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Comparative Study
. 2011 Aug 19;109(5):543-53.
doi: 10.1161/CIRCRESAHA.111.241299. Epub 2011 Jun 30.

Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease

Hibo Noureddine  1 , Guillaume Gary-Bobo, Marco Alifano, Elisabeth Marcos, Mirna Saker, Nora Vienney, Valérie Amsellem, Bernard Maitre, Ari Chaouat, Christos Chouaid, Jean-Luc Dubois-Rande, Diane Damotte, Serge Adnot
Affiliations
Comparative Study

Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease

Hibo Noureddine et al. Circ Res. .

Abstract

Rationale: Senescence of pulmonary artery smooth muscle cells (PA-SMCs) caused by telomere shortening or oxidative stress may contribute to pulmonary hypertension associated with chronic lung diseases.

Objective: To investigate whether cell senescence contributes to pulmonary vessel remodeling and pulmonary hypertension in chronic obstructive pulmonary disease (COPD).

Methods and results: In 124 patients with COPD investigated by right heart catheterization, we found a negative correlation between leukocyte telomere length and pulmonary hypertension severity. In-depth investigations of lung vessels and derived cultured PA-SMCs showed greater severity of remodeling and increases in senescent p16-positive and p21-positive PA-SMCs and proliferating Ki67-stained cells in 14 patients with COPD compared to 13 age-matched and sex-matched control subjects who smoke. Cultured PA-SMCs from COPD patients displayed accelerated senescence, with fewer cell population doublings, an increased percentage of β-galactosidase-positive cells, shorter telomeres, and higher p16 protein levels at an early cell passage compared to PA-SMCs from controls. Both in situ and in vitro PA-SMC senescence criteria correlated closely with the degree of pulmonary vessel wall hypertrophy. Because senescent PA-SMCs stained for p16 and p21 were virtually confined to the media near the Ki67-positive cells, which predominated in the neointima and hypertrophied media, we evaluated whether senescent cells affected normal PA-SMC functions. We found that senescent PA-SMCs stimulated the growth and migration of normal target PA-SMCs through the production and release of paracrine soluble and insoluble factors.

Conclusion: PA-SMC senescence is an important contributor to the process of pulmonary vascular remodeling that underlies pulmonary hypertension in chronic lung disease.

