The effect of TGFβRI inhibition on fibroblast heterogeneity in hypertrophic scar 2D in vitro models

Rajiv S Raktoe; Marion H Rietveld; Jacoba J Out-Luiting; Marianna Kruithof-de Julio; Paul P M van Zuijlen; Remco van Doorn; Abdoelwaheb El Ghalbzouri

doi:10.1016/j.burns.2021.01.004

The effect of TGFβRI inhibition on fibroblast heterogeneity in hypertrophic scar 2D in vitro models

Burns. 2021 Nov;47(7):1563-1575. doi: 10.1016/j.burns.2021.01.004. Epub 2021 Jan 23.

Authors

Rajiv S Raktoe¹, Marion H Rietveld², Jacoba J Out-Luiting², Marianna Kruithof-de Julio³, Paul P M van Zuijlen⁴, Remco van Doorn², Abdoelwaheb El Ghalbzouri²

Affiliations

¹ Department of Dermatology, Leiden University Medical Centre (LUMC), Leiden, the Netherlands. Electronic address: rs.raktoe@gmail.com.
² Department of Dermatology, Leiden University Medical Centre (LUMC), Leiden, the Netherlands.
³ Department of Urology, LUMC, Leiden, the Netherlands; Department of Urology, University of Bern, Bern, Switzerland.
⁴ Amsterdam UMC Location VUmc, Department of Plastic, Reconstructive and Hand Surgery, Amsterdam Movement Sciences, Amsterdam, the Netherlands; Burn Center and Department of Plastic and Reconstructive Surgery, Red Cross Hospital, Beverwijk, the Netherlands.

PMID: 33558094
DOI: 10.1016/j.burns.2021.01.004

Abstract

In burn patients, wound healing is often accompanied by hypertrophic scarring (HTS), resulting in both functional and aesthetic problems. HTSs are characterized by abundant presence of myofibroblasts (MFs) residing in the dermis. HTS development and MF persistence is primarily regulated by TGF-β signalling. A promising method to target the transforming growth factor receptor I (TGFβRI; also known as activin-like kinase 5 (ALK5)) is by making use of exon skipping through antisense oligonucleotides. In HTS the distinguishing border between the papillary dermis and the reticular dermis is completely abrogated, thus exhibiting a one layered dermis containing a heterogenous fibroblast population, consisting of papillary fibroblasts (PFs), reticular fibroblasts (RFs) and MFs. It has been proposed that PFs, as opposed to RFs, exhibit anti-fibrotic properties. Currently, it is still unclear which fibroblast subtype is most affected by exon skipping treatment. Therefore, the aim of this study was to investigate the effect of TGFβRI inhibition by exon skipping in PF, RF and HTS fibroblast monocultures. Morphological analyses revealed the presence of a PF-like population after exon skipping in the different fibroblast cultures. This observation was further confirmed by the expression of genes specific for PFs, demonstrated by qPCR analyses. Further investigations on mRNA and protein level revealed that indeed MFs and to a lesser extent RFs are targeted by exon skipping. Furthermore, collagen gel contraction analysis showed that ALK5 exon skipping reduced TGF-β- induced contraction together with decreased alpha-smooth muscle actin expression levels. In conclusion, we show for the first time that exon skipping primarily targets pro-fibrotic fibroblasts. This could be a promising step towards reduced HTS development of burn tissue.

Keywords: ALK5; Exon skipping; Fibroblast heterogeneity; Hypertrophic scars; Papillary fibroblast; Reticular fibroblast.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actins / genetics
Burns* / pathology
Burns* / therapy
Cells, Cultured
Cicatrix, Hypertrophic* / genetics
Cicatrix, Hypertrophic* / pathology
Cicatrix, Hypertrophic* / therapy
Exons
Fibroblasts / pathology
Fibrosis
Humans
Myofibroblasts / pathology
Oligonucleotides, Antisense
Receptor, Transforming Growth Factor-beta Type I / antagonists & inhibitors*

Substances

Actins
Oligonucleotides, Antisense
Receptor, Transforming Growth Factor-beta Type I
TGFBR1 protein, human