Wounds heal better in skin with terminal hair follicles (large and pigmented) as opposed to those with vellus hair follicles (small and unpigmented), while dermal fibroblasts from different anatomical regions also exhibit phenotypical differences. Tissue repair requires a tight control of cell proliferation, migration and apoptosis, and recent studies have shown the importance of inhibitors of apoptosis proteins (IAPs), which are proteins that prevent the process of apoptosis via their interaction with caspase molecules in wound healing. Oestrogens improve the rate and quality of wound healing, but their relationship with IAPs in human skin has not been studied. Therefore, terminal (scalp) and vellus (facial) hair bearing skin from the same donor was compared in situ and matching primary cultures of dermal fibroblasts were established from terminal (DF(T)) and vellus (DF(V)) hair bearing skin. Using immunofluorescent staining, the expression of IAPs and their antagonists was compared at different stages of the hair cycle following depilation using a murine model and then in terminal and vellus hair bearing human skin. The size and granularity of matching DF(T) and DF(V) cultures was compared by FACS analysis and mRNA and protein expression of Apollon, cIAP2, NAIP and XIAP and their antagonists DIABLO and Xaf1 analysed by qRT-PCR and immunocytochemistry in unwounded and mechanically wounded fibroblast cultures. Differences in proliferation, migration, viability and caspase 3 activity in the presence of 17β-oestradiol and changes in mRNA expression of the oestrogen receptors (GPR30, ERα and ERβ) were compared between the two cell types. IAP protein expression was generally found higher during mid anagen of the hair cycle in murine skin and hair follicles. Overall, expression was slightly higher in human terminal hair bearing skin compared to corresponding vellus hair bearing skin. IAP protein expression was similar in unwounded DF(T) and DF(V) cells with the exception of Apollon which was higher in DF(V) cells. With the exception of XIAP and its direct antagonist Xaf1, mRNA expression was higher in DF(V) cells compared to corresponding DF(T) cells. FACS analysis demonstrated that DF(V) cells were more granular than matching DF(T) cells and proliferated faster. 17β-oestradiol accelerated migration of DF(T) cells only. Mechanical wounding decreased XIAP mRNA in DF(T) and increased it in DF(V) cells, while simultaneously decreasing Xaf1 expression. In unwounded cells, 17β-oestradiol stimulated the expression of XIAP mRNA in both DF(T) and DF(V) cells, but in scratched monolayers, while it also increased expression in DF(T) cells it decreased it in DF(V) cells. A XIAP inhibitor reduced cell viability in both DF(T) and DF(V) cells, which was rescued by 17β-oestradiol in unwounded and mechanically wounded DF(T) cells, but only in unwounded DF(V) cells. 17β-oestradiol decreased caspase 3 activity in the presence of a XIAP inhibitor only in DF(T) cells. These results demonstrate significant differences between dermal fibroblasts cultured from terminal and vellus hair bearing skin of the same individual. The correlation between an increase in XIAP in response to 17β-oestradiol and a higher number of viable cells, along with a reduction in caspase 3 activity suggests that the protective effect of 17β-oestradiol may be modulated via the regulation of XIAP. Further elucidation of these different signalling pathways in dermal fibroblasts from hair bearing skin may lead to improved therapies for chronic non-healing wounds, particularly in postmenopausal females.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:732076 |
| Date | January 2014 |
| Creators | Kamala, Ola |
| Publisher | University of Bradford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10454/13841 |
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