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Figures

Figure 1
Figure 1
(A) Immunolocalization and quantification of α-SMA-positive smooth muscle cells (blue staining) stained for p16, p21, and Ki67 (brown staining) in sections of pulmonary vessels from patients with COPD and controls. Scale bar: 100 μm. Bar graphs represent the percentages of p16-, p21-, and Ki67-positive cells in pulmonary vessels from patients with COPD and controls. Values are means±SEM. *P<0.01 compared with values from controls. Correlations between the vascular wall area ratio and the percentage of p16-positive cells (r=0.54, P<0.01), p21 (r=0.56, P<0.01) and Ki67-positive cells (r=0.67, P<0.001 ) in patients with COPD and controls. (B) Similar representations for vWF positive endothelial cells stained for p16- and p21. Correlation between vascular wall thickness, and p16- (r=0.61, P<0.01), and p21-positive cells (r=0.68, P<0.001). (C) Similar representations for Masson trichrome staining where collagen appears in blue and cells in red. Correlation with collagen content (r=0.69, P<0.001).
Figure 1
Figure 1
(A) Immunolocalization and quantification of α-SMA-positive smooth muscle cells (blue staining) stained for p16, p21, and Ki67 (brown staining) in sections of pulmonary vessels from patients with COPD and controls. Scale bar: 100 μm. Bar graphs represent the percentages of p16-, p21-, and Ki67-positive cells in pulmonary vessels from patients with COPD and controls. Values are means±SEM. *P<0.01 compared with values from controls. Correlations between the vascular wall area ratio and the percentage of p16-positive cells (r=0.54, P<0.01), p21 (r=0.56, P<0.01) and Ki67-positive cells (r=0.67, P<0.001 ) in patients with COPD and controls. (B) Similar representations for vWF positive endothelial cells stained for p16- and p21. Correlation between vascular wall thickness, and p16- (r=0.61, P<0.01), and p21-positive cells (r=0.68, P<0.001). (C) Similar representations for Masson trichrome staining where collagen appears in blue and cells in red. Correlation with collagen content (r=0.69, P<0.001).
Figure 1
Figure 1
(A) Immunolocalization and quantification of α-SMA-positive smooth muscle cells (blue staining) stained for p16, p21, and Ki67 (brown staining) in sections of pulmonary vessels from patients with COPD and controls. Scale bar: 100 μm. Bar graphs represent the percentages of p16-, p21-, and Ki67-positive cells in pulmonary vessels from patients with COPD and controls. Values are means±SEM. *P<0.01 compared with values from controls. Correlations between the vascular wall area ratio and the percentage of p16-positive cells (r=0.54, P<0.01), p21 (r=0.56, P<0.01) and Ki67-positive cells (r=0.67, P<0.001 ) in patients with COPD and controls. (B) Similar representations for vWF positive endothelial cells stained for p16- and p21. Correlation between vascular wall thickness, and p16- (r=0.61, P<0.01), and p21-positive cells (r=0.68, P<0.001). (C) Similar representations for Masson trichrome staining where collagen appears in blue and cells in red. Correlation with collagen content (r=0.69, P<0.001).
Figure 2
Figure 2
Immunolocalization of p16-, p21-, and Ki67-positive cells and collagen accumulation visualized by Masson trichrome staining in sections of remodeled pulmonary vessels from patients with COPD. Senescent PA-SMCs stained for p16 and p21 were virtually confined to the media, whereas Ki67-stained cells predominated in the neointima and hypertrophied media. Some p16 and p21 immunostaining was also associated with endothelial cells or PA-SMCs near the vessel lumen. In Masson trichrome-stained sections, the collagen is blue and the embedded cells are red.
Figure 3
Figure 3
(A) Replicative senescence of PA-SMCs from patients with COPD and controls. Cells were subjected to repeated passages and counted at each passage, and the population doubling level (PDL) was calculated for patients with COPD and controls. (B) Data are means±SEM. *P<0.01, versus controls. (C) PA-SMCs were stained for senescence-associated β-galactosidase (β-Gal) activity at passage 2 and at senescence when cells began to exhibit proliferative arrest. (C): Representative photographs of cells stained for senescence-associated β-Gal activity at passage 2 and at senescence. (D) Percentage of β-Gal-positive cells. Data are means±SEM. *P<0.01 versus controls; §P<0.05, versus corresponding values at passage 2. (E) Correlations between the vascular wall area ratio and the PDL (r=−0.61; P<0.001).
Figure 3
Figure 3
(A) Replicative senescence of PA-SMCs from patients with COPD and controls. Cells were subjected to repeated passages and counted at each passage, and the population doubling level (PDL) was calculated for patients with COPD and controls. (B) Data are means±SEM. *P<0.01, versus controls. (C) PA-SMCs were stained for senescence-associated β-galactosidase (β-Gal) activity at passage 2 and at senescence when cells began to exhibit proliferative arrest. (C): Representative photographs of cells stained for senescence-associated β-Gal activity at passage 2 and at senescence. (D) Percentage of β-Gal-positive cells. Data are means±SEM. *P<0.01 versus controls; §P<0.05, versus corresponding values at passage 2. (E) Correlations between the vascular wall area ratio and the PDL (r=−0.61; P<0.001).
