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Annals of Vascular Diseases logoLink to Annals of Vascular Diseases
. 2010 Sep 10;3(2):117–126. doi: 10.3400/avd.AVDoa01008

T-Type Ca2+ Channel Blockers Increase Smooth Muscle Progenitor Cells and Endothelial Progenitor Cells in Bone Marrow Stromal Cells in Culture by Suppression of Cell Death

Ryota Hashimoto 1,✉, Youichi Katoh 2,✉, Seigo Itoh 3,✉, Takafumi Iesaki 1,✉, Hiroyuki Daida 3,✉, Yuji Nakazato 2,✉, Takao Okada 1,✉
PMCID: PMC3595759  PMID: 23555398

Abstract

Objective: To examine the expression patterns and roles of voltage-dependent Ca2+ channels in bone marrow stromal cells (BMSCs).

Materials and Methods: Ca2+ currents of BMSCs were measured by the whole-cell patch clamp method. The number and percentage of deaths of BMSCs cultured for 14 days with or without Ca2+ channel blockers were evaluated using a MTT assay and an LDH assay, respectively.

Results: T-type Ca2+ channel current was recorded in 0, 2, 10, and 4% of BMSCs on days 3, 10, 17, and 24 in culture, respectively. L-type Ca2+ channel current was first recorded on day 24 in 6% of BMSCs. Addition of the T-type Ca2+ channel blocker mibefradil but not the L-type Ca2+ channel blocker nifedipine significantly increased the cell count. Immunocytochemical analysis revealed increases in the counts of smooth muscle progenitor cells (SMPCs) and endothelial progenitor cells (EPCs). Mibefradil but not nifedipine significantly decreased the rate of cell death.

Conclusion: T-type Ca2+ channel blockers increased the numbers of SMPCs and EPCs in cultured BMSCs, partly through suppression of cell death. Thus, T-type Ca2+ channel blockers may have the potential to provide an increased number of both BMSC-derived SMCs and ECs of potential use in cell and gene therapy.

Keywords: bone marrow stromal cells, endothelial progenitor cells, smooth muscle progenitor cells, T-type Ca2+ channel blocker

Introduction

Bone marrow stromal cells (BMSCs)1) are capable of differentiation into various lineages including osteoblasts, chondroblasts, adipocytes, endothelial cells,2) cardiomyocytes,3) smooth muscle cells,4, 5) and skeletal muscle cells.6) These findings have raised the hope of developing new stem cell strategies for tissue repair and regeneration, particularly since accumulating evidence suggests that BMSCs contribute to vascular healing and remodeling under physiological and pathological conditions. Although there is growing enthusiasm for therapeutic and diagnostic application of BMSCs, there are concerns that transplanted BMSCs may contribute to the pathogenesis of diseases such as cancer, retinopathy, and atherosclerosis. Thus, it is important to determine the mechanisms through which BMSCs proliferate and differentiate.

Voltage-dependent Ca2+ channels in the plasma membrane open in response to changes in cell membrane potential, providing an important pathway for regulated entry of extracellular Ca2+. Voltage-dependent Ca2+ channels are classified as high or low voltage-activated channels and can be subdivided further based on their pharmacological properties. High voltage-activated channels include L-, N-, P-/Q-, and R-type Ca2+ channels, and the low voltage-activated channel is designated as a T-type Ca2+ channel. T-type Ca2+ channels are expressed in many developing tissues and may be important in regulating cellular phenotypic modulations that lead to cell proliferation, differentiation, growth and death. The T-type Ca2+ current is first detectable in BMSCs after 6–7 days in culture and reaches a peak at 8 days, after which the current falls rapidly.7) However, the role of T-type Ca2+ channels in BMSCs has not been established. Therefore, the purpose of this study was to examine the expression pattern and role of T-type Ca2+ channels in BMSCs during cell development and differentiation.

