Mibefradil suppresses the proliferation of pulmonary artery smooth muscle cells
- Hong-Hong Li,
- Li-Jian Xie,
- Ting-Ting Xiao,
- Min Huang,
- Jie Shen
- Department of Cardiology, Shanghai Children's Hospital, Shanghai Jiaotong University, Shanghai, People's Republic of China
- Correspondence to Jie Shen, Department of Cardiology, Shanghai Children's Hospital, Shanghai Jiaotong University, 355 Luding Road, Shanghai 200062, People's Republic of China; she6nt{at}163.com
Abstract
Intracellular Ca2+ levels play a critical role in the regulation of vasodilation and vasoconstriction by stimulating pulmonary artery smooth muscle cell (PASMC) proliferation, which is important in the pathogenesis of pulmonary arterial hypertension (PAH); however, L-type Ca2+ channel antagonists are useful in only few patients with PAH. The present study sought to assess the effect of mibefradil, which blocks T-type Ca2+ channels, on PASMC proliferation and Ca2+ channel profile. Human PASMCs were stimulated with 25 ng/mL platelet-derived growth factor-BB (PDGF-BB) with and without 10 µM mibefradil or 100 nM sildenafil. After 48 or 72 h, PASMC proliferation and Ca2+ channel expression were assessed by MTT assays and western blot analysis, respectively. PDGF-BB-induced PASMC proliferation at 72 h (p<0.01), which was inhibited by both sildenafil and mibefradil (p<0.01). Transient receptor potential Ca2+ channel 6 (TRPC6) expression was significantly increased with PDGF-BB stimulation (p=0.009); however, no changes in TRPC1, TRPC3, CAV1.2, and CAV3.2 levels were observed. Although both TRPC1 and CAV1.2 expression levels were increased in PDGF-stimulated PASMCs on mibefradil and sildenafil treatment, it was not statistically significant (p=0.086 and 1.000, respectively). Mibefradil inhibits PDGF-BB-stimulated PASMC proliferation; however, the mechanism through which it functions remains to be determined. Further studies are required to elucidate the full therapeutic value of mibefradil for PAH.
- Ca2+ channel
- mibefradil
- proliferation
- pulmonary arterial hypertension
- vasoconstriction
Introduction
Pulmonary arterial hypertension (PAH) is an incurable, progressive disease characterized by shortness of breath, dizziness, fainting, and heart failure.1 In the pathogenesis of PAH, vasoconstriction and inflammatory damage are followed by vascular remodeling of the pulmonary vessel wall, and thrombosis, resulting in fibrosis of blood vessels connected to and within the lungs.2 Although the molecular mechanism underlying PAH remains unclear, the process of pulmonary vascular remodeling in PAH involves all layers of the vessel wall, as evidenced by medial hypertrophy, intimal proliferation and adventitial thickening.3 Proliferation of pulmonary artery smooth muscle cells (PASMCs) is also a key step in the pathogenesis of PAH. These changes culminate in severe vasoconstriction and smooth muscle hypertrophy characteristic of PAH.4
Recent studies have provided compelling evidence that peptide growth factors, including platelet-derived growth factor (PDGF), are important contributors to pulmonary vascular smooth muscle cell proliferation and vascular remodeling.5 In addition, intracellular Ca2+ levels play a critical role in the regulation of vasodilation6 by stimulating PASMC proliferation,7 ,8 and abnormal Ca2+ channel expression on PASMCs may play a critical pathogenesis role in PAH development as increased transient receptor potential Ca2+ channels (TRPC) 1, 3, and 6 levels were observed in PASMCs from patients with PAH.9 ,10
Although no randomized controlled trial has been performed to demonstrate the beneficial effects of Ca2+ channel blockers in the treatment of patients with PAH, uncontrolled studies have suggested that long-term administration of high-dose Ca2+ channel antagonists dramatically improves survival in a small subset of patients who respond acutely to those drugs.11–13 However, most of the currently used Ca2+ antagonists block L-type Ca2+ channels and, therefore, may not provide efficient therapeutic effects due to the altered Ca2+ channel profile in PAH. Thus, studies have evaluated the effects of Ca2+ channel blockers that target other Ca2+ channel types. For example, sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, increased cyclic GMP and inhibited TRPC6 activity and expression in cardiomyocytes,14 and its administration with fasudil, a Rho kinase inhibitor, increased survival in an animal model of PAH.15
Because the effects of mibefradil, a T-type Ca2+ channel inhibitor,16 have not been well-characterized in PASMCs, the objective of this study was to determine if mibefradil can (1) downregulate activated PASMC proliferation and (2) alter the Ca2+ channel profile in PASMCs. Given the altered Ca2+ channel profile in the PASMCs of patients with PAH compared to healthy control (ie, increased T-type Ca2+ channels and TRPC1, 3, and 6),9 ,10 drugs, such as mibefradil, that block Ca2+ channels other than the L-type channels, may have therapeutic value.
