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Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension.

Sahara M, Sata M, Morita T, Hirata Y, Nagai R - PLoS ONE (2012)

Bottom Line: Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH.These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME.Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan. saharam-tky@umin.ac.jp

ABSTRACT

Background: An antianginal K(ATP) channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.

Materials and methods: Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg(-1)·day(-1)) alone; or nicorandil as well as either a K(ATP) channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).

Results: Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.

Conclusions: Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.

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Nicorandil prevents the induction of vascular endothelial cell apoptosis in vitro.(A) The HUVECs were incubated in the serum-free medium in the absence or presence of nicorandil (10–1000 µmol/L) for 48 h. The serum-starved HUVECs exhibited apoptotic morphology, which is characterized by cell shrinkage (see “serum starved”). (B) The viability of the HUVECs was measured with the MTS assay, and the percent cell death was calculated. Stimulation with nicorandil and diazoxide partially restored cell viability in a concentration-dependent manner. VEGF, vascular endothelial growth factor (positive control). *P<0.05 and **P<0.01 vs. control (no drug). (C) TUNEL staining revealed that a large number of the serum-starved HUVECs exhibited apoptotic morphology (left), and the apoptotic effects induced by serum starvation were attenuated by nicorandil (middle). TUNEL (green); nuclei (blue). (D) The TUNEL-positive nuclei in the serum-starved HUVECs were counted in 10 randomly selected fields and expressed as a percentage of the total number of nuclei. **P<0.01 vs. control (no drug).
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pone-0033367-g007: Nicorandil prevents the induction of vascular endothelial cell apoptosis in vitro.(A) The HUVECs were incubated in the serum-free medium in the absence or presence of nicorandil (10–1000 µmol/L) for 48 h. The serum-starved HUVECs exhibited apoptotic morphology, which is characterized by cell shrinkage (see “serum starved”). (B) The viability of the HUVECs was measured with the MTS assay, and the percent cell death was calculated. Stimulation with nicorandil and diazoxide partially restored cell viability in a concentration-dependent manner. VEGF, vascular endothelial growth factor (positive control). *P<0.05 and **P<0.01 vs. control (no drug). (C) TUNEL staining revealed that a large number of the serum-starved HUVECs exhibited apoptotic morphology (left), and the apoptotic effects induced by serum starvation were attenuated by nicorandil (middle). TUNEL (green); nuclei (blue). (D) The TUNEL-positive nuclei in the serum-starved HUVECs were counted in 10 randomly selected fields and expressed as a percentage of the total number of nuclei. **P<0.01 vs. control (no drug).

Mentions: Next, we examined the in vitro anti-apoptotic effects of nicorandil for vascular endothelial cells. HUVECs were cultured in serum-free medium and then exhibited apoptotic morphology that was characterized by cell shrinkage (Figure 7A), and there was a decrease in the viability of these cells as determined by the MTS assay (Figure 7B). In addition, there was an increase in the number of serum-starved HUVECs that exhibited apoptotic morphology as identified by TUNEL staining (Figure 7C and 7D). Stimulation with nicorandil and the KATP channel opener diazoxide partially inhibited the serum starvation-induced endothelial cell apoptosis in a concentration-dependent manner, while these effects of nicorandil were also inhibited by glibenclamide and l-NAME (Figure 7A–D).


Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension.

Sahara M, Sata M, Morita T, Hirata Y, Nagai R - PLoS ONE (2012)

Nicorandil prevents the induction of vascular endothelial cell apoptosis in vitro.(A) The HUVECs were incubated in the serum-free medium in the absence or presence of nicorandil (10–1000 µmol/L) for 48 h. The serum-starved HUVECs exhibited apoptotic morphology, which is characterized by cell shrinkage (see “serum starved”). (B) The viability of the HUVECs was measured with the MTS assay, and the percent cell death was calculated. Stimulation with nicorandil and diazoxide partially restored cell viability in a concentration-dependent manner. VEGF, vascular endothelial growth factor (positive control). *P<0.05 and **P<0.01 vs. control (no drug). (C) TUNEL staining revealed that a large number of the serum-starved HUVECs exhibited apoptotic morphology (left), and the apoptotic effects induced by serum starvation were attenuated by nicorandil (middle). TUNEL (green); nuclei (blue). (D) The TUNEL-positive nuclei in the serum-starved HUVECs were counted in 10 randomly selected fields and expressed as a percentage of the total number of nuclei. **P<0.01 vs. control (no drug).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316574&req=5

pone-0033367-g007: Nicorandil prevents the induction of vascular endothelial cell apoptosis in vitro.(A) The HUVECs were incubated in the serum-free medium in the absence or presence of nicorandil (10–1000 µmol/L) for 48 h. The serum-starved HUVECs exhibited apoptotic morphology, which is characterized by cell shrinkage (see “serum starved”). (B) The viability of the HUVECs was measured with the MTS assay, and the percent cell death was calculated. Stimulation with nicorandil and diazoxide partially restored cell viability in a concentration-dependent manner. VEGF, vascular endothelial growth factor (positive control). *P<0.05 and **P<0.01 vs. control (no drug). (C) TUNEL staining revealed that a large number of the serum-starved HUVECs exhibited apoptotic morphology (left), and the apoptotic effects induced by serum starvation were attenuated by nicorandil (middle). TUNEL (green); nuclei (blue). (D) The TUNEL-positive nuclei in the serum-starved HUVECs were counted in 10 randomly selected fields and expressed as a percentage of the total number of nuclei. **P<0.01 vs. control (no drug).
Mentions: Next, we examined the in vitro anti-apoptotic effects of nicorandil for vascular endothelial cells. HUVECs were cultured in serum-free medium and then exhibited apoptotic morphology that was characterized by cell shrinkage (Figure 7A), and there was a decrease in the viability of these cells as determined by the MTS assay (Figure 7B). In addition, there was an increase in the number of serum-starved HUVECs that exhibited apoptotic morphology as identified by TUNEL staining (Figure 7C and 7D). Stimulation with nicorandil and the KATP channel opener diazoxide partially inhibited the serum starvation-induced endothelial cell apoptosis in a concentration-dependent manner, while these effects of nicorandil were also inhibited by glibenclamide and l-NAME (Figure 7A–D).

Bottom Line: Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH.These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME.Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan. saharam-tky@umin.ac.jp

ABSTRACT

Background: An antianginal K(ATP) channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.

Materials and methods: Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg(-1)·day(-1)) alone; or nicorandil as well as either a K(ATP) channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).

Results: Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.

Conclusions: Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.

Show MeSH
Related in: MedlinePlus