Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

Scars, Burns & Healing
Rajiv S RaktoeAbdoelwaheb El Ghalbzouri

Abstract

In burn patients, wound healing is often accompanied by hypertrophic scar (HS) development, resulting in both functional and aesthetic problems. HSs are characterised by abundant presence of myofibroblasts that contribute to overproduction of extracellular matrix (ECM) that is regulated by the TGF-β signalling pathway. Studies have shown that inhibition of TGF-β receptors in fibrotic diseases reduces the fibrotic load. In the present study, we aim to inactivate ALK5, also known as TGF-β receptor I, in human HS fibroblasts by exon skipping using antisense oligonucleotides (AONs). HS biopsies were used to isolate and set up fibroblast monocultures. AONs targeting ALK5 were supplemented to the fibroblast cultures to induce exon skipping, while pharmacological ALK5 inhibition was induced using SB431542. AON delivery in HS fibroblasts was examined using immunofluorescence (IF), while TGF-β signalling downstream targets, such as Smad2/3, PAI-1, ACTA2, COL1A1 and COL3A1, were analysed using touchdown polymerase chain reaction (PCR), quantitative PCR (qPCR), IF or western blotting. Our data clearly demonstrate that AONs were successfully delivered in the nuclei of HS fibroblasts and that functional exon skipping of ALK5 took place as c...Continue Reading

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Citations

Feb 10, 2021·Burns : Journal of the International Society for Burn Injuries·Rajiv S RaktoeAbdoelwaheb El Ghalbzouri

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Methods Mentioned

BETA
antisense oligonucleotides
biopsies
PCR
nuclear translocation
biopsy

Software Mentioned

GraphPad Prism
GraphPad
GeneTools
TScratch
ImageJ
CellScan

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