Pro-apoptotic effects of imatinib on PDGF-stimulated pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

doi:10.1016/j.ijcard.2011.02.024

International Journal of Cardiology

Volume 159, Issue 2, 23 August 2012, Pages 100-106

https://doi.org/10.1016/j.ijcard.2011.02.024 Get rights and content

Abstract

Background

Remodeling of the pulmonary artery by an inappropriate increase of pulmonary artery smooth muscle cells (PASMCs) is problematic in the treatment of idiopathic pulmonary arterial hypertension (IPAH). Effective treatment that achieves reverse remodeling is required. The aim of this study was to assess the pro-apoptotic effects of imatinib, a platelet-derived growth factor (PDGF)-receptor tyrosine kinase inhibitor, on PASMCs obtained from patients with IPAH.

Methods

PASMCs were obtained from 8 patients with IPAH undergoing lung transplantation. Cellular proliferation was assessed by ³H-thymidine incorporation. Pro-apoptotic effects of imatinib were examined using TUNEL and caspase-3,7 assays and using transmission electron microscopy.

Results

Treatment with imatinib (0.1 to 10 μg/mL) significantly inhibited PDGF-BB (10 ng/mL)-induced proliferation of PASMCs from IPAH patients. Imatinib (1 μg/mL) did not induce apoptosis in quiescent IPAH-PASMCs, but it had a pro-apoptotic effect on IPAH-PASMCs stimulated with PDGF-BB. Imatinib did not induce apoptosis in normal control PASMCs with or without PDGF-BB stimulation. PDGF-BB induced phosphorylation of Akt at 15 min, and Akt phosphorylation was inhibited by imatinib in IPAH-PASMCs. Akt-I-1/2 (1 μmol/L), an Akt inhibitor, in the presence of PDGF-BB significantly increased apoptotic cells compared with the control condition. Thus, Akt-I-1/2 could mimic the effects of imatinib on PASMCs.

Conclusion

Imatinib has anti-proliferative and pro-apoptotic effects on IPAH-PASMCs stimulated with PDGF. The inhibitory effect of imatinib on Akt phosphorylation induced by PDGF plays an important role in the pro-apoptotic effect.

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease characterized by progressive elevation of pulmonary vascular resistance and pulmonary artery pressure. Increased pulmonary vascular resistance is induced by pulmonary vasoconstriction, vascular remodeling by intimal and medial hypertrophy, and thrombosis [1], [2]. Pulmonary vascular medial hypertrophy is caused by an inappropriate increase in pulmonary artery smooth muscle cells (PASMCs). Treatment with several vasodilators such as calcium channel blockers, prostaglandin I₂ and endothelin receptor antagonists was found to improve survival of patients with IPAH, but 5-year survival remains at 50% [3], [4]. Effective treatment that achieves reverse remodeling is needed. This will require anti-proliferative and pro-apoptotic agents for PASMCs.

We have reported that platelet-derived growth factor (PDGF)-BB stimulation causes a higher growth rate of cultured PASMCs from patients with IPAH than that of control cells [5], [6], [7]. Recently, the use of a PDGF-receptor inhibitor such as imatinib (STI571) is starting to garner attention as a targeted therapy for pulmonary hypertension (PH) [8], [9], [10], [11]. Imatinib is a drug used to treat certain types of cancer such as chronic myelogenous leukemia and gastrointestinal stromal tumors. In laboratory settings, imatinib is used as an experimental agent to suppress PDGF by inhibiting PDGF receptor ß (PDGF-Rß). It is an agent that acts by specifically inhibiting a certain enzyme, tyrosine kinase, that is characteristic of a particular cancer cell, rather than non-specifically inhibiting the proliferation of and killing all rapidly dividing cells. Schermuly et al. reported that imatinib reverses pulmonary vascular remodeling and cor pulmonale in rats with monocrotaline-induced PH and in mice with chronic hypoxia-induced PH [8]. Perros et al. reported that PDGF-BB-induced proliferation and migration of PASMCs from patients with IPAH were inhibited by imatinib [10].

Not only inhibition of proliferation but also induction of apoptosis of PASMCs is needed to actively reduce stenosis due to vascular remodeling at small pulmonary arteries of patients with IPAH. These two effects may lead to reverse remodeling of the pulmonary vasculature. Expression of PDGF-B is up-regulated in the medial layer of small pulmonary arteries of rats with monocrotaline-induced PH and imatinib induces apoptosis in the small pulmonary arteries [8]. However, imatinib does not induce apoptosis in cultured IPAH-PASMCs without PDGF treatment [10]. Thus, imatinib may not be able to induce apoptosis in quiescent cells. We hypothesized that imatinib in the presence of PDGF-BB induces apoptosis of PASMCs from patients with IPAH, but that imatinib cannot induce apoptosis in PASMCs without PDGF stimulation. We therefore investigated whether imatinib in the presence and absence of PDGF-BB induces apoptosis of PASMCs from patients with IPAH.

