Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot