Cellular-self Extracellular Matrix fingerprint of Dystrophic Epidermolysis Bullosa patients

Extracellular matrix (ECM), although long viewed to have a primordial structural function, has a vital role in overall tissue homeostasis and disease. Dystrophic epidermolysis bullosa (DEB) is a genetic skin disease caused by gene mutations on COL7A1 that encodes collagen VII protein, the major component of the anchoring fibrils in the dermal-epidermal junction. The impaired function of collagen VII leads to a decreased resistance of the skin against friction and, consequently, results in skin blistering and in the development of chronic wounds with extensive associated fibrosis and scaring. To date, very little information exists on the consequences of the COL7A1 mutations in the cell microenvironment and particularly on the ECM. Hence, this work aims to unravel the main alterations in the ECM produced by fibroblasts obtained from DEB patients in comparison to that from healthy individuals. For that purpose, we used cell sheet engineering to mimic the biological nature of the dermal compartment in normal and pathologically altered skin. Healthy primary fibroblasts and immortalized cell lines of three different variants of DEB (representing different aggressiveness degrees of the disease), provided by EB house Austria, were cultured for 14 days with ascorbic acid in order to promote maximum ECM deposition. Gene expression (q-PCR) analysis showed that mutations in COL7A1 lead to a downregulation of both collagen VII and I – important ECM players in skin homeostasis. Additionally, analysis of protein content (western blot and immunocytochemistry) showed a reduction in basement membrane proteins that are present in the derma-epidermal junctional zone, such as collagen IV and laminin, together with an increase in dermal matrix proteins greatly associated with wound healing and scarring, namely tenascin C and vimentin. Our preliminary results pointed out that the impaired function of collagen VII impacts ECM composition of important proteins that are closely altered in the diseased scenario.

Acknowledgements: The authors would like to acknowledge FCT for grant SFRH/BD/137766/2018 (MDM), the ERC Consolidator Grant – ECM_INK (ERC-2016-COG-726061), NORTE-01-0145-FEDER-000021 (MTC), the European Union for The Discoveries Centre for Regenerative and Precision Medicine (H2020-WIDESPREAD-2014-1-739572)