Evidence of telomere attrition and a potential role for DNA damage in systemic sclerosis

Abstract

Background: To investigate the role of cell senescence in systemic sclerosis (SSc), we analyzed telomere shortening (TS) in SSc patients and the effect of targeting DNA damage in the bleomycin model of skin fibrosis.

Results: Telomere length (TL) in blood leukocytes of 174 SSc patients and 68 healthy controls was measured by Southern blot, and we found shorter age-standardized TL in SSc patients compared to healthy controls. TL was shorter in SSc patients with ILD compared to those without ILD and in anti-topoisomerase I positive compared to anti-centromere positive patients. To analyze the potential role of DNA damage in skin fibrosis, we evaluated the effects of the DNA protective GSE4 peptide in the bleomycin mouse model of scleroderma and the fibrotic response of cultured human dermal fibroblasts. Administration of GSE4-nanoparticles attenuated bleomycin-induced skin fibrosis as measured by Masson's staining of collagen and reduced Acta2 and Ctgf mRNA expression, whereas transduction of dermal fibroblasts with a lentiviral GSE4 expression vector reduced COL1A1, ACTA2 and CTGF gene expression after stimulation with bleomycin or TGF-β, in parallel to a reduction of the phospho-histone H2A.X marker of DNA damage.

Conclusions: SSc is associated with TS, particularly in patients with lung disease or anti-topoisomerase I antibodies. Administration of GSE4 peptide attenuated experimental skin fibrosis and reduced fibroblast expression of profibrotic factors, supporting a role for oxidative DNA damage in scleroderma.

Keywords: Fibroblast; Systemic sclerosis; Telomere length.

Fig. 1

Telomere length in SSc patients and healthy controls. A TL in bp as determined in whole blood DNA by TRF Southern blot in healthy controls (blue, n 68) and SSc patients (red, n 174) plotted against age. r indicates the TL/age correlation coefficient by Spearman rank test, SSc p = 0.0001. HC p < 0.0001. B TL z-score of healthy controls, SSc patients, and SSc clinical and serological subsets. Median and IQR [25,75%] are represented. HC: healthy controls, lcSSc: limited cutaneous SSc, dcSSc: diffuse cutaneous SSc, ILD: Interstitial lung disease, ATA+: anti-topoisomerase-I positive, ACA+: anti-centromere protein B positive, Comorb.: Comorbid conditions, NS: Non significant, *p < 0.05, ** p < 0.0001

Fig. 2

Effect of GSE4-nanoparticles on the development of bleomycin-induced skin fibrosis. C3H mice received daily subcutaneous injections of bleomycin, and were also injected with either scramble (Bleo + Scr) or GSE4 (Bleo + GSE4) nanoparticles every other day for 4 weeks. Control mice were daily injected with saline. A Representative image of Masson’s stained skin biopsy for each group. B Fibrosis was measured as the fold increase in the collagen Masson stained area adjusted to the normal area (saline group). Quantification of mRNA expression by quantitative RT-PCR of Acta2 (C) and Ctgf (D). *p < 0.05, **P < 0.01, ***p < 0.001 (Median and IQR, Mann-Whitney test). Graphic shows data of 3 independent experiments with 10 mice per group. Bar 50 μm

Fig. 3

Effect of GSE4-lentiviral transduction to cultured dermal fibroblasts on profibrotic genes and phospho-H2A.X protein expression. Fibroblasts were transduced with scramble or GSE4 expression lentiviral vectors, and treated with TGF-β or bleomycin for 24 h as indicated. A Quantification of mRNA expression by quantitative RT-PCR of COL1A1, CTGF and ACTA2 (n = 11). (B) Representative image of Western blot analysis of phospho-H2A.X protein expression in a dermal fibroblast line (upper panel). Densitometric quantification shown as the ratio pH2A.X/β-actin (n = 10) (lower panel). *p < 0.05, **p < 0.01, ***p < 0.0001 (Median and IQR, Wilcoxon matched-pairs signed Rank Test)