three cell types and increased the secretion of collagen by DF and DS cells. All three secreted similar basal levels of vascular endothelial growth factor (VEGF), which was increased by wounding DF and DS cells, but not DP cells. DP cells required estradiol to increase VEGF secretion; by contrast VEGF secretion was decreased by estradiol in wounded DS cells. These results highlight differences in the responses of DF, DP, and DS cells to estradiol in a scratch-wound assay, providing further support for the dichotomy of cellular functions in the hair follicle. Further understanding of the role of estrogen in cutaneous wound healing may have important implications for the management of chronic wounds and scarring.Skin appendages play a key role in epithelial regeneration following cutaneous wounding. Specifically, the regenerative properties of the adult hair follicle suggest that hair follicle stem cells may play an important role in the wound healing process. While epidermal stem cells of the hair follicle allow for re-epithelialization, 1 the hair follicle mesenchyme, namely the dermal papilla (DP) and dermal sheath (DS), may play a part in dermal repair following cutaneous wounding. 2The importance of the hair follicle in cutaneous wound healing is provided by animal models. For example, full-thickness wounds heal more quickly in hairy animals than in humans, with greater contraction and less scar formation. Furthermore, a relationship between wound healing and the hair cycle has been demonstrated, with skin containing anagen (growing) follicles healing more rapidly. In humans, it has also been suggested that cutaneous wounds heal better in hair-bearing skin. When used for split-thickness skin grafting, rapid donor site healing has been reported in scalp skin with low infection rates and minimal scar formation.3 Furthermore, in a prospective study it was shown that following split-thickness skin graft harvesting, donor sites on hair-bearing scalp skin heal significantly better than those on nonhairy thigh skin. 4 Evidence for the stem cell potential of both DP and DS cells comes from a study demonstrating that cells der...

Guided Regeneration of the Human Skin : in vitro and in vivo studies

“…One of the simplest models is the monolayer scratch wound. By altering the environment, the cells, or the media, researchers can study different effects of the stimuli [80,81].…”

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts

“…Previous dose-response assays using this technique have shown that a range of concentrations of 17b-estradiol (10 27 -10 29 M) and DHEA (10 25 -10 28 M) all stimulated cell migration to a similar level (37,39). The ability of the cells to metabolize these steroids was determined by including 100 nM Arimidex (to block aromatase activity) in the presence and absence of DHEA or testosterone, and 100 nM STX64 (to block STS activity) in the presence and absence of DHEA-S. Migration was quantitated as previously described (28,30). The addition of 10 mg/ml of mitomycin C to block proliferation did not alter the migration of keratinocytes (n = 3) or fibroblasts (n = 5; data not shown).…”

“…Briefly, cells were seeded into 6-well plates at a density of 1 3 10 5 per well and grown to confluence, then mechanically wounded as previously described (28,30,37). After washing with PBS, they were incubated with serum-free medium containing 0.5 mCi [1b 3 H]-androstenedione (NEN Perkin Elmer, Waltham, MA, USA) for 2 or 24 h at 37°C.…”

“…The effect of steroid hormones on fibroblast and keratinocyte migration was assessed using the scratch wound assay as previously described (28,30,37). Mechanically wounded cells were assayed in phenol red-free, serum-free medium containing 10 mg/ml mitomycin C (to block proliferation) and the following steroids: DHEA-S (10 mM), DHEA (10 mM), testosterone (50 nM), 17b-estradiol (1 nM), or vehicle control (0.0001% ethanol).…”

“…For RNA extraction, each sample was transported in RNA later or frozen in liquid nitrogen; for cell culture, biopsies were kept in DMEM (GIBCO, Invitrogen, Milan, Italy, stored at 4°C and processed within 24 h of surgery. Primary cultures of dermal fibroblasts and epidermal keratinocytes were established as described previously (28)(29)(30). Briefly, small pieces of skin were washed in PBS containing amphotericin B (750 ml/ml); excess fat and dermis were removed before incubating overnight in dispase (2.4 U/ml) at 4°C, then at 37°C for 1 h. The epidermis was separated from the dermis with forceps, washed, and incubated with 0.05% trypsin/EDTA at 37°for 5-6 min.…”