Materials and Methods

Cell culture

Methods for cell culture have been described previously.4,8) Briefly, male mice of the C57Bl/6 strain (Charles River Japan, Kanagawa, Japan) were sacrificed by cervical dislocation under diethyl ether anesthesia. Bone marrow cells were collected from the tibia and femur and cultured in Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS, Equitech-Bio, TX, USA) at 37°C in 5% CO2/95% air. We selectively maintained adherent cells (BMSCs) by removing floating cells during a change in medium. The study conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. The experimental protocol was approved by the Animal Care and Use Committee of Juntendo University.

Electrophysiological measurements

BMSCs were plated on coverslips. Patch pipettes were pulled from plain capillary tubes made of soda lime glass (Chase Scientific Glass, Rockwood, TN, USA); resistances were 2–5 MΩ when the pipettes were filled with solution. Membrane currents were measured using a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA, USA).8,9) Data acquisition and analysis were carried out using pClamp software (Axon Instruments). Current signals were filtered at 1 kHz and sampled at a frequency of 5 kHz. Cells were voltage clamped at a holding potential of −80 mV and currents were evoked by +10 mV increment depolarizing steps of 500 ms up to +80 mV. Ca2+ currents were recorded in Cs Tyrode (CsT) solution, which contained 135 mM NaCl, 5.4 mM CsCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, and 10 mM glucose, with the pH adjusted to 7.4 with NaOH. The pipette solution used for recording Ca2+ currents contained 125 mM CsCl, 1.0 mM MgCl2, 5 mM HEPES, 3 mM Mg-ATP, and 5 mM Cs-BAPTA, with the pH adjusted to 7.3 with CsOH.

Cell number and cell death

BMSCs (100 μl) were seeded on 96-well plates at 5000 cells/well. After one day, various concentrations of Ca2+ channel blockers or solvent were added to the wells. The channel blockers were efonidipine (T-type + L-type, a generous gift from Nissan Chemical Industries, Tokyo, Japan), mibefradil (T-type, Sigma-Aldrich, St. Louis, MO, USA), and nifedipine (L-type, Sigma-Aldrich). At defined times from days 1 to 14, the cell count was estimated using a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay,10) which is a modification of the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay. In brief, 10 μl of WST-8 reagent (Cell Counting Kit, Dojindo Laboratories, Kumamoto, Japan) was added to the wells. After a 4 h-incubation at 37°C, the absorbance was recorded at 450 nm with a microtiter plate reader. On day 14, the percentage cell death was assessed using a lactate dehydrogenase (LDH) assay. In brief, 50 μl of culture medium were transferred to a new 96 well plate, 100 μl of reaction solution (Cytotoxicity Detection KitPLUS, Roche, Mannheim, Germany) was added, and absorption was measured after 1 h at 490 nm with a microtiter plate reader. The percentage cell death was calculated according to the following equation: percentage cell death (%) = (experimental value − background control) / (positive control − background control) × 100. For this assay, a positive control of 100% cytotoxicity caused by lysing the cells completely was included. The positive control was treated with 0.1% Triton X-100 solution 1 hour before the LDH assay.

Immunocytochemistry

BMSCs were plated on coverslips and Ca2+ channel blockers were added to the cells after one day. The cells were then cultured for another 13 days before being fixed in 4% paraformaldehyde for 20 min, washed in PBS, permeabilized in 0.5% Triton X-100 for 5 min, washed in PBS, and blocked with 10% FBS for 1 h. They were then incubated for 2 h with indocarbocyanine (Cy3)-conjugated mouse monoclonal anti-α-smooth muscle actin (α-SMA) antibody (Sigma-Aldrich) diluted with 1% FBS (1/100). The coverslips were washed in PBS and mounted in anti-fading solution (Vector Laboratories, Burlingame, CA, USA) before observation by fluorescent microscopy. The count of α-SMA positive cells [smooth muscle progenitor cells (SMPCs)11)] was analyzed using image J software and expressed as the number per area and the percentage (%) of the total number of BMSCs.

