The effect of skin tension on the formation of keloid scars

Keloid scars (KS) are a type of abnormal scarring which is unique to humans. They extend beyond the confines of the original wound margins, do not regress over time and invade the surrounding unaffected skin. The mechanisms involved in the formation of KS remain largely unknown. Clinical observation has shown that in areas where increased tension occurs, such as the sternum, there is a greater propensity for developing KS. However, the precise relationship between skin tension and KS development is yet to be identified. In view of this, I hypothesize that skin tension plays a significant role in KS development by affecting tension-related biomarkers that may alter the phenotype of KS. Therefore, the objective of this research was to investigate the effect of skin tension in the formation of KS. To this end, the first aim was to identify possible targets among biomarkers that might contribute to the differentiation between KS and hypertrophic scars in tissue and cells obtained from diverse anatomical locations. The second aim was to investigate the effect of tension-related biomarkers on extracellular matrix (ECM) steady-state synthesis in keloid fibroblasts (KF) extracted from a highly tensioned body region (the sternum). The third aim was to develop a 3D in-vitro model to mimic in-vivo tension and to evaluate KF behaviour and ECM synthesis under tension. To achieve these aims 21 biomarkers were selected from published microarray and in-house microarray studies, the inclusion criteria was based on up-regulation of the genes in KS in relation to fibrosis, apoptosis and tension. For this purpose, samples from normal skin and KS were used to perform qRT-PCR screening in tissue and cells, as well as protein analysis by Western and In-cell Western blot. The siRNA knockdown technique was employed to evaluate the functional role of the tension-related markers in keloid fibroblasts. Finally, a photogrammetry technique was employed to evaluate skin tension in-vivo; the results from this evaluation were used in the development and design of a novel in-vitro 3D-model. The first biomarker screening in tissue showed convincing up-regulation of five tension-related targets (Hsp27, PAI-2 and ?2?1-integrin, MMP-19 and CPRP). In addition, the expression of the above-mentioned targets was significantly higher in samples from the sternum compared to samples from other anatomical locations. To further validate these findings, the screening of the 21 biomarkers was assessed in KS and KF taken from the sternum. The results demonstrated over expression of 3 of the 5 tension-related targets (Hsp27, PAI-2 and ?2?1-Integrin). It was also demonstrated that Hsp27, PAI-2 and ?2?1-Integrin performed a functional role in terms of regulation of extracellular matrix production and deposition in KF when their expression was down-regulated by siRNA knockdown. Using the newly created 3D model, it was shown that mechanical tension significantly induced the expression of Hsp27, PAI-2 and ?2?1-Integrin as well as ECM components such as Collagen I. Furthermore, the results showed that the knockdown of the expression of Hsp27, PAI-2 and ?2?1-integrin in fibroblast populated collagen lattices subjected to tension influenced not only the ECM synthesis but also adhesion and spreading genes in keloid and normal fibroblasts. In summary, this research convincingly shows that skin tension alters keloid fibroblast behaviour, morphology, mechano-responsive gene expression and extracellular matrix production. The findings from my thesis offer insight into keloid pathobiology and provide options for targeted treatment of specific genes affected in keloids by biomechanical stress.