Mutations in the COL7A1 gene, which encodes collagen VII protein, the major
component of the anchoring fibrils in the dermal-epidermal junction, cause all forms of
dystrophic epidermolysis bullosa (DEB). Different clinical variants have been described
with both dominant and recessive inheritance. However, information regarding the
consequences of different COL7A1 mutations in the cell microenvironment, particularly
on extracellular matrix (ECM), is still scarce. Moreover, several studies found the
spectrum of biologic and clinical phenotypes of DEB to be wider than initially
anticipated. Hence, this work aims to unravel the main differences in ECM composition
between DEB patients and healthy individuals, as well as between representative
variants of the disease. 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 DEB variants
(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. Mass spectrometry-based label-free quantification was used to
assess changes in the ECM deposited by the different cell populations. Then a
combination of western blot, quantitative real-time PCR and histological methods were
used to confirm the proteomic results and investigate the biological pathways linked to
the obtained results.
Analysis of the extracellular proteome revealed that fibroblasts from each DEB variant
have their own proteomic signature. Independently of the DEB variant - and its
associated clinical aggressiveness - the different COL7A1 mutations studied impacted
dermal ECM organization through the down-regulation of major ECM players such as
collagen XII, decorin, biglycan and fibulin-5. Furthermore, ECM organization-
associated proteins were found to be differently expressed between DEB variants. For
the phenotypes associated to increased severity of disease, a down-regulation of
proteins linked to ECM structure and remodelling, namely collagens I, III and V and
matrix metalloproteinases 1 and 2, was observed.
Our results corroborate previous studies showing that total loss of collagen VII has an
enormous impact on dermal ECM dynamics. Additionally, our results also
demonstrated that a partial loss of type VII collagen impacts cell microenvironment,
affecting mostly the ECM structural proteins. Overall, our work contributes to the
generation of further knowledge on DEB variants molecular features.
Acknowledgements: The authors would like to acknowledge FCT for grant SFRH/BD/137766/2018 (MDM), contract
Grant No.Norte-01-0145-FEDER-02219015 (MTC) and ERC Consolidator Grant – ECM_INK (ERC-2016-COG-726061).
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