Uptake of acetylated low density lipoprotein (acLDL) and binding of isolectin B4

BMSCs were plated on coverslips and Ca2+ channel blockers were administered to the cells after one day. The cells were cultured for another 13 days and then incubated with 5 μg/ml 1,19-dioctadecyl-3,3,3939-tetramethylindocarbocyanine perchlorate (Dil)-labeled acLDL (DiI-acLDL, Biomedical Technologies, Stoughton, MA, USA) and 2.5 μg/ml fluorescein isothiocyanate (FITC)-isolectin B4 (Vector Laboratories) at 37°C for 4 h. The cells were then washed in PBS before observation by fluorescent microscopy. The count of Dil-acLDL and FITC-isolectin B4 double-positive cells was analyzed using image J software and expressed as the number per area and the percentage (%) of the total number of BMSCs. Adherent cells with an endothelial phenotype defined by uptake of acLDL and binding of lectin are commonly referred to as endothelial progenitor cells (EPCs).12)

Statistical analysis

Data are expressed as means ± standard deviation (SD). Homogeneity of variances and means were confirmed by a Bartlett test and one-way ANOVA, respectively. Significance was evaluated by Tukey test, with differences considered significant at P < 0.05.

Results

BMSCs in culture have T- and L-type Ca2+ channel currents

A whole-cell patch-clamp method was used to detect Ca2+ currents in BMSCs in culture. We defined low and high voltage-activated Ca2+ currents in BMSCs as those blocked by mibefradil and nitrendipine, respectively, indicating that they were T-type and L-type Ca2+ channel currents, respectively (Figs. 1A, B). T-type Ca2+ channel currents were recorded in 0, 2, 10, and 4% of BMSCs on days 3, 10, 17, and 24 in culture, respectively (Figs. 1A, C). An L-type Ca2+ channel current was first recorded on day 24 in 6% of BMSCs (Figs. 1B, C).

Fig. 1.

Fig. 1

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Bone marrow stromal cells in culture have T- and L-type Ca2+ channel currents.

Graphs of (A) low voltage-activated Ca2+ current (LVA) and (B) high voltage-activated Ca2+ current (HVA) in bone marrow stromal cells (BMSCs) in culture. Panel a represent current-voltage (I-V) curves for BMSCs with or without treatment with channel blockers. Cells were voltage clamped at a holding potential of –80 mV and currents were evoked by +10 mV increment depolarizing steps up to +80 mV. Panels b and c show typical traces of ICa elicited by depolarization from –80 mV to (A) –30 mV or (B) +10 mV with or without channel blockers. (C) The percentages of T- and L- type Ca2+ channels in BMSCs in culture. Data are expressed as the mean ± SD of at least 10 independent experiments at each day.

T-type Ca2+ channel blockers increase the number of BMSCs in culture

We investigated the effects of several Ca2+ channel blockers on the number of BMSCs in culture. Efonidipine (10 μM), a T- and L-type Ca2+ channel blocker, significantly increased the number of BMSCs on day 14 (Fig. 2A; 19238 ± 2806 vs. 14219 ± 4784 cells for control treatment). Mibefradil (100 nM, 1 μM), a T-type Ca2+ channel blocker, also significantly increased the cell number on day 14 (Fig. 2B; 17786 ± 7269 (100 nM) and 31070 ± 3624 (1 μM) vs. 14219 ± 4784 cells for control treatment). In contrast, 10 μM mibefradil significantly decreased the number of BMSCs on days 5–14 (Fig. 2B). Nifedipine (10 nM–10 μM), an L-type Ca2+ channel blocker, had little effect on the number of BMSCs on days 5–14 (Fig. 2C). These results show that T-type Ca2+ channel blockers increase the BMSC count in culture.

Fig. 2.

Fig. 2

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T-type Ca2+ channel blockers increase the number of BMSCs in culture.

Growth curves showing the effects of Ca2+ channel blockers (A: efonidipine T-+ L-type; B: mibefradil T-type; C: nifedipine L-type) on the number of BMSCs in culture. Seeded cells (5000 per well) were exposed to different concentrations of each Ca2+ channel blocker after incubation for 1 day. The cell count was evaluated on the indicated day. Data are expressed as the mean ± SD of at least 8 independent experiments. *P < 0.05 **P < 0.01 vs. control.

