Topical application of an irreversible small molecule inhibitor of lysyl oxidases ameliorates skin scarring and fibrosis.

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.

A Methylome and Transcriptome Analysis of Normal Human Scar Cells Reveals a Role for FOXF2 in Scar Maintenance.

Scars are maintained for life and increase in size during periods of growth such as puberty. Epigenetic changes in fibroblasts after injury may underpin the maintenance and growth of scars. In this study, we combined methylome and transcriptome data from normotrophic mature scar and contralateral uninjured normal skin fibroblasts to identify potential regulators of scar maintenance. In total, 219 significantly differentially expressed and 1,199 significantly differentially methylated promoters were identified, of which there were 12 genes both significantly differentially methylated and expressed. Of these, the two transcription factors, FOXF2 and MKX, were selected for further analysis. Immunocytochemistry and qPCR suggested that FOXF2 but not MKX had elevated expression in scar fibroblasts. Using RNA sequencing, FOXF2 knockdown was shown to significantly reduce the expression of extracellular matrix‒related genes, whereas MKX did not appear to affect similar pathways. Finally, FOXF2 knockdown was also shown to significantly decrease collagen I production in scar and keloid fibroblasts. This study provides insights into the maintenance of normotrophic scar, suggesting that FOXF2 is an important regulator of this process. Targeting genes responsible for maintenance of scar phenotype may ameliorate scar appearance and improve patient outcomes in the future.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-ß.

BACKGROUND: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. OBJECTIVES: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. METHODS: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-ß (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. RESULTS: KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-ß. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-ß. TGF-ß1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. CONCLUSIONS: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

Secreted Factors from Keloid Keratinocytes Modulate Collagen Deposition by Fibroblasts from Normal and Fibrotic Tissue: A Pilot Study.

Interactions between keratinocytes and fibroblasts in the skin layers are crucial in normal tissue development, wound healing, and scarring. This study has investigated the role of keloid keratinocytes in regulating collagen production by primary fibroblasts in vitro. Keloid cells were obtained from removed patients' tissue whereas normal skin cells were discarded tissue obtained from elective surgery procedures. Fibroblasts and keratinocytes were isolated, cultured, and a transwell co-culture system were used to investigate the effect of keratinocytes on collagen production using a 'scar-in-a-jar' model. Keloid fibroblasts produced significantly more collagen than normal skin fibroblasts in monoculture at the RNA, secreted protein, and stable fibrillar protein level. When keloid keratinocytes were added to normal skin fibroblasts, expression of collagen was significantly upregulated in most samples, but when added to keloid fibroblasts, collagen I production was significantly reduced. Interestingly, keloid keratinocytes appear to decrease collagen production by keloid fibroblasts. This suggests that signaling in both keratinocytes and fibroblasts is disrupted in keloid pathology.

A versatile transgenic allele for mouse overexpression studies.

For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.