Akt is a member of the serine/threonine-specific kinase family known for facilitating cell survival via the inhibition of apoptotic pathways [12]. Therefore, induction of apoptosis of IPAH-PASMCs may be related to Akt inactivation. We also investigated whether imatinib inhibits Akt activation.

Section snippets

Isolation, culture and identification of PASMCs

Peripheral segments of the pulmonary artery were obtained at lung transplantation [13] from 8 patients with IPAH as previously described [5], [6], [14], [15] (2 males and 6 females; mean age, 22 ± 4 years; age range 11–43 years) (Table 1). For normal control experiments, samples of pulmonary arteries were also obtained at lung lobectomy from a patient with bronchogenic carcinoma (male, 58 years old) who showed no evidence of PAH and received no systemic chemotherapy or radiation therapy before lung

Inhibitory effect of imatinib on proliferation of PASMCs from IPAH patients

Treatment with imatinib inhibited PDGF-BB-induced proliferation of PASMCs from IPAH patients as assessed by ³H-thymidine incorporation (n = 5–12 experiments in each cell) (Fig. 1). This result is consistent with recent findings of other investigators [10].

Effect of imatinib on apoptosis of PASMCs from IPAH patients

We performed a TUNEL assay using an ApopTag fluorescein to assess the effect of imatinib on apoptosis of PASMCs from IPAH patients. Fig. 2 shows representative cases of the TUNEL assay. TUNEL-positive cell (green) was observed after 24-hour

Discussion

Two major new findings were obtained in the present study. First, imatinib did not induce apoptosis in quiescent IPAH-PASMCs and normal PASMCs, but it had a pro-apoptotic effect on IPAH-PASMCs stimulated with PDGF. Second, inhibition of Akt is related to the anti-proliferative and pro-apoptotic effects of imatinib on PDGF-stimulated IPAH-PASMCs.

Imatinib alone did not induce apoptosis in IPAH-PASMCs. This result is consistent with recent findings of other investigators [10]. However, the

Conflict of interest

There are no relationships with industry.

Acknowledgments

The authors thank Kaoru Akazawa, Masayo Ohmori, and Miyuki Fujiwara for their excellent technical assistance. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [23].

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The results showed that mPAP, RVSP and RVHI were significantly increased in MCT group, WA% and WT% were increased, and cell proliferation rate was also significantly increased in PDGF-BB group, which strongly indicated that the relevant PAH model and cell proliferation model were successfully established. Recently, in vitro and in vivo experiments have shown that 18β-GA is a pentacyclic triterpenoid derivative of the b-amyrin type produced from the hydrolysis of glycyrrhizic acid [33,34], confer beneficial effects on cell proliferation, which arousing our interest in the protective effect of 18β-GA on PAH. In the present study, hemodynamics and histological analysis showed that abnormal increases in mPAP, RVSP and RVHI were inhibited in the 18β-GA group, suggesting that 18β-GA can reduce mPAP, RVSP and RVHI in PAH rats.
Excessive proliferation, migration and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the basis for the development of pulmonary vascular remodeling, and it is the driving force for pulmonary arterial hypertension (PAH). 18β-glycyrrhetinic acid (18β-GA) is the main active substance extracted from Chinese herbal medicine licorice, with outstanding anti-inflammatory, anti-oxidation and anti-proliferative effects. Our team found in previous studies that 18β-GA has protective effects on monocrotaline-induced PAH in rats. However, the anti-angiogenic effect of 18β-GA on PAH remains unclear. Therefore, in order to further investigate whether the beneficial effects of 18β-GA on PAH are related to its antiproliferative effect, we conducted experiments in vivo and in vitro.
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^☆: Dr. Nakamura was supported by the Research Grant for Cardiovascular Diseases (19–9) from the Ministry of Health, Labour and Welfare, Japan.

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Pro-apoptotic effects of imatinib on PDGF-stimulated pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension☆

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Isolation, culture and identification of PASMCs

Inhibitory effect of imatinib on proliferation of PASMCs from IPAH patients

Effect of imatinib on apoptosis of PASMCs from IPAH patients

Discussion

Conflict of interest

Acknowledgments

J Am Coll Cardiol

J Am Coll Cardiol

Int J Cardiol

Biochem Biophys Res Commun

Int J Cardiol

Lancet

Int J Cardiol

Primary pulmonary hypertension: a vascular biology and translational research "Work in progress"

Circulation

Three-dimensional structure of pulmonary capillary vessels in patients with pulmonary hypertension

Circulation

PDGF signaling in pulmonary arterial hypertension

J Clin Invest

Prednisolone inhibits proliferation of cultured pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension

Circulation