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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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Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A: cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. ***P < 0.001. B: this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C: PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. **P < 0.005. D: PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. *P < 0.05; **P < 0.005. E: PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. ***P < 0.005. F: PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. ***P < 0.001.
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Figure 1: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A: cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. ***P < 0.001. B: this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C: PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. **P < 0.005. D: PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. *P < 0.05; **P < 0.005. E: PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. ***P < 0.005. F: PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. ***P < 0.001.

Mentions: Our group and others have shown previously that fibroblasts isolated from chronic hypoxic animals have undergone a phenotypic switch, which results in constitutive activation of p38 MAPK and a proproliferative phenotype. Whether this effect is seen in other models of pulmonary hypertension is unknown. Therefore, we examined the proliferative potential of fibroblasts derived from MCT animals and compared them to that of fibroblasts isolated from both normal and chronic hypoxic animals (Fig. 1A). This clearly showed an increased predisposition to proliferation by the MCT-derived fibroblasts in relation to low dose serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition.


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)

Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A: cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. ***P < 0.001. B: this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C: PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. **P < 0.005. D: PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. *P < 0.05; **P < 0.005. E: PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. ***P < 0.005. F: PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. ***P < 0.001.
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Related In: Results  -  Collection

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Figure 1: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A: cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. ***P < 0.001. B: this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C: PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. **P < 0.005. D: PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. *P < 0.05; **P < 0.005. E: PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. ***P < 0.005. F: PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. ***P < 0.001.
Mentions: Our group and others have shown previously that fibroblasts isolated from chronic hypoxic animals have undergone a phenotypic switch, which results in constitutive activation of p38 MAPK and a proproliferative phenotype. Whether this effect is seen in other models of pulmonary hypertension is unknown. Therefore, we examined the proliferative potential of fibroblasts derived from MCT animals and compared them to that of fibroblasts isolated from both normal and chronic hypoxic animals (Fig. 1A). This clearly showed an increased predisposition to proliferation by the MCT-derived fibroblasts in relation to low dose serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition.

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