Inhibition of Human Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Sabiporide, a New Specific NHE-1 Inhibitor : Journal of Cardiovascular Pharmacology

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Original Article

Inhibition of Human Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Sabiporide, a New Specific NHE-1 Inhibitor

Wu, Dongmei MD, PhD*; Doods, Henri PhD†; Stassen, Jean Marie PhD†

Author Information

From the *Department of Research, Mount Sinai Medical Center, Miami Beach, FL; and †Boehringer Ingelheim Pharma KG, Biberach, Germany.

Received for publication April 18, 2006; accepted June 30, 2006.

Reprints: Dongmei Wu, MD, PhD, Department of Research, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140 (E-mail: [email protected]).

Journal of Cardiovascular Pharmacology 48(2):p 34-40, August 2006. | DOI: 10.1097/01.fjc.0000239691.69346.6a
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Abstract

Abnormal growth of vascular smooth muscle cells is seen in various pathological conditions such as hypertension, atherosclerosis, and restenosis. Na+/H+ exchanger (NHE) activation appears to play a permissive role in vascular smooth muscle cell proliferation and vascular remodeling. The present study investigated the effect of a new specific NHE-1 inhibitor, sabiporide, on human pulmonary artery smooth muscle cell proliferation and migration. Concentrations of sabiporide as low as 20 μmol/L in the culture medium containing growth factors inhibited cell proliferation, as measured by cell counting, and also inhibited the rate of DNA synthesis, as examined by measuring BrdU incorporation into DNA. Cell growth inhibition was not caused by cell death, as demonstrated by the measurement of intracellular lactate dehydrogenase release and by the reversibility of inhibition upon washing. By fluorescent-activated cell sorting analysis, we are the first to demonstrate that NHE-1 inhibition arrests the cell cycle progression at G0/G1 phase, suggesting that NHE activation plays a permissive role in entrance of cells into the cell cycle. Sabiporide also concentration-dependently inhibited human pulmonary artery smooth muscle cell migration. The present study showed that sabiporide inhibits vascular smooth muscle cell proliferation and migration by blocking the cell cycle progression at G0/G1 phase.

Abnormal growth of vascular smooth muscle cells (SMC) is seen in various pathological conditions such as essential hypertension, atherosclerosis, restenosis after coronary angioplasty, and diabetes.1-4 Many extracellular and intracellular factors are involved in the modulation of vascular SMC growth.5,6 Stimulation of Na+/H+ exchanger (NHE) plays a permissive role in optimal growth.7,8 The NHE is an electroneutral transporter that mediates the exchange of extracellular sodium for intracellular hydrogen ion. The only ubiquitously expressed isoform, NHE-1, is the predominant type in vascular SMCs and is thought to be an important regulator of intracellular pH (pHi).9 Increase in NHE-1 activity can contribute to increase in pHi and provide either a permissive or an obligatory signal for cell proliferation and differentiation.10,11 Increased NHE-1 activity is associated with pathological conditions, such as neoplastic transformation,12,13 tumor invasion,14 and essential hypertension.15,16 The NHE is activated by all growth factors as one of the earliest events associated with entrance of cell into the cell cycle.7 In addition, NHE appears to be important for cell migration. It has been shown that NHE is activated when polymorphonuclear white blood cells migrate and that NHE inhibitors block chemotaxis.17 NHE inhibitors such as HOE 694 and EIPA have been shown to inhibit vascular SMC growth and prevent neointima formation after percutaneous transluminal coronary angioplasty.18,19 Sabiporide {(N-(Aminoiminomethyl)-4-[4-(1H-pyrrol-2-ylcarbonyl)-1-piperazinyl]-3-(trifluormethyl)-benzamid) was synthesized by Boehringer Ingelheim Pharma KG, Ingelheim, Germany} is a new specific NHE-1 inhibitor possessing remarkable cardioprotective properties.20 Inhibition of initial rates of 22Na+ uptake shows that sabiporide is a potent NHE-1 inhibitor (Ki of 5 ± 1.2 × 10-8 mol/L), and discriminates efficiently between the NHE-1,2,3 isoforms (Ki for NHE-2: 3 ± 0.9 × 10−6 mol/L, and Ki > 1 mmol/L for NHE-3). Sabiporide greatly reduced infarct size against myocardial ischemia-reperfusion injury.20 Sabiporide also significantly improved left ventricular function, and attenuated myocardial apoptosis and fibrosis in rabbits with pacing-induced heart failure.21,22 In the present study, we investigated the effects of sabiporide on human pulmonary vascular SMC proliferation and migration.

