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. 2020 Aug;19(8):e13196.
doi: 10.1111/acel.13196. Epub 2020 Jul 21.

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Nunzia Caporarello  1 , Jeffrey A Meridew  1 , Aja Aravamudhan  1 , Dakota L Jones  1 , Susan A Austin  2 , Tho X Pham  3 , Andrew J Haak  1 , Kyoung Moo Choi  1 , Qi Tan  1 , Adil Haresi  3 , Steven K Huang  4 , Zvonimir S Katusic  2 , Daniel J Tschumperlin  1 , Giovanni Ligresti  3   1
Affiliations

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Nunzia Caporarello et al. Aging Cell. 2020 Aug.

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non-resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro-fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non-resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ-induced pro-fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ-induced lung fibroblast activation in 2D and 3D co-cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Keywords: aging; eNOS; fibroblast activation; lung fibrosis; vascular dysfunction.

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Conflict of interest statement

None declared.

Figures

FIGURE 1
FIGURE 1
Delayed fibrosis resolution in aged mice following bleomycin challenge. (a) Young and aged mice were exposed to bleomycin and sacrificed after 30 and 75 days. Lungs were harvested and prepared for FACS sorting. (b) Col1a1 transcriptional analysis of FACS‐sorted GFP+/CD31−/CD45−/EpCAM− lung fibroblasts isolated from young and aged animals after bleomycin‐induced injury (young sham, N = 5; young 30 days, N = 8; young 75 days, N = 5; aged sham, N = 7; aged 30 days, N = 9; aged 75 days, N = 5). (c) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (young sham, N = 8; young 14 days, N = 7; young 30 days, N = 9; young 75 days, N = 7). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (d) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (aged sham, N = 8; aged 14 days, N = 5; aged 30 days, N = 11; aged 75 days, N = 8). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (e, f). Representative immunohistochemistry images and quantification of Collagen I by automated image analysis (young 75 days, N = 4; aged 75 days, N = 4). Data are non‐normally distributed, are expressed as median and IQR, and analyzed using non‐parametric Mann–Whitney test (*p < 0.05; **p < 0.01).
FIGURE 2
FIGURE 2
Vascular rarefaction accompanies persistent fibrosis in aged mice challenged with bleomycin. (a) Quantification of vascular density by automated image analysis (young sham, N = 4; young 30 days, N = 8; young 75 days, N = 9; aged sham, N = 4; aged 30 days, N = 6; aged 75 days, N = 11). Data passed Shapiro–Wilk normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (b) Representative IF images of mouse lung tissue stained with PECAM‐1 antibody. Scale bars: 100 μm. (c) Immunostaining of human tissue derived from normal or IPF lung for PECAM‐1 counterstained with hematoxylin. Magnifications: upper row, 4X, scale bars: 250 μm, lower row, 10X, scale bars: 100 μm. FF = fibroblastic foci. Arrows show areas occupied by microvessels in regions bordering FF. (d) Schematic for ex vivo lung tissue culture. Pieces of lungs from young and aged mice were embedded in collagen for 7 days in presence of 20 ng/ml VEGFA. (e) Collagen gel culture of lung explants derived from young and aged mice. (f) Vessel counts demonstrate reduction of sprouting outgrowth in aged mice. Data are non‐normally distributed, are expressed as median and IQR, and analyzed using non‐parametric Mann–Whitney test (*p < 0.05; ***p < 0.001).
FIGURE 3
FIGURE 3
Loss of endothelial cell identity in the lungs of aged mice following bleomycin challenge. (a) Young and aged mice were exposed to bleomycin and sacrificed after 30 and 75 days. Lungs were harvested and prepared for FACS sorting. (b) FACS‐sorted CD31+/GFP−/CD45−/EpCAM− lung ECs from young and aged mice (30 days) were analyzed by using an endothelial cell biology profiler PCR Array (N = 4 mice). The heatmap was generated by averaging n = 4 mice for each condition. Sham animals were harvested at the same time of bleomycin‐treated animals. The data represent fold changes relative to the young sham and normalized to the housekeeping gene Actb. (c) Transcriptional analysis of FACS‐sorted CD31+/GFP−/CD45−/EpCAM− lung ECs isolated from young and aged mice after bleomycin challenge (young sham, N = 5, young 30 days, N = 10; aged sham, N = 4; aged 30 days, N = 9). Data passed D’Agostino and Pearson omnibus or Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test) (*p < 0.05; **p < 0.01; ***p < 0.001).
FIGURE 4
FIGURE 4
Loss of eNOS leads to sustained lung fibrosis in young animals following bleomycin challenge. (a) Nos3 transcriptional analysis of FACS‐sorted CD31+/GFP−/CD45−/EpCAM− lung ECs isolated from young and aged mice after bleomycin‐induced lung injury (young sham, N = 6; young 30 days, N = 10; young 75 days, N = 8; aged sham, N = 7; aged 30 days, N = 9; aged 75 days, N = 7). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (b) Lung homogenates from WT and eNOS−/− mice were analyzed via Western blot using anti eNOS and anti GAPDH antibodies. (c) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (WT sham, N = 7; WT 11 days, N = 3; WT 60 days, N = 10; eNOS−/− sham, N = 7; eNOS−/− 11 days, N = 3; eNOS−/− 60 days, N = 14). Data passed Shapiro–Wilk normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (d) Masson's trichrome assay was used to stain lung tissue. (e) Transcriptional analysis of whole lung homogenates obtained from WT and eNOS−/− mice (WT 60 days, N = 7; eNOS−/− 60 days, N = 4). The reference group throughout all the genes analyzed in this panel is WT 60 days after bleomycin. Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using Student's t test (*p < 0.05; **p < 0.01).
FIGURE 5
FIGURE 5
eNOS promotes lung fibroblast deactivation through activation of the NO/sGC pathway. (a, b) Pro‐fibrotic gene and protein analysis of HLFs treated with TGFβ (2 ng/ml) and BAY 41‐2272 (5 μM) or TGFβ (2 ng/ml) and BAY 60‐2770 (1 μM) for 48 hr. N = 5 independent experiments. Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (c) Schematic of 2D co‐culture system. TGFβ‐primed HLFs and control‐ or NOS3‐silenced HLMECs were seeded for co‐cultures in μ‐Slide 2 well Co‐culture. (d, e) Immunofluorescence images (10x objective magnification) of HLFs primed 24 hr with 2 ng/ml TGFβ and then co‐cultured with control or NOS3 siRNA transfected HLMECs (72 hr). Scale bars: 1000 μm. αSMA intensity was determined using automated imaging software. N = 4 independent experiments. Data are non‐normally distributed, are expressed as median and IQR, and analyzed using non‐parametric Mann–Whitney test. (f) Control‐ and NOS3‐silenced HLMECs (72 hr) were analyzed via Western blot using anti eNOS and anti GAPDH antibodies. (g) Transcriptional analysis of control and NOS3‐silenced HLMECs (72 hr). N = 4 independent experiments. Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD, and analyzed using Student's t test. (h, i) Schematic of 3D co‐cultures generation. Visualization of DiI‐labeled HLMECs (red) and Col1α1‐GFP mouse fibroblasts (green) within an endothelial cell fibroblast 3D co‐culture. Scale bar: 500 μm. (j) Gene expression analysis of fibroblasts transcripts from mouse fibroblasts alone versus co‐cultures with control‐ and NOS3 siRNA transfected HLMECs at day 3. N = 3 independent experiments. Data passed Shapiro–Wilk normality test, are expressed as mean ± SD, and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (**p < 0.01; ***p < 0.001).
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