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

Raktoe, Rajiv S; Rietveld, Marion H; Out-Luiting, Jacoba J; Kruithof-de Julio, Marianna; van Zuijlen, Paul Pm; van Doorn, Remco; Ghalbzouri, Abdoelwaheb El (2020). Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts. Scars, burns & healing, 6, p. 2059513120908857. Sage 10.1177/2059513120908857

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Background

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).

Methods

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.

Results

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 confirmed with touchdown PCR and qPCR. In addition, exon skipping affected the expression of ECM-related genes, such as type I/III collagens, PAI-1 and CCN2. Moreover, AON treatment did not affect the migration of HS fibroblasts in a model for wound healing.

Conclusion

Exon skipping is a promising tool to modulate the TGF-β signalling pathway in HS. This would open a therapeutic window for the treatment of patients suffering from HSs.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Dermatology, Urology, Rheumatology, Nephrology, Osteoporosis (DURN) > Clinic of Urology

UniBE Contributor:

Kruithof-de Julio, Marianna

Subjects:

600 Technology > 610 Medicine & health

ISSN:

2059-5131

Publisher:

Sage

Language:

English

Submitter:

Jeannine Wiemann

Date Deposited:

04 Aug 2020 08:53

Last Modified:

12 Sep 2020 01:57

Publisher DOI:

10.1177/2059513120908857

PubMed ID:

32528734

Uncontrolled Keywords:

(myo)fibroblasts ALK5 Hypertrophic scars TGF-β antisense oligonucleotides exon skipping

BORIS DOI:

10.7892/boris.145536

URI:

https://boris.unibe.ch/id/eprint/145536

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