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The interaction of endothelin-1 and TGF-β1 mediates vascular cell remodeling.

Lambers C, Roth M, Zhong J, Campregher C, Binder P, Burian B, Petkov V, Block LH - PLoS ONE (2013)

Bottom Line: This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)).Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria.

ABSTRACT

Background: Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective: To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods: PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results: ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and clinical relevance: ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

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The mitogenic-supportive effect of Et-1 on PASMC.(A) ET-1 alone has no mitogenic effect, while 5% foetal calf serum (FCS) induced proliferation of PASMC within 3 days (n = 6). (B) When combined with 5% FCS, ET-1 dose-dependently increased cell numbers within 3 days (n = 6). Similar results were obtained after 5 days. (C) ET-receptor blockade by Bosentan and chemical Erk1/2 MAPK inhibition abolished the supportive effect of ET-1 on serum-induced PASMC proliferation (n = 3). (D) ET-1 induced accumulation of S/G2-phase cells is prevented by Bosentan (n = 3). (A–D) Bars represent mean ± SEM. (E) Representative immune-blot of the effect of ET-1 and Bosentan on cell compartment distribution of p27(Kip), similar results were obtained in 3 additional experiments. (F) TGF-ß alone did not induce proliferation, whereas the addition of ET-1 significantly increased PASMC proliferation, which was abolished by the inhibition of Erk1/2 MAPK.
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pone-0073399-g001: The mitogenic-supportive effect of Et-1 on PASMC.(A) ET-1 alone has no mitogenic effect, while 5% foetal calf serum (FCS) induced proliferation of PASMC within 3 days (n = 6). (B) When combined with 5% FCS, ET-1 dose-dependently increased cell numbers within 3 days (n = 6). Similar results were obtained after 5 days. (C) ET-receptor blockade by Bosentan and chemical Erk1/2 MAPK inhibition abolished the supportive effect of ET-1 on serum-induced PASMC proliferation (n = 3). (D) ET-1 induced accumulation of S/G2-phase cells is prevented by Bosentan (n = 3). (A–D) Bars represent mean ± SEM. (E) Representative immune-blot of the effect of ET-1 and Bosentan on cell compartment distribution of p27(Kip), similar results were obtained in 3 additional experiments. (F) TGF-ß alone did not induce proliferation, whereas the addition of ET-1 significantly increased PASMC proliferation, which was abolished by the inhibition of Erk1/2 MAPK.

Mentions: ET-1 (0.1-1 μM) did not induce proliferation of growth arrested, serum starved PASMC; while 5% FCS stimulation increased the cell number by ~1.8 fold over 3 days (Figure 1A). When combined, ET-1 dose-dependently further increased the FCS (5%) induced proliferation of PASMC up to 1.43 fold (Figure 1B). Bosentan (100 μM) significantly reduced the ET-1 induced proliferation of PASMCs (Figure 1C). The p38 MAPK inhibitor SB203580 (10 µM) had no significant effect on ET-1 induced PASMC proliferation, while the ERK1/2 inhibitor U0126 significantly reduced both ET-1 and 5% FCS-induced proliferation (Figure 1C). To further characterize the effect of ET-1 on proliferation control, we determined the cell cycle distribution and found that ET-1 reduced the number of cells in the G1-phase in a dose-dependent manner, while it increased the number of cells in G2-phase from 39.2% to 48.6%. When pre-incubated with Bosentan, the percentage of cells in the G2-phase significantly decreased to 33.5% (Figure 1D). The inhibitory effect of Bosentan on ET-1 induced proliferation was paralleled by increased nuclear accumulation of the cell cycle inhibitor p27(Kip1) (Figure 1E) while p21(Waf1/Cip1) was not affected (data not shown). TGF-ß (0, 0.5, 1 and 5 ng/ml) had no effect on proliferation (data only shown for 5 ng/ml, Figure 1F). In contrast, the addition of ET-1 (10 nM) significantly induced proliferation, while the ERK1/2 inhibitor U0126 significantly reduced the combined effect of ET-1 and TGF-ß-induced proliferation (Figure 1F).


