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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 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B: supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. *P < 0.05 **P < 0.005. C: PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D: PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. **P < 0.01; ***P < 0.01, for C and D. E: PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. *P < 0.05; **P < 0.005; ***P < 0.0001. F: PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. ****P < 0.0001. G: PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. **P < 0.01. H and I: PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. ***P < 0.005; ****P < 0.001, for I.
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Figure 6: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B: supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. *P < 0.05 **P < 0.005. C: PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D: PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. **P < 0.01; ***P < 0.01, for C and D. E: PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. *P < 0.05; **P < 0.005; ***P < 0.0001. F: PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. ****P < 0.0001. G: PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. **P < 0.01. H and I: PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. ***P < 0.005; ****P < 0.001, for I.

Mentions: Exposure of RPAF to hypoxia for up to 48 h led to upregulation of IL-6 and release of IL-6 from the cells. Secreted levels of IL-6 were significantly increased at 48 h of exposure. Treatment of cells with SB203580 prevented the release of the IL-6 from hypoxia-exposed fibroblasts (Fig. 6, A and B). This was time dependent with peak mRNA effect observed at 12 h after hypoxic exposure (Fig. 6C). This was reduced in the presence of p38 inhibition (data not shown). Further studies showed that pulmonary artery fibroblasts proliferate in response to IL-6, which was not significantly increased in the presence of the soluble IL-6R-α subunit (testing transactivation of the IL-6 pathway) but was more pronounced under hypoxic conditions (Fig. 6D). In PASMCs, the addition of IL-6 also proved to be a strong mitogen (Fig. 6E) and incubation with anti-IL-6 blocked the PASMC proliferation (Fig. 6F). In addition, the proliferation via IL-6 seemed to be mediated by the classical STAT-3 pathway with increased phosphorylation in a time-dependent manner as shown (Fig. 6, G and H).


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 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B: supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. *P < 0.05 **P < 0.005. C: PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D: PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. **P < 0.01; ***P < 0.01, for C and D. E: PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. *P < 0.05; **P < 0.005; ***P < 0.0001. F: PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. ****P < 0.0001. G: PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. **P < 0.01. H and I: PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. ***P < 0.005; ****P < 0.001, for I.
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Figure 6: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B: supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. *P < 0.05 **P < 0.005. C: PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D: PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. **P < 0.01; ***P < 0.01, for C and D. E: PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. *P < 0.05; **P < 0.005; ***P < 0.0001. F: PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. ****P < 0.0001. G: PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. **P < 0.01. H and I: PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. ***P < 0.005; ****P < 0.001, for I.
Mentions: Exposure of RPAF to hypoxia for up to 48 h led to upregulation of IL-6 and release of IL-6 from the cells. Secreted levels of IL-6 were significantly increased at 48 h of exposure. Treatment of cells with SB203580 prevented the release of the IL-6 from hypoxia-exposed fibroblasts (Fig. 6, A and B). This was time dependent with peak mRNA effect observed at 12 h after hypoxic exposure (Fig. 6C). This was reduced in the presence of p38 inhibition (data not shown). Further studies showed that pulmonary artery fibroblasts proliferate in response to IL-6, which was not significantly increased in the presence of the soluble IL-6R-α subunit (testing transactivation of the IL-6 pathway) but was more pronounced under hypoxic conditions (Fig. 6D). In PASMCs, the addition of IL-6 also proved to be a strong mitogen (Fig. 6E) and incubation with anti-IL-6 blocked the PASMC proliferation (Fig. 6F). In addition, the proliferation via IL-6 seemed to be mediated by the classical STAT-3 pathway with increased phosphorylation in a time-dependent manner as shown (Fig. 6, G and H).

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