Site-Specific Keloid Fibroblasts Alter the Behaviour of Normal Skin and Normal Scar Fibroblasts through Paracrine Signalling

Kevin J Ashcroft,Ardeshir Bayat,Farhatullah Syed,Effie C Tsilibary

doi:10.1371/journal.pone.0075600

Kevin J Ashcroft, Ardeshir Bayat + Show 2 more

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https://doi.org/10.1371/journal.pone.0075600

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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.

Highlights

Keloid disease (KD) is a complex fibroproliferative disorder of the skin characterised by formation of raised dermal lesions following an abnormal wound healing response [1]
normal skin fibroblasts (NF) cells treated with peri-lesional keloid fibroblasts (PKF) conditioned media (Figure 4A) produced steady increases in Cell Index (CI) from 60–240 hrs, indicating an enhanced NF cell spreading, proliferation and changes in cellular morphology
The highest overall CI was from PKF (Figure 4C) followed by PKF, intralesional keloid fibroblasts (IKF) and NF media treatments in which PKF media elicited consistently higher CI for the duration of 240 hrs

Summary

Introduction

Keloid disease (KD) is a complex fibroproliferative disorder of the skin characterised by formation of raised dermal lesions following an abnormal wound healing response [1]. KD scars are characterised by rich vasculature [3,4], a thickened epidermis and a high mesenchymal cell density [5]. These features are accompanied by thick compact hyalinised collagen fibres [6] forming whirl-like nodular structures in the reticular dermis [7,8]. This irregular collagen distribution forms the basis for the dense ECM meshwork within the KD lesion [5] and is distinct from normal skin, in which collagen bundles appear parallel to the epidermis [9]. The exact mechanisms by which KF potentiate keloid scar formation and invasion, remain to be fully characterised

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