MATERIALS AND METHODS

Cell Culture

Human primary pulmonary artery smooth muscle cells were purchased from Clonetics Human Cell Systems (Heidelberg, Germany). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2/95% air. The growth medium was SmBM3 (smooth muscle cell basal medium-3) supplemented with 10% fetal bovine serum, 10 pg/mL human recombinant epidermal growth factor, 2 pg/mL human recombinant fibroblast growth factor, 0.8 μg/mL dexamethasone, 50 pg/mL gentamicin, and 50 pg/mL amphotericin-B. Medium was changed twice per week and cell passages 4 to 8 were used for all experiments.

Growth Inhibition Assay

Human pulmonary artery SMCs were seeded into 6-well cluster plates (2 × 105 cells/well) in growth medium without or with sabiporide (20, 30, 40 μmol/L). Medium was changed on days 1, 3, and 5. Cell growth was determined by using CASY Cell Analysis System (Schaerfe System GmbH, Reutlingen, Germany) on days 1, 3, and 5. Triplicate plates were used for each concentration. The same experiment was repeated twice.

Determination of DNA Synthesis

DNA synthesis in SMC culture was determined by BrdU assay (Boehringer Mannheim, Indianapolis, IN). Human pulmonary artery SMCs were plated into 96-well microtiter plates (104 cells/well) in growth medium. After 24 h, medium was replaced with growth medium without or with sabiporide (20, 30, 40, 50, 60 μmol/L). After 3 d of incubation, BrdU was added and the measurement was performed according to the manufacturer's instructions. BrdU incorporation into DNA was determined by a standard enzyme-linked immunosorbent assay reader. Triplicate wells were used for each concentration. The same experiment was repeated twice.

Flow Cytometry Fluorescence-activated Cell Sorting (FACS) Analysis

Human pulmonary artery SMCs were seeded at a density of 4000 cells/cm2 in 75-cm2 culture flasks. After 48 h of incubation, medium was replaced with growth medium in the absence or presence of sabiporide (60 μmol/L). After a further 48 h of incubation, cells were collected, washed with phosphate-buffered saline containing RNase, and fixed in 70% ethanol. Following staining of DNA with PI buffer (50 μg/mL propidium iodide, 10 mmol/L Tris, pH 7.5, 5 mmol/L MgCl2, 200 μg/mL RNase A) for 30 min at 37°C, the cell-cycle distribution of each sample was determined by FACS analysis using BD Bioscience software (Becton Dickinson, Heidelberg, Germany). For another set of experiments, human pulmonary artery SMCs were seeded at a density of 4000 cells/cm2 in 75-cm2 culture flasks. After 48 h, cell growth was arrested by incubation for 48 h in serum-free SmBM3. The quiescent cells were stimulated by the growth medium and then incubated for a further 24 h in the absence or presence of sabiporide (60 μmol/L). The cell-cycle distribution of each sample was then determined as described above. The same experiment was repeated twice.

Caspase 3 Activity Assay

Human pulmonary artery SMCs were grown in 75-cm2 culture flasks in growth medium. When cells were 90% confluent, medium was replaced with growth medium in the absence or presence of sabiporide (20, 40, 60, 80 μmol/L). After 3 d of incubation, cells were collected and cellular lysate was prepared. The caspase 3 activity was measured according to the manufacturer's instructions (Boehringer Mannheim).