Materials and methods
PASMC culture and stimulation with PDGF-BB
Human PASMCs were purchased from ScienCell Research Laboratories (Carlsbad, California, USA) and were cultured on poly-l-lysine-coated culture dishes in Smooth Muscle Cell Medium (Vascular Cell Basal Medium plus Vascular Smooth Muscle Cell Growth Kit containing the following components: recombinant human (rh) FGFb, rh insulin, ascorbic acid, l-glutamine, rh EGF, and FBS; penicillin (10 units/mL)–streptomycin (10 μg/mL)–amphotericin B (25 ng/mL) solution (all, ATCC)) at a density of 5000 cells/cm2 following the manufacturer's instructions.
PASMCs were activated with PDGF-BB in the presence and absence of mibefradil or sildenafil as previously described.17 ,18 Briefly, 5000 cells were cultured in 96-well culture dishes in triplicate for 48 and 72 h, after which they were stimulated with 25 ng/mL PDGF-BB (Sigma-Aldrich, St Louis, Missouri, USA) with and without 10 µM mibefradil (Sigma-Aldrich) or 100 nM sildenafil (Pfizer, Sandwich, Kent, UK), which is known to inhibit PASMC proliferation19 for 48 or 72 h.
Cell proliferation analysis
After 48 or 72 h post-PDGF-BB activation, PASMC proliferation was determined using the MTT assay, as previously described.17 In brief, the plates were washed, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added. After an incubation period of 2 h at 37°C, 5% CO2, acidified isopropanol was added to dissolve the precipitated formazan. Absorbance was determined by a spectrophotometer at 550 nm.
Western blot analysis
After treatment of the PDGF-BB-activated cells with mibefradil or sildenafil for either 10 min (for analysis of the signaling proteins) or 72 h (for analysis of the Ca2+ channels), the cells were lysed in a RIPA buffer (Beyotime Biotechnology, Jiangsu, China), as previously described.17 Protein concentration was determined by Bradford assay, and 50 mg of each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Temecula, California, USA). After the membranes were blocked in 5% BSA for 2 h, they were incubated in the following primary antibodies: p-ERK1/2 (Abcam, Cambridge, Massachusetts, USA), total ERK1/2 (Life Technologies, Grand Island, New York, USA), p-AKT (Cell Signaling, Danvers, Massachusetts, USA), pan AKT (Life technologies), TRPC1 (Santa Cruz Biotechnology, Santa Cruz, California, USA), TRPC3 (Alomone Labs, Jerusalem, Israel), TRPC6 (Alomone Labs), L-type Cav1.2 (Alomone Labs), T-type Cav3.2 (Antibodies-online, Atlanta, Georgia, USA), and β-actin (Abcam), overnight, at 4°C. The membranes were next incubated in the appropriate HRP-labeled secondary antibodies (MultiSciences) for 1 h at room temperature, and signals were detected using Supersignal (Pierce Biotechnology, Rockford, Illinois USA) following the manufacturer's instructions.
Statistical analysis
Data were expressed as means±SD for each condition (n=3 per condition). Differences among conditions were compared using one-way analysis of variance (ANOVA) with Bonferroni post hoc pair-wise comparisons. For the cell proliferation analysis, data were presented as means±SD, and two-sample t tests were used to compare the values between the 48 and 72 h time points for each given condition. The statistical assessments were two-tailed, and p values <0.05 were considered significant. For cell viability, an adjusted significance level of p=0.01 was applied, as was done for the multiple comparisons. All statistical analyses were carried out with IBM SPSS statistical software V.22 for Windows (IBM Corp., New York, New York, USA).
Results
Mibefradil treatment reduced PASMC proliferation
To evaluate the effect of mibefradil on PASMC proliferation, cells were stimulated with PDGF-BB with or without the Ca2+ channel blockers, mibefradil and sildenafil, for 48 h and 72 h, after which proliferation was determined by the MTT assay. As shown in figure 1, the addition of PDGF-BB significantly induced cell proliferation at 72 h (p<0.01), which was significantly inhibited by sildenafil at both 48 and 72 h (p<0.01). Similarly, mibefradil inhibited PDGF-induced PASMC proliferation without obvious cytotoxicity at 72 h (p<0.01; figure 1). Moreover, two-sample t tests for each condition revealed that there was no significant difference between the 48 and 72 h time points.
Mibefradil treatment has no obvious effect on mitogen-activated protein kinase activation in PASMCs
Because PDGF activates mitogen-activated protein kinases, such as ERK and Akt,5 we next examined whether their activation was inhibited by mibefradil, given its antiproliferative effect on PASMCs. As shown in figure 2, although the phosphorylation of Akt and ERK obviously increased after 10 min of PDGF-BB stimulation, it did not reach statistical significance (p=0.881 and 0.060, respectively). Furthermore, although one-way ANOVA revealed that P-Akt/total Akt and P-ERK/total ERK levels were significantly different among groups (p=0.023 for P-Akt/total Akt; p=0.009 for P-ERK/total ERK), their activation was not altered by mibefradil or sildenafil (all p>0.05).
