Pflügers Archiv - European Journal of Physiology

, Volume 465, Issue 2, pp 221–232

Involvement of large conductance Ca2+-activated K+ channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation

Authors

  • Xiaoling Jia
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Jingyun Yang
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Wei Song
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Ping Li
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Xia Wang
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Changdong Guan
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Liu Yang
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Yan Huang
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Xianghui Gong
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Meili Liu
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
  • Lisha Zheng
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
    • Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang University
Ion Channels, Receptors and Transporters

DOI: 10.1007/s00424-012-1182-z

Cite this article as:
Jia, X., Yang, J., Song, W. et al. Pflugers Arch - Eur J Physiol (2013) 465: 221. doi:10.1007/s00424-012-1182-z

Abstract

The large conductance Ca2+-activated K+ (BKCa) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BKCa channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BKCa channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BKCa protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BKCa in SS-induced inhibition of proliferation was examined by BKCa inhibition using a BKCa specific blocker, iberiotoxin (IBTX), and by BKCa transfection in BKCa non-expressing CHO cells. The changes in [Ca2+]i were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC4(3). We found that laminar SS inhibited VSMC proliferation and stimulated BKCa channel expression. Furthermore, laminar SS induced an increase in [Ca2+]i and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BKCa channel activity on cell proliferation in response to SS was also confirmed in BKCa-transfected CHO cells showing a decline in proliferation. Blocking BKCa channel reversed its inhibitory effect, providing additional support for the involvement of BKCa in SS-induced proliferation reduction. Our results suggest, for the first time, that BKCa channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Keywords

Laminar shear stressBKCa channelVSMCProliferation

Introduction

Vascular smooth muscle cells (VSMCs) found in the tunica media in blood vessels exhibit two distinct phenotypes — contractile and proliferative. In response to abnormal environmental cues, contractile VSMC revert to a proliferative state, and then proliferate excessively and migrate into the intima of blood vessels, and further lead to many cardiovascular diseases [28]. Studies have shown that alteration in local hemodynamic forces is an important factor related to cardiovascular diseases [32, 33].

Fluid shear stress (SS) mainly affects vascular endothelial cells (EC), but VSMCs can also be exposed to SS following iatrogenic endothelial denudation [33]. Since Kraiss and co-workers [16] showed that SS affected VSMC proliferation in vascular prosthetic grafts, the majority of studies have focused on further exploring the impacts of SS on VSMC proliferation and investigating the underlying mechanism [10, 12, 19, 27, 38]. So far, it has been demonstrated that laminar subnormal SS (≤5 dyn/cm2) or oscillatory SS promotes VSMC proliferation [12, 27], while normal laminar SS has an inhibitory effect [10, 38]. The mechanisms underlying that promotion or inhibition have been mainly focused on the activation of a complex cascade of kinases and transcription factors [10, 12, 19, 27, 38]. To date, few reports have focused on the direct contribution of K+ channels to VSMC proliferation in response to SS, although K+ channels have been accentuated as mechanosensors [17, 19] or cell proliferation modulators [14].

K+ channels, through altering membrane potential and intracellular [Ca2+]i, impact multiple processes in VSMC, such as phenotype switching, migration, and proliferation [14]. Beside voltage-dependent K+ (Kv) channels, the BKCa channels are also ubiquitously expressed in VSMCs, and comprise of four α-subunits and accessory β-subunits [5, 11]. The pore-forming α-subunit is encoded by a single gene, KCNMA1, whereas the tissue-specific β-subunit is encoded by several genes. Only the β1-subunit (KCNMB1) has been identified in smooth muscle, where it performs an important vasoregulatory function [6, 24, 42, 43]. In the vasculature, BKCa channel operates by limiting Ca2+ entry and mediating membrane hyperpolarization [24]. Recently numerous reports have demonstrated that abnormalities in BKCa channel expression or activity are closely related to cardiovascular diseases, such as hypertension, aging, diabetes and others [3, 20, 23, 30]. Thereupon, some vasoactive factors, such as exercise training or special diet, are targeted to activate BKCa channel [2, 8]. Moreover, some intriguing reports have shown that the BKCa channel is involved in cell proliferation [34], and the activator of BKCa channel can inhibit cancer cell migration [15].