Lactate Dehydrogenase (LDH) Assay

LDH assay (Boehringer Mannheim) was used to determine the cytotoxicity of sabiporide on human pulmonary artery SMCs. A cytotoxicity detection kit was used to measure the LDH activity released from the cytosol of damaged cells into the medium. Human pulmonary artery SMCs were plated into 96-well microtiter plates (104 cells/well) in growth medium. After 4 d, the medium was replaced with SmBM3 containing 1% bovine serum albumin in the absence or presence of sabiporide (20, 40, 60, 80 μmol/L). After 24 h of incubation, aliquots of the medium from each well were removed for LDH assay. LDH activity in the medium and the cell lysate was determined according to manufacturer's instructions. Triplicate wells were used for each concentration. The same experiment was repeated twice.

Reversibility of Cell Growth Inhibition

The reversibility of cell growth by sabiporide was also determined. Human pulmonary artery SMCs were seeded into 6-well cluster plates (2 × 105 cells/well) in growth medium in the absence or presence of sabiporide (40 μmol/L). The compound was removed by washing, and the cells were further incubated in growth medium for up to 6 d. Cell proliferation in control culture and in cultures treated with sabiporide for 6, 24, and 48 h or throughout the incubation period was determined each day for 6 d. Triplicate wells were used for each concentration. The same experiment was repeated twice.

Cell Migration Assay

Human pulmonary artery SMCs were seeded into 6-cm plates. When the culture was confluent, cells were removed from half of each plate by scraping with a surgical blade. Cultures were washed twice with phosphate-buffered saline and incubated with growth medium in the absence or presence of sabiporide (20, 40, 60 μmol/L). The cell migration was observed and photographed over a 3-d period. Triplicate wells were used for each concentration. The same experiment was repeated twice. Migrated cells were fixed and stained with May-Grunwald giemsa and quantitated using a scanning densitometer.

Statistical Analysis

Data are presented as mean ± SEM for the number of experiments indicated. Analysis of variance and Duncan's multiple-range test were used to determine significant differences (P < 0.05) between the control value and the various experimental conditions.

RESULTS

Effect of Sabiporide on Human Pulmonary Artery SMC Proliferation

Sabiporide (20-40 μmol/L) inhibited the proliferation of human pulmonary artery SMC in a concentration-dependent manner (Fig. 1A). At concentrations of 30 and 40 μmol/L, sabiporide significantly inhibited the growth of the human pulmonary artery SMCs from day 3. After 5 d in culture, the number of cells present was ≈22% of control at a sabiporide concentration of 40 μmol/L.

F1-6
FIGURE 1:
A, Inhibition of human pulmonary artery SMC proliferation by sabiporide. Human pulmonary artery SMCs were seeded into 6-well cluster plates (2 × 105 cells/well) in growth medium without or with sabiporide (20, 30, 40 μmol/L). Cell growth was determined on days 1, 3, and 5. Each point indicates the mean ± SEM, n = 3, *P < 0.05 vs control. B, Effect of sabiporide on DNA synthesis in human pulmonary artery SMCs. Human pulmonary artery SMCs were plated into 96-well microtiter plates (104 cells/well) in growth medium. After 24 h, medium was replaced with growth medium without or with sabiporide (20, 30, 40, 50, 60 μmol/L). After 3 d of incubation, BrdU incorporation into DNA was determined.

Effect of Sabiporide on DNA Synthesis in Human Pulmonary Artery SMCs

The effect of sabiporide on cell growth was also determined by DNA synthesis. Sabiporide produced concentration-dependent inhibition of DNA synthesis (Fig. 1B). Fifty percent inhibition of DNA synthesis (IC50) was achieved at 42 μmol/L.

Cell-cycle Arrest at G0/G1

A slight increase in the cell population at the G0/G1 phase of the cell cycle (73.8%-80.2%) was observed after the addition of sabiporide (60 μmol/L) to growing-phase cells (Fig. 2A). In the quiescent cultures (nonstimulated cells), the populations in G0/G1, S, and G2/M were 86.7%, 3.2%, and 10.1%, respectively, of the total population. By contrast, in cells stimulated for 24 h by replacement of growth medium, the corresponding values were 69.2%, 24.9%, and 5.8%. At 60 μmol/L, sabiporide blocked the G0/G1-to-S transition completely. The corresponding values for sabiporide-treated cells (60 μmol/L) were 88.6%, 3.7%, and 7.7% (Fig. 2B). Sabiporide completely arrested the cell cycle progression at G0/G1 phase.

