Speaker
Description
Introduction: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in the COL7A1 gene, in which patients exhibit mechanically fragile skin (1). The underlying mutation determines the clinical phenotype, which can range from a severe condition characterized by widespread blistering associated with the development of chronic wounds-RDEB (recessive subtype) - to a relatively mild disorder associated with localized blistering-DDEB (dominant subtype). To date, the impact of the different COL7A1 mutations on ECM biological and mechanical properties is largely unexplored. Hence, this work aimed to investigate the differences in ECM that occur amongst DEB subtypes and, for the first time, to establish a correlation between these alterations and ECM mechanical properties.
Methodology: Immortalized cells from patients representing three DEB subtypes (different COL7A1 mutations and disease aggressiveness) were obtained from EB House Austria. As a control, healthy primary fibroblasts were used. The cells were cultured for 14 days with 50µg/mL ascorbic acid to promote maximum ECM deposition. Mass spectrometry-based label-free quantification was used to assess changes in the deposited ECM. Western blot and immunocytochemistry were used to further dissect the abnormal ECM features in the different DEB subtypes. Furthermore, the influence of distinct COL7A1 mutations in ECM mechanical properties was addressed at nanoscale by Atomic Force Microscopy (AFM) and at macroscale by uniaxial tensile testing.
Results: Extracellular proteome analysis revealed that fibroblasts from each DEB subtype have their own unique proteomic fingerprint. Independently of the disease subtype - and its associated clinical aggressiveness - down-regulation of structural proteins that impact ECM tensile strength and compression to resistance were identified. Additionally, all DEB subtypes demonstrated a decrease in the dermal-epidermal junctional proteins collagen IV and laminin, as well as an increase in ECM proteins closely associated with wound healing and scarring (tenascin C and vimentin). Furthermore, those differences strongly impact the ECM mechanical properties. At the nanoscale, data indicated a significant reduction in the mechanical stability of the native ECM produced by the different DEB subtypes, which was simultaneously associated with a significant decrease in macroscale stiffness. Interestingly, the more severe subtype (RDEB) results in a significant loss in ECM tensile strength when compared to a healthy control, whereas the milder form (DDEB) shows no difference.
Conclusion: This study demonstrates that different COL7A1 mutations in DEB have a significant impact on the overall dermal ECM features, with structural proteins involved in the mechanical stability being down-expressed and proteins involved in pathological ECM remodeling of wound healing and scarring – hallmarks of DEB - being over-expressed. Furthermore, these changes in the biological features of the ECM have a major effect on the native ECM’s mechanical integrity at both the macro and nano scales. Overall, this work contributes to the advancement of DEB knowledge, being the first to correlate alterations in ECM composition with its mechanical properties in disease scenario.
Acknowledgements: Financially supported by ERC Consolidator Grant ERC-2016-COG-726061,FCT project MImBI-PTDC/EME-APL/29875/2017 and LAETA project UIDB/50022/2020. FCT for grant SFRH/BD/137766/2018(MDM),grant SFRH/BD/147807/2019(AA) and contract Grant No.Norte-01-0145-FEDER-02219015(MTC).
References:
1.G.Tartaglia et al. Int. J. Mol. Sci. 22 (2021).
73296308004