The interaction of endothelin-1 and TGF-β1 mediates vascular cell remodeling.

Lambers C, Roth M, Zhong J, Campregher C, Binder P, Burian B, Petkov V, Block LH - PLoS ONE (2013)

The mitogenic-supportive effect of Et-1 on PASMC.(A) ET-1 alone has no mitogenic effect, while 5% foetal calf serum (FCS) induced proliferation of PASMC within 3 days (n = 6). (B) When combined with 5% FCS, ET-1 dose-dependently increased cell numbers within 3 days (n = 6). Similar results were obtained after 5 days. (C) ET-receptor blockade by Bosentan and chemical Erk1/2 MAPK inhibition abolished the supportive effect of ET-1 on serum-induced PASMC proliferation (n = 3). (D) ET-1 induced accumulation of S/G2-phase cells is prevented by Bosentan (n = 3). (A–D) Bars represent mean ± SEM. (E) Representative immune-blot of the effect of ET-1 and Bosentan on cell compartment distribution of p27(Kip), similar results were obtained in 3 additional experiments. (F) TGF-ß alone did not induce proliferation, whereas the addition of ET-1 significantly increased PASMC proliferation, which was abolished by the inhibition of Erk1/2 MAPK.
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pone-0073399-g001: The mitogenic-supportive effect of Et-1 on PASMC.(A) ET-1 alone has no mitogenic effect, while 5% foetal calf serum (FCS) induced proliferation of PASMC within 3 days (n = 6). (B) When combined with 5% FCS, ET-1 dose-dependently increased cell numbers within 3 days (n = 6). Similar results were obtained after 5 days. (C) ET-receptor blockade by Bosentan and chemical Erk1/2 MAPK inhibition abolished the supportive effect of ET-1 on serum-induced PASMC proliferation (n = 3). (D) ET-1 induced accumulation of S/G2-phase cells is prevented by Bosentan (n = 3). (A–D) Bars represent mean ± SEM. (E) Representative immune-blot of the effect of ET-1 and Bosentan on cell compartment distribution of p27(Kip), similar results were obtained in 3 additional experiments. (F) TGF-ß alone did not induce proliferation, whereas the addition of ET-1 significantly increased PASMC proliferation, which was abolished by the inhibition of Erk1/2 MAPK.
Mentions: ET-1 (0.1-1 μM) did not induce proliferation of growth arrested, serum starved PASMC; while 5% FCS stimulation increased the cell number by ~1.8 fold over 3 days (Figure 1A). When combined, ET-1 dose-dependently further increased the FCS (5%) induced proliferation of PASMC up to 1.43 fold (Figure 1B). Bosentan (100 μM) significantly reduced the ET-1 induced proliferation of PASMCs (Figure 1C). The p38 MAPK inhibitor SB203580 (10 µM) had no significant effect on ET-1 induced PASMC proliferation, while the ERK1/2 inhibitor U0126 significantly reduced both ET-1 and 5% FCS-induced proliferation (Figure 1C). To further characterize the effect of ET-1 on proliferation control, we determined the cell cycle distribution and found that ET-1 reduced the number of cells in the G1-phase in a dose-dependent manner, while it increased the number of cells in G2-phase from 39.2% to 48.6%. When pre-incubated with Bosentan, the percentage of cells in the G2-phase significantly decreased to 33.5% (Figure 1D). The inhibitory effect of Bosentan on ET-1 induced proliferation was paralleled by increased nuclear accumulation of the cell cycle inhibitor p27(Kip1) (Figure 1E) while p21(Waf1/Cip1) was not affected (data not shown). TGF-ß (0, 0.5, 1 and 5 ng/ml) had no effect on proliferation (data only shown for 5 ng/ml, Figure 1F). In contrast, the addition of ET-1 (10 nM) significantly induced proliferation, while the ERK1/2 inhibitor U0126 significantly reduced the combined effect of ET-1 and TGF-ß-induced proliferation (Figure 1F).

Bottom Line: This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)).Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria.

ABSTRACT

Background: Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective: To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods: PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results: ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and clinical relevance: ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

Show MeSH
Related in: MedlinePlus