Mibefradil increases TRPC1 expression
PASMCs express many Ca2+ channel isotypes, such as L-type, T-type, receptor-operated Ca2+ channels (ROC), store operated Ca2+ channels (SOC), and TRPCs, and their relative distribution was altered in PAH.9 ,10 To examine whether the inhibitory effect on PASMC proliferation by mibefradil and sildenafil treatment is mediated through altering Ca2+ channel expression levels, candidate Ca2+ channel protein expression was evaluated via western blotting. As shown in figure 3C, TRPC6 expression was significantly increased in PASMCs on PDGF stimulation (p=0.039) as compared to the untreated control cells; however, no changes in TRPC1, TRPC3, CAV1.2, and CAV3.2 levels in response to PDGF were noted (figure 3A,B,D,E, respectively). In addition, TRPC1 (figure 3A) and CAV1.2 (figure 3D) expression levels were increased in PDGF-stimulated PASMCs on mibefradil, but not at levels that reached statistical significance (p=0.086 and 0.283, respectively). Furthermore, no differences in gene expression were observed between mibefradil or sildenafil.
Discussion
Of patients with PAH treated with Ca2+ channel blockers, only approximately 5–6% are considered long-term responders.13 Most of the currently used antagonists used for PAH block L-type Ca2+ channels and, therefore, may not provide efficient therapeutic effects due to the altered Ca2+ channel profile in patients with PAH.9 ,10 Thus, we evaluated the effects of mibefradil, a T-type Ca2+ channel inhibitor,16 on PDGF-BB-stimulated PASMC proliferation. At 72 h, both sildenafil and mibefradil inhibited PDGF-BB-induced PASMC proliferation. However, these effects were not mediated by Akt or ERK signaling or by significantly altering Ca2+ channel expression.
Previous studies have shown that sildenafil can repress PASMC proliferation induced by hypoxia,19 and improve the histological appearance of the lung tissue and blood vessels in an in vivo model of PAH.20 Inhibition of PASMC proliferation by mibefradil has also been reported.21 Similarly, in the present study, both sildenafil and mibefradil suppressed PDGF-BB-induced PASMC proliferation.
In the present study, neither sildenafil nor mibefradil altered Akt or ERK phosphorylation in PASMCs stimulated with PDGF-BB. This is in contrast to a report by Kiss et al,20 in which sildenafil reduced ERK and p38 activation, and enhanced Akt phosphorylation in a monocrotaline-induced rat PAH model. It is possible that these inconsistencies are due to the different models employed. To fully examine whether both sildenafil and mibefradil work in an Akt/ERK-independent manner to reduce PASMC proliferation, additional time points need to be assessed. Additional studies will also evaluate the involvement of the Stat3/c-jun signaling pathway as it is activated in PDGF-induced PASMC proliferation.8
Previous studies have shown that the Ca2+ channel profile is altered in the PASMCs of patients with PAH compared to healthy control patients; increased TRPC1, 3 and 6 as well as T-type Ca2+ channels were observed in the PASMCs from patients with PAH.9 ,10 ,22 However, only increased TRPC6 expression was noted in PASMCs stimulated with PDGF, which is similar to that reported previously, in which PDGF-mediated PASMC proliferation was mediated by upregulation of TRPC6 expression.8 Similarly, TRPC6 levels correlated with Ki67 expression in normal human PASMCs, suggesting that it is associated with proliferation of this cell type.22 However, no effects of sildenafil or mibefradil on Ca2+ channel expression were noted in the present study. This is inconsistent with a study by Wang et al,19 in which sildenafil attenuated hypoxia-induced upregulation of TRPC1 expression. It remains possible that the inconsistencies were due to differences in the PAH model. While mibefradil may have no effect on Ca2+ channel expression by PASMCs, its selective inhibition of T-type Ca2+ currents may still have a role, as suggested in a study by Rodman et al;18 this will, therefore, be explored in further studies.
The present study is limited by the in vitro nature of the model used. Thus, the effects of mibefradil on PAH have to be examined with in vivo studies, especially given the involvement of other cell types in its pathogenesis. Indeed, inhibition of T-type voltage-gated Ca2+ channels in endothelial cells by mibefradil has been observed.23 In addition, the mechanism through which mibefradil suppresses PDGF-induced PASMC proliferation remains unknown. Thus, further studies will assess the effects of mibefradil on the Stat3/c-jun signaling pathway, which is activated in PDGF-stimulated PASMCs.8 Also, further studies will compare the effects of mibefradil with those of imatinib mesylate, a PDGF receptor inhibitor that increases pulmonary haemodynamics in patients with PAH.5
Conclusions
Mibefradil inhibits PDGF-BB-stimulated PASMC proliferation; however, this effect was not mediated by ERK or Akt signaling or by altered Ca2+ channel expression. Thus, the mechanism through which mibefradil suppresses PASMC proliferation remains to be determined. In addition, further studies are required to elucidate the full therapeutic value of mibefradil for PAH.
Acknowledgments
This paper was supported by ‘the Key Project of Science and Technology Commission of Shanghai’ Study on mechanisms and therapies of severe pulmonary hypertension associated with congenital heart diseases (NO.12411952400,124111952403).
Footnotes
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.