Autor:innen:
V. Schmidt (Bad Oeynhausen, DE)
J. Ohmes (Bad Oeynhausen, DE)
T. Ly (Bad Oeynhausen, DE)
B. Fischer (Bad Oeynhausen, DE)
A. Kleine (Bad Oeynhausen, DE)
C. Knabbe (Bad Oeynhausen, DE)
I. Faust-Hinse (Bad Oeynhausen, DE)
Introduction: Fibrosis represents a dysregulated wound healing process in which extracellular matrix (ECM)-producing myofibroblasts, after completion of the repair process, do not induce apoptosis or cellular senescence but persist in the tissue. Cellular senescence of myofibroblasts during the physiologic wound healing process is referred to as acute senescence and serves to protect against fibrosis. Cellular senescence, by definition, is an irreversible form of cell cycle arrest preventing damaged cells from undergoing proliferation and precluding potential neoplastic transformation. The xylosyltransferase isoform XT-I represents a biomarker of myofibroblast differentiation. The isoforms XT-I and XT-II catalyze the initial rate-limiting step of proteoglycan (PG) biosynthesis by transferring xylose from UDP-xylose to specific serine residues of the PG core protein. Known mediators of XT-I regulation in context to fibrotic processes are transforming growth factor β1 (TGF-β1), activin A or interleukin 1β. Although the regulation of XT- I in profibrotic processes is mainly understood, little is known about the XT-I regulation during the transition of myofibroblasts into an acute senescent cell state. The aim of this study was to determine whether XT-I is regulated during this transition and, consequently, has an impact on ECM remodeling at the end of the physiological wound healing process.
Methods: To investigate XT-I regulation in acute senescent myofibroblasts, primary normal human dermal fibroblasts (NHDF) were treated with H2O2 and cultured in a myofibroblast cell culture model. XT-I regulation was examined at gene, protein and enzyme activity levels by quantitative real-time PCR, western blot analysis and a XT-I selective mass spectrometric activity assay. The induction of an acute senescence state was verified by a senescence-associated (SA) β-galactosidase activity assay and the determination of p21 and p16 protein expressions by immunofluorescence microscopy.
Results: Treatment of NHDF with H2O2 significantly reduced XYLT1 and XYLT2 mRNA expression in comparison to control. Based on the results of the mass spectrometry activity assay, the significantly decreased XT protein expression determined by western blot, was assigned to the XT-I isoform. Characterization of the SA secretory phenotype revealed significantly increased gene expressions of interleukin 1β and matrix metalloproteinase 1. In contrast, collagen I, fibronectin and decorin expressions were significantly reduced. However, on TGF-β1 gene expression senescence induction had no physiologically relevant effect.
Conclusion: In summary, our study demonstrated an anti-fibrotic effect in acute senescent myofibroblasts leading to a suppressed XYLT1 and XT-I expression. It can be concluded that XT-I plays an important role in the switch between physiological and pathological wound healing process. Nevertheless, further studies are needed to elucidate the underlying signaling pathways.