Global gene expression analysis of systemic sclerosis myofibroblasts demonstrates a marked increase in the expression of multiple NBPF genes

Giuseppina Abignano,Francesco Del Galdo,Sergio A Jimenez,Sonsoles Piera-Velazquez,Heidi Hermes,Sankar Addya

doi:10.1038/s41598-021-99292-y

Abstract

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-β signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

Highlights

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc)
To determine the global transcriptome of α-smooth muscle actin (α-SMA) positive SSc myofibroblasts we examined monolayer cell cultures that had been expanded from biopsies excised from the leading edge of forearm skin lesions of four patients with dcSSc of recent onset
The results showed that α-SMA expression was an average of 370% greater in the α-SMA-positive myofibroblasts compared to the α-SMA-negative fibroblasts as described elsewhere[26]

Summary

Introduction

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). The cells responsible for tissue fibrosis and fibroproliferative processes are myofibroblasts, a population of mesenchymal cells displaying unique biological functions including cell mobility and tissue contraction, and the high expression of α-smooth muscle actin (α-SMA) and numerous molecules associated with fibrotic processes[5,6,7,8]. It has been shown that the global gene expression profile of SSc myofibroblasts present in lungs from patients with SSc-associated interstitial lung disease examined employing single cell RNA sequencing assays displays unique gene expression signatures compared to two different types of fibroblasts[25]. The purpose of this study was to analyze the specific transcriptome of pure SSc myofibroblasts To accomplish this goal myofibroblasts expressing α-SMA actin were isolated employing laser capture microdissection (LCM) from monolayer cultures of cells derived from skin biopsies from affected SSc skin

Objectives

Methods

Results

Conclusion