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 α2β1-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 α2β1-Integrin). It was also demonstrated that Hsp27, PAI-2 and α2β1-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 α2β1-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 α2β1-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.

The migration and invasion of cancer cells through 3D confined extracellular matrices is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell mechanics is mainly determined by both the mechanics of the largest organelle in the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal and nuclear mechanics are the major contributors to cell mechanics. Among other factors, steric hindrances of the extracellular matrix confinement are supposed to affect nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that the percentage of invasive cells and their invasion depths into loose and dense 3D extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order to investigate the effect of both nuclear and cytoskeletal mechanics on the overall cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased their invasion depth in dense 3D extracellular matrices, whereas the invasion depths in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF- 7 human breast cancer cells in dense matrices were significantly increased compared to loose matrices, where the invasion depths were decreased. These results are also valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB- 231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that cytoskeletal mechanics seem to be regulated by nuclear mechanics.

Besides biochemical and molecular regulation, the migration and invasion of cells is controlled by the environmental mechanics and cellular mechanics. Hence, the mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the migratory capacity in confined 3D extracellular matrices. Recently, we have shown that the migratory and invasive capacity of mouse embryonic fibroblasts depends on the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration and invasion through the polymerization of actin filaments. Since Rac1 deficient cells become mechanically softer than controls, we investigated the effect of group I PAKs and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore, we determined whether mouse embryonic fibroblasts, in which Rac1 was knockedout, and control cells, displayed cell mechanical alterations after treatment with group I PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1 inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence of Rac1 independent of their adhesive state. However, in the absence of Rac1 the effect was abolished in the adhesive cell state for both inhibitors and in their nonadhesive state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3 inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I (PAK1, 2, and 3) as major players in cell mechanics.

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