T-type Ca2+ channel blockers increase SMPCs and EPCs in a BMSC culture

BMSCs included about 3% SMPCs (Fig. 3C) and 80% EPCs (Fig. 4C). To identify the cell types contributing to the changes in the BMSC count, the numbers of SMPCs and EPCs were determined. First, cells were stained with Cy3-conjugated anti-α-SMA antibody on day 14. The number of α-SMA positive cells (SMPCs) significantly increased with mibefradil treatment compared to the control (Fig. 3C; 1182 ± 573 vs. 476 ± 238 cells/mm2), but the ratio of α-SMA-positive cells to BMSCs showed little change (Fig. 3D). The number and ratio of α-SMA-positive cells changed little with nifedipine (Fig. 3C,D). The cells were also treated with Dil-Ac-LDL and FITC-isolectin B4 on day 14. Mibefradil increased the number of double-positive cells compared to the control (Fig. 4C; 25735 ± 5890 vs. 12141 ± 1762 cells/mm2), but had little effect on the ratio of double-positive cells to BMSCs (Fig. 4D). Nifedipine had little effect on the number and ratio of double-positive cells (Figs. 4C, D). These results indicate that T-type Ca2+ channel blockers increase the numbers of SMPCs and EPCs in BMSCs in culture.

Fig. 3.

Fig. 3

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T-type Ca2+ channel blockers increase the number of smooth muscle progenitor cells in BMSCs in culture.

Representative (A) bright field and (B) fluorescence photomicrographs of BMSCs stained with Cy3-conjugated anti-α-SMA antibody. (C) Number of α-SMA-positive cells and (D) the percentage (%) of α-SMA-positive cells relative to total BMSCs. Data are expressed as the mean ± SD of at least 5 independent experiments. Seeded cells were exposed to 1 μM mibefradil or 1 μM nifedipine after incubation for 1 day. After another 13 days the cells were stained with anti-α-SMA antibody. **P < 0.01 vs. control.

Fig. 4.

Fig. 4

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T-type Ca2+ channel blockers increase the number of endothelial progenitor cells in BMSCs in culture.

Representative (A) bright field and (B) fluorescence photomicrographs of BMSCs stained with Dil-Ac-LDL and FITC-isolectin B4. (C) Number of double-positive cells and (D) the percentage (%) of double-positive cells relative to total BMSCs. Data are expressed as the mean ± SD of at least 5 independent experiments. Seeded cells were exposed to 1 μM mibefradil or 1 μM nifedipine after incubation for 1 day. After another 13 days the cells were treated with ac-LDL and isolectin B4. *P < 0.05 vs. control.

T-type Ca2+ channel blockers suppress the cell death of BMSCs in culture

To determine the effects of Ca2+ channel blockers on BMSC survival, the percentage cell death was examined on day 14 using a LDH assay. Cell death decreased significantly with mibefradil treatment and hardly changed with nifedipine (Fig. 5; 7 ± 9%, 11 ± 6%, and 16 ± 9% for mibefradil, nifedipine, and control treatment, respectively). With 10 μM mibefradil, 63 ± 10 % of the cells died (Fig. 5). These results suggest that T-type Ca2+ channel blockers increase the number of BMSCs by suppressing cell death of BMSCs in culture.

Fig. 5.

Fig. 5

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T-type Ca2+ channel blockers suppress the death of BMSCs in culture.

Graph showing the effects of mibefradil or nifedipine on the cell death of BMSCs in culture. Seeded cells were exposed to 1 μM or 10 μM mibefradil or 1 μM nifedipine after 1 day. After another 13 days, LDH activity in the cell culture supernatant was assessed. Data are expressed as the mean ± SD of at least 8 independent experiments. **P < 0.01 vs. control.

Discussion

The results of this study showed functional expression of T- and L-type Ca2+ channels during development and differentiation of BMSCs. Treatment with T-type Ca2+ channel blockers increased the number of BMSCs, with the increase in cell count due to suppression of cell death. Increases in both SMPCs and EPCs were observed. T-type Ca2+ channel blockers have been shown to decrease the counts of endothelial cells,13) smooth muscle cells,14, 15) tumor cells,16) epithelial cells,17) and blood mononuclear cells.18) In sharp contrast, our study showed that treatment with T-type Ca2+ channel blockers increases the number of BMSCs.

