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The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension.

Church AC, Martin DH, Wadsworth R, Bryson G, Fisher AJ, Welsh DJ, Peacock AJ - Am. J. Physiol. Lung Cell Mol. Physiol. (2015)

Bottom Line: Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod).Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod.This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6.

View Article: PubMed Central - PubMed

Affiliation: Scottish Pulmonary Vascular Unit, University of Glasgow, Glasgow, United Kingdom; colinchurch@nhs.net.

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p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A: lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B: lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05 **P < 0.01. C and D: lungs from CH 28-day controls and SB203580-treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. **P < 0.001. E: lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05; ns is not significant by ANOVA. F: PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. ***P < 0.005. G: PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. *P < 0.01; ***P < 0.005. H: fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. **P < 0.01, ***P < 0.001.
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Figure 7: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A: lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B: lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05 **P < 0.01. C and D: lungs from CH 28-day controls and SB203580-treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. **P < 0.001. E: lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05; ns is not significant by ANOVA. F: PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. ***P < 0.005. G: PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. *P < 0.01; ***P < 0.005. H: fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. **P < 0.01, ***P < 0.001.

Mentions: In the prevention study of the chronic hypoxic-associated pulmonary hypertension we found a reduction in IL-6 levels in the lungs of SB203580 drug-treated animals compared with vehicle-treated hypoxic controls (Fig. 7A). Similarly, in the reversal study with SB203580, IL-6 levels were reduced in the lung and the amount of phospho-STAT3 signaling was reduced (Fig. 7, B–D).


The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension.

Church AC, Martin DH, Wadsworth R, Bryson G, Fisher AJ, Welsh DJ, Peacock AJ - Am. J. Physiol. Lung Cell Mol. Physiol. (2015)

p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A: lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B: lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05 **P < 0.01. C and D: lungs from CH 28-day controls and SB203580-treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. **P < 0.001. E: lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05; ns is not significant by ANOVA. F: PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. ***P < 0.005. G: PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. *P < 0.01; ***P < 0.005. H: fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. **P < 0.01, ***P < 0.001.
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Figure 7: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A: lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B: lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05 **P < 0.01. C and D: lungs from CH 28-day controls and SB203580-treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. **P < 0.001. E: lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. *P < 0.05; ns is not significant by ANOVA. F: PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. ***P < 0.005. G: PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. *P < 0.01; ***P < 0.005. H: fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. **P < 0.01, ***P < 0.001.
Mentions: In the prevention study of the chronic hypoxic-associated pulmonary hypertension we found a reduction in IL-6 levels in the lungs of SB203580 drug-treated animals compared with vehicle-treated hypoxic controls (Fig. 7A). Similarly, in the reversal study with SB203580, IL-6 levels were reduced in the lung and the amount of phospho-STAT3 signaling was reduced (Fig. 7, B–D).

Bottom Line: Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod).Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod.This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6.

View Article: PubMed Central - PubMed

Affiliation: Scottish Pulmonary Vascular Unit, University of Glasgow, Glasgow, United Kingdom; colinchurch@nhs.net.

Show MeSH
Related in: MedlinePlus