Figure 4
Figure 4
Levels of IL-6, IL-8, TNF-α, MCP-1, and TGF-β in PA-SMC media from the 14 patients with COPD and 13 controls, collected at passage 2 and at senescence. Each bar is the mean±SEM. * P<0.01 compared with values for PA-SMCs from controls. § P<0.05 compared with corresponding values for PA-SMCs at passage 2.
Figure 5
Figure 5
Growth stimulation of normal PA-SMCs by soluble (medium) or insoluble (matrix) factors secreted by presenescent (gray bars) or senescent (black bars) PA-SMCs. Presenescent cells were passage 3–4 cells from controls and senescent cells were passage 3–4 cells from patients with COPD. (A) Percentage of cells after exposure to the medium of presenescent or senescent PA-SMCs. (B) Percentage of cells plated onto matrices deposited by presenescent or senescent PA-SMCs. Values are mean±SEM of 12 values obtained from six independent experiments. (C) PA-SMC proliferation (OD: optical density, in arbitrary units) in a typical experiment where cells were stimulated by PDGF (20 ng/mL), medium, matrix, or medium combined with matrix from presenescent or senescent cells. (D) Percentage of cells after exposure to the medium of presenescent or senescent PA-SMCs in the presence of control or neutralizing antibodies to IL-8, TNF-α, IL-6, and MCP-1. *P<0.05 compared with values corresponding to stimulation by presenescent cells; § p<0.05 versus values with control antibodies.
Figure 5
Figure 5
Growth stimulation of normal PA-SMCs by soluble (medium) or insoluble (matrix) factors secreted by presenescent (gray bars) or senescent (black bars) PA-SMCs. Presenescent cells were passage 3–4 cells from controls and senescent cells were passage 3–4 cells from patients with COPD. (A) Percentage of cells after exposure to the medium of presenescent or senescent PA-SMCs. (B) Percentage of cells plated onto matrices deposited by presenescent or senescent PA-SMCs. Values are mean±SEM of 12 values obtained from six independent experiments. (C) PA-SMC proliferation (OD: optical density, in arbitrary units) in a typical experiment where cells were stimulated by PDGF (20 ng/mL), medium, matrix, or medium combined with matrix from presenescent or senescent cells. (D) Percentage of cells after exposure to the medium of presenescent or senescent PA-SMCs in the presence of control or neutralizing antibodies to IL-8, TNF-α, IL-6, and MCP-1. *P<0.05 compared with values corresponding to stimulation by presenescent cells; § p<0.05 versus values with control antibodies.
Figure 6
Figure 6
Stimulation of migration of normal PA-SMCs by soluble (medium) or insoluble (matrix) factors secreted by presenescent (gray bars) or senescent (black bars) PA-SMCs. Presenescent cells were passage 3–4 cells from controls and senescent cells were passage 3–4 cells from patients with COPD. (A) Migration area of cells measured 24 h after stimulation by 0% or 5% fetal calf serum. (B) Migration area of cells after exposure to the medium of presenescent or senescent PA-SMCs. (C) Migration area of cells onto matrices deposited by presenescent or senescent PA-SMCs. Values are mean±SEM of six values obtained from three independent experiments. Photomicrographs represent corresponding cells migrating from agarose droplets. (D) Migration area of cells after exposure to the medium of presenescent or senescent PA-SMCs in the presence of control or neutralizing antibodies to IL-8, TNF-α, IL-6, and MCP-1. *P<0.05, versus values corresponding to stimulation by presenescent cells; § P<0.05 versus values with control antibodies.
Figure 6
Figure 6
Stimulation of migration of normal PA-SMCs by soluble (medium) or insoluble (matrix) factors secreted by presenescent (gray bars) or senescent (black bars) PA-SMCs. Presenescent cells were passage 3–4 cells from controls and senescent cells were passage 3–4 cells from patients with COPD. (A) Migration area of cells measured 24 h after stimulation by 0% or 5% fetal calf serum. (B) Migration area of cells after exposure to the medium of presenescent or senescent PA-SMCs. (C) Migration area of cells onto matrices deposited by presenescent or senescent PA-SMCs. Values are mean±SEM of six values obtained from three independent experiments. Photomicrographs represent corresponding cells migrating from agarose droplets. (D) Migration area of cells after exposure to the medium of presenescent or senescent PA-SMCs in the presence of control or neutralizing antibodies to IL-8, TNF-α, IL-6, and MCP-1. *P<0.05, versus values corresponding to stimulation by presenescent cells; § P<0.05 versus values with control antibodies.
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