F2-6
FIGURE 2:
A, Effect of sabiporide on cell-cycle distribution in growing-phase cells. Human pulmonary artery SMCs were seeded at a density of 4000 cells/cm2 in 75-cm2 culture flasks. After 48 h, medium was replaced with growth medium in the absence or presence of sabiporide (60 μmol/L). Cells were then incubated for a further 48 h. The cell-cycle distribution of each sample was determined by means of flow cytometer. Each figure indicates the result of representative chart. B, Effect of sabiporide on the cell-cycle progression in quiescent cells stimulated by growth medium. Growing-phase cells were arrested by incubation for 48 h in serum-free SmBM3. The quiescent cells were stimulated by the growth medium and then incubated for a further 24 h in the absence or presence of sabiporide (60 μmol/L). The cell-cycle distribution of each sample was then determined by means of flow cytometer. Each figure indicates the result of representative chart from 3 separate experiments. Data are mean ± SEM, n = 3, *P < 0.05 vs control.

Effect of Sabiporide on Viability of Human Pulmonary Artery SMCs

To investigate whether the inhibition of DNA synthesis in vascular SMCs resulted from the inhibition of cell proliferation or from a reduction in cell number caused by cytotoxicity, we determined the release of intracellular LDH in the culture medium. Figure 3A shows that the amount of LDH release from the cells treated with varying concentrations of sabiporide was similar to that of control culture, which suggests that drug treatment under this condition was not associated with an excessive cell death. Figure 3B shows the caspase 3 activity in the cells treated with varying concentrations of sabiporide. At a concentration that produces >90% inhibition of DNA synthesis, sabiporide did not significantly increase caspase 3 activity.

F3-6
FIGURE 3:
A, Effect of sabiporide on cell viability. Confluent cultures of human pulmonary artery SMCs were treated with the indicated concentrations of sabiporide for 24 h. LDH release in the culture medium was determined. Data are mean ± SEM, n = 3. B, Effect of sabiporide on caspase 3 activity. Confluent cultures of human pulmonary artery SMCs were treated with the indicated concentrations of sabiporide for 3 d. The caspase 3 activity was measured. Data are mean ± SEM, n = 3.

The effect of sabiporide on human pulmonary artery SMC viability was also determined by the reversibility of cell growth inhibition. As shown in Figure 4, a brief exposure of cells to sabiporide (40 μmol/L) was not sufficient to produce growth inhibition. Cell growth was inhibited in cultures treated with sabiporide for 24 and 48 h. In these cultures, cell proliferation was resumed upon removal of the compound by washing.

F4-6
FIGURE 4:
Reversibility of cell growth inhibition by sabiporide. Human pulmonary artery SMCs were incubated with 40-μmol/L sabiporide for 6, 24, and 48 h. The compound was then removed by washing, and the cells were further incubated in growth medium for up to 6 d. Cell proliferation in control cultures and in cultures treated with sabiporide for 6, 24, and 48 h or through the incubation period was determined at the indicated intervals. Data are mean ± SEM, n = 3, *P < 0.05 vs control.

Effect of Sabiporide on Migration of Human Pulmonary Artery SMCs

Confluent, scrape-wounded human pulmonary artery SMC monolayers were incubated with sabiporide (20, 40, 60 μmol/L) and the rate of migration was observed over a 3-d period. As shown in Figures 5A and 5B, all concentrations of sabiporide inhibited migration of human pulmonary artery SMCs into the denuded area, with complete inhibition at 60 μmol/L.

F5-6
FIGURE 5:
Effect of sabiporide on cell migration. Human pulmonary artery SMCs were seeded into 6-cm plates. When the cultures were confluent, cells were removed from half of each plate by scraping with a surgical blade. Cultures were washed and then incubated with growth medium in the absence or presence of sabiporide (20, 40, 60 μmol/L). The cell migration was observed and photographed over a 3-d period. A, Data are mean ± SEM, n = 3, *P < 0.05 vs control. B, Each figure indicates the result of representative chart from 3 separate experiments.

