title: Site-specific keloid fibroblasts alter the behaviour of normal skin and normal scar fibroblasts through paracrine signalling.
abstract: Keloid disease (KD) is an abnormal cutaneous fibroproliferative disorder of unknown aetiopathogenesis. Keloid fibroblasts (KF) are implicated as mediators of elevated extracellular matrix deposition. Aberrant secretory behaviour by KF relative to normal skin fibroblasts (NF) may influence the disease state. To date, no previous reports exist on the ability of site-specific KF to induce fibrotic-like phenotypic changes in NF or normal scar fibroblasts (NS) by paracrine mechanisms. Therefore, the aim of this study was to investigate the influence of conditioned media from site-specific KF on the cellular and molecular behaviour of both NF and NS enabled by paracrine mechanisms. Conditioned media was collected from cultured primary fibroblasts during a proliferative log phase of growth including: NF, NS, peri-lesional keloid fibroblasts (PKF) and intra-lesional keloid fibroblasts (IKF). Conditioned media was used to grow NF, NS, PKF and IKF cells over 240 hrs. Cellular behavior was monitored through real time cell analysis (RTCA), proliferation rates and migration in a scratch wound assay. Fibrosis-associated marker expression was determined at both protein and gene level. PKF conditioned media treatment of both NF and NS elicited enhanced cell proliferation, spreading and viability as measured in real time over 240 hrs versus control conditioned media. Following PKF and IKF media treatments up to 240 hrs, both NF and NS showed significantly elevated proliferation rates (p<0.03) and migration in a scratch wound assay (p<0.04). Concomitant up-regulation of collagen I, fibronectin, α-SMA, PAI-1, TGF-β and CTGF (p<0.03) protein expression were also observed. Corresponding qRT-PCR analysis supported these findings (P<0.03). In all cases, conditioned media from growing marginal PKF elicited the strongest effects. In conclusion, primary NF and NS cells treated with PKF or IKF conditioned media exhibit enhanced expression of fibrosis-associated molecular markers and increased cellular activity as a result of keloid fibroblast-derived paracrine factors.
authors.affiliation: Plastic & Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, United Kingdom ; Institute of Inflammation & Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Ashcroft
authors.affiliation: Plastic & Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, United Kingdom ; Institute of Inflammation & Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Farhatullah
authors.initials: F
authors.lastname: Syed
authors.affiliation: Plastic & Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, United Kingdom ; Institute of Inflammation & Repair, University of Manchester, Manchester, United Kingdom ; Department of Plastic and Reconstructive Surgery, University Hospital South Manchester Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom ; The University of Manchester, Manchester Academic Health Science Centre, University Hospital South Manchester Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom.
authors.forename: Ardeshir
authors.initials: A
authors.lastname: Bayat
mesh_terms: D000328:Adult; D000368:Aged; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002921:Cicatrix / Q000378:metabolism* / Q000473:pathology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007627:Keloid; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology* / Q000378:metabolism / Q000473:pathology*
chemical_list: D017077:Culture Media, Conditioned