Suppression of cell death and stimulation of cell division can cause an increase in cell count. Mibefradil has been shown to inhibit loss of cell viability following acute oxygen radical generation in endothelial cells19) and efonidipine inhibits glomerular cell apoptosis.20) In contrast, pimozide, another T-type Ca2+ channel blocker, and mibefradil have been suggested to have cytotoxic effects in retinoblastoma and MCF7 cells21) and mibefradil has also been found to induce neuronal death.22) Mibefradil also has an antiproliferative effect in smooth muscle cells14, 15) and blood mononuclear cells.18) In the present study, we found that T-type Ca2+ channel blockers suppressed BMSC death, and this is likely to be the mechanism through which these channel blockers increase the number of BMSCs. It has also been shown that 1 μM mibefradil can both increase19) and decrease21, 22) cell viability. We found increased BMSC viability after treatment at 1 μM mibefradil but decreased viability at 10 μM mibefradil. Thus, mibefradil may enhance or reduce cell viability depending on the concentration and cell type. Mibefradil contributes to biophysical interactions with the cell membrane that underlie antioxidant and cytoprotective activities in models of oxidative stress.19) However, the protective mechanism of mibefradil against BMSC death may not depend upon direct cytoprotective effects against oxidative stress, because the L-type Ca2+ channel blocker nifedipine, which is also known to have an antioxidative stress effect,23, 24) did not show a protective effect against BMSC death. Therefore, the protection mechanism of mibefradil may, at least in part, be due to the suppression of T-type Ca2+ channel current. A further study is required to examine this finding in more detail.

Numerous studies have suggested that abundant accumulation of smooth muscle cells in the subendothelial intima plays a principal role in the pathogenesis of occlusive vascular diseases. Control of proliferation and migration of smooth muscle cells is a therapeutic strategy for atherosclerosis. Neointimal hyperplasia may be mediated not only by the migration or proliferation of preexisting vascular smooth muscle cells in the media but also by the homing of bone marrow-derived smooth muscle-like progenitor cells.25) Efonidipine26) and mibefradil15, 27) have been shown to have antiatherosclerotic effects. Mibefradil decreases the count of vascular smooth muscle cells, leading to prevention of neointima formation.15, 27) However, our results clearly showed that T-type Ca2+ channel blockers increase the number of bone marrow-derived SMPCs. This discrepancy may be due to the difference between vessel-derived and bone marrow-derived cells or the concentrations of the T-type Ca2+ channel blockers.

The therapeutic potential of EPCs in treating ischemic vascular disease is promising,28, 29) but limited by the availability of EPCs. Thus, efforts to increase EPC numbers are underway. Our results show that T-type Ca2+ channel blockers increase the EPC count, indicating that these channel blockers may be a novel option for modulation of EPCs in treatment of ischemic vascular diseases. The construction of stable blood vessels consisting mainly of endothelial cells and smooth muscle cells is a fundamental challenge for tissue engineering in regenerative medicine. We8) and others30) have reported that bone marrow-derived smooth muscle cells have physiological properties similar to those of native smooth muscle cells. Furthermore, Jin et al.30) have constructed functional tissue-engineered blood vessels from bone marrow progenitor cells including SMPCs and EPCs. Our results show that T-type Ca2+ channel blockers increase the numbers of SMPCs and EPCs, which suggests that these channel blockers may offer a new approach to obtaining large numbers of cells as a source for construction of tissue-engineered vessels.

Conclusion

Functional expression of T- and L-type Ca2+ channels was observed during development and differentiation of BMSCs. T-type Ca2+ channel blockers increased the numbers of SMPCs and EPCs in the BMSCs, at least in part by suppression of cell death. Our results suggest that use of T-type Ca2+ channel blockers may allow large numbers of cells to be obtained as a source for construction of tissue-engineered vessels.

Acknowledgements

This work was supported by a Japan Heart Foundation Young Investigator's Research Grant.

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