DISCUSSION

The present study has shown that a new cardioprotective NHE-1 inhibitor, sabiporide, inhibits human pulmonary artery SMC proliferation and migration. Sabiporide inhibits DNA synthesis and prevents the quiescent cells to enter the cell cycle. Enhanced NHE activity of erythrocytes, leukocytes, platelets, and fibroblasts has been found in both essential hypertension and type 1 diabetes mellitus.23 NHE activity is elevated in both cultured vascular SMCs24 and vessels from genetically hypertensive rats.25 Both hyperplastic and hypertrophic agents, including growth factors7 and vasoactive agents26,27 stimulate NHE, leading to intracellular alkalinization. Compelling evidence that links increased pHi to vascular SMC growth comes from the observation that DNA synthesis in vascular SMCs can be induced by cellular alkalinization in the absence of mitogens.28 In addition, in mutant mouse lung fibroblasts that lack NHEs, growth factors fail to cause cell proliferation unless the cell pHi is artificially raised to >7.2,29 thereby adding further evidence that activation of the cell membrane NHE is a necessary step in cell proliferation and growth.

In the present study, NHE-1 inhibition by sabiporide inhibited human pulmonary artery SMC proliferation and DNA synthesis in the presence of human recombinant epidermal growth factor and human recombinant fibroblast growth factor. We have shown that the inhibition of cell proliferation by sabiporide was not caused by cytotoxicity. The amount of intracellular LDH release from the cells treated with varying concentrations of sabiporide was similar to that of control culture, suggesting that drug treatment under these conditions was not associated with excessive cell death. In addition, at a concentration that produces >90% inhibition of DNA synthesis in human pulmonary artery SMCs, sabiporide did not significantly increase caspase 3 activity. Furthermore, we have shown that the inhibition of cell proliferation by sabiporide was reversible. In exponentially growing cultures of human pulmonary artery SMCs, removal of the drug led to the resumption of cell growth.

Vascular cells are at a nonproliferation state under physiological conditions. Abnormal growth of vascular SMCs is seen in various pathological conditions such as essential hypertension, atherosclerosis, restenosis, and diabetes.1-4 NHE activation has been suggested as one of the earliest events associated with entrance of cell into the cell cycle.7 Using FACS analysis, we are the first to confirm that NHE-1 inhibition arrests cell cycle progression at G0/G1, completely blocking the cell to enter the S phase. Results from the present study confirm the hypothesis that NHE activation plays a permissive role in the entrance of cells into the cell cycle.

Vascular SMC migration is one of the processes of vascular hyperplasia. Cell migration has been extensively studied because it is known to occur during tumor metastasis, in-stent stenosis, and restenosis. Although not fully understood, cell migration is known to involve regulated attachment, detachment, contraction of nonmuscle myosin and actin, and cytoskeletal plasticity.30-32 The extracellular signals include physical force33 and soluble regulators such as vasoactive hormones,34,35 polypeptide growth factors,31 and probably Ca2+, Mg2+, pH, and pO2.36 Simchowitz and Cragoe found that the NHE activation is required for migration of white blood cells.17 Investigators have also suggested that changes in intracellular sodium are important in migration, especially in the shape changes that are required for cell motility.37 The present study has shown that sabiporide inhibited human pulmonary artery SMC migration in a concentration-dependent manner, suggesting that NHE-1 activation is involved in human pulmonary artery SMC migration.

In summary, we have shown that a new specific NHE-1 inhibitor, sabiporide, inhibits human pulmonary artery SMC proliferation, migration, and DNA synthesis. NHE-1 inhibition arrests the cell cycle progression at G0/G1 phase, suggesting that NHE activation plays a permissive role in entrance of cell into the cell cycle. Accordingly, NHE-1 inhibitors as cardioprotective drugs under development38 may have therapeutic benefits in diseases related to abnormal growth of vascular SMCs.

ACKNOWLEDGMENT

This work was supported by Boehringer Ingelheim Pharma KG, Biberach, Germany.

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Keywords:

Na+/H+ exchanger; cell proliferation; cell migration; cell cycle regulation

© 2006 Lippincott Williams & Wilkins, Inc.