Cellular senescence-like features of lung fibroblasts derived from idiopathic pulmonary fibrosis patients - PubMed (original) (raw)

Cellular senescence-like features of lung fibroblasts derived from idiopathic pulmonary fibrosis patients

Hagai Yanai et al. Aging (Albany NY). 2015 Sep.

Erratum in

Abstract

Idiopathic pulmonary fibrosis (IPF) is an age-related fatal disease with unknown etiology and no effective treatment. In this study, we show that primary cultures of fibroblasts derived from lung biopsies of IPF patients exhibited (i) accelerated replicative cellular senescence (CS); (ii) high resistance to oxidative-stress-induced cytotoxicity or CS; (iii) a CS-like morphology (even at the proliferative phase); and (iv) rapid accumulation of senescent cells expressing the myofibroblast marker α-SMA. Our findings suggest that CS could serve as a bridge connecting lung aging and its quite frequent outcome -- pulmonary fibrosis, and be an important player in the disease progression. Consequently, targeting senescent cells offers the potential of being a promising therapeutic approach.

Keywords: aging; cellular senescence; fibroblasts; idiopathic pulmonary fibrosis; myofibroblasts.

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Conflict of interest statement

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1. Growth curves and response to oxidative stress of IPF-derived and normal human pulmonary fibroblasts

(A) Lung fibroblasts derived from IPF patients (IPF-PF) and healthy subjects (N-PF) during routine culture. PD stands for Population Doublings. Note that IPF-derived fibroblasts cease to proliferate after passage 16, whereas the N-PF ones are still in the logarithmic phase of cell growth. The difference between N-PF and IPF-PF growth curves is highly significant (Mann–Whitney _U_-test; p < 0.001). (B) Lung fibroblasts derived from IPF patients (IPF-PF) and healthy (N-PF) were treated with indicated doses of H2O2 for two hours, and tested for viability by Neutral Red assay (LD50 was 28.7 μM and 136 μM for N-PF and IPF-PF, respectively; p < E-06). Results represent 3 independent experiments. (C) SA-β-gal staining of normal (left panel) and IPF-derived fibroblasts (right panel) treated with H2O2 (the doses that caused 20% cytotoxicity were used).

Figure 2

Figure 2. Morphology analysis of lung fibroblasts by ImageStreamX

BF – bright field. Phalloidin, DAPI and DiD refer to staining of actin, nucleus and cell membrane, respectively. (A) Selected representative images. (B) Distribution of cell populations according to indicated criterion. The meaning of each variable is explained next to the images. Red lines indicate the cells in which actin is co-localized with cell membrane (score > 1.5). Note: IPF-PF vs. N-PF represent a more heterogeneous (more variable) cell population as evident by CV values (Table 1).

Figure 3

Figure 3. Morphology analysis of lung fibroblasts by Z-stack confocal microscopy

(A) Representative image of lung fibroblasts derived from IPF patients (IPF-PF) and healthy (N-PF). Background subtracted. Phalloidin stains for actin; DiD for membrane and all pictures show DAPI staining of the nucleus (B) Quantification of cell morphology using the Imaris software (see methods) of confocal z-stack images taken for fibroblasts of early passage (passage 7). Whiskers indicate standard error [; bars indicate range (min/max); middle line indicates mean.

Figure 4

Figure 4. Immunostaining for SA-β-gal and α-SMA in primary cultures of pulmonary fibroblasts

(A) early passages (upper left panels) and at cellular senescence (upper right panels). SA-β-gal – green staining; α-SMA – red staining. (B) Representative co-stained cells. (C) Distribution of cells expressing α-SMA, SA-β-gal, or both in senescent primary cultures of normal pulmonary (N-PF), IPF-derived fibroblasts (IPF-PF) and normal dermal Fibroblasts (HDF). The difference between the two pulmonary cell types was insignificant (p > 0.05).

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