Considering that both SS and BKCa, also an SS-sensing channel [6], are related to cell proliferation, we hypothesized that BKCa may play a role in SS-induced alteration of VSMC proliferation. To investigate this possibility, we exposed rat aortic VSMC in a parallel-plate chamber to SS at 0, 6, 12 and 24 dyn/cm2 for 6, 12 and 24 h, respectively. The changes in VSMC proliferation and BKCa channel protein and gene expression were investigated. The influence of BKCa blockade and BKCa transfection on cell proliferation in response to SS was confirmed. To further explore the possible cause or result of BKCa activation, the changes in intracellular [Ca2+]i and membrane potential were also determined. Our data suggested that BKCa channel mediates laminar SS-induced inhibition of VSMC proliferation, which may be partly due to membrane hyperpolarization induced by BKCa activation in the presence of SS.

Methods

Isolation and culture of VSMC

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996), and the protocol was approved by the Animal Research Committee of Peking University Health Science Center.

Male Sprague–Dawley rats (200–250 g) were anesthetized with pentobarbital sodium 100 mg/kg (i.p.) and sacrificed by cervical dislocation. Thoracic aorta VSMCs were isolated and cultured by the methods described in the Supplementary material online, Methods. VSMC of purity more than 90 % were characterized by immunofluorescence staining for smooth muscle cell-specific α-actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) (see Fig. S1). Cells between passages 3 and 5 were used for experiments.

CHO cell transfection

CHO cells (from the ATCC) were transiently cotransfected with human BKCa channel α-subunit and β1-subunit cDNA in a mammalian expression vector (pTarget), a kind gift from Dr. Hucheng Zhao (Institute of Biomechanics and Medical Engineering, Tsinghua University, China). Transfections were performed using DNA-LipofectamineTM 2000 (Invitrogen) according to manufacturer`s instructions, and fresh medium was changed after 6 h. For another 48 h of culture, BKCa channel expression was determined using the methods described below.

Shear application

VSMCs or CHO cells were seeded on glass slides and grown to 80 % confluence. Then the cell monolayer was exposed to laminar SS of 6 dyn/cm2 (SS-6), 12 dyn/cm2 (SS-12) and 24 dyn/cm2 (SS-24) for 6, 12, and 24 h, respectively, in a parallel-plate flow chamber described in the Supplementary material online (Methods) and Fig. S3. CHO cells transfected with BKCa were subjected to laminar SS of 12 dyn/cm2.

In BKCa blockade cases, VSMCs were incubated with 100 nM iberiotoxin (IBTX) at 37 °C for 1 h prior to shear exposure or static culture.

Flow cytometry for protein expression determination

The protein expression of proliferating cell nuclear antigen (PCNA) or the two subunits of the BKCa channel were determined by flow cytometry as described in the Supplementary material online, Methods. Cells were incubated with an irrelevant IgG1 isotype antibody instead of a specific-binding primary antibody as a negative control (NC).

Flow cytometry for cell cycle analysis

VSMCs were synchronized with serum-free medium for 24 h. Then the synchronized cells were exposed to SS or static culture in media containing 10 % FBS. After 12 or 24 h, the cells were harvested. Cells (1 × 106) were fixed with 70 % ethanol overnight. After the cells were digested with RNase for 30 min at 37 °C, the DNA was stained with propidium iodide (PI) and analyzed with FACSCalibur (Becton Dickinson) on FL-2 channel. The results were analyzed using the ModFit software.

RT-PCR

Total RNA was isolated using a Trizol RNA-prep kit (Invitrogen) according to manufacturer's instructions. The method for RT-PCR is further described in the Supplementary material online, Methods.

Immunofluorescent microscopy

VSMC identification, confirmation of BKCa transfection in CHO cells, as well as measurements of PCNA expression in the cases of BKCa blockade or BKCa transfection were performed by immunofluorescent microscopy and further quantified by ImageJ software described in the Supplementary material online, Methods.

Intracellular [Ca2+]i analysis

VSMCs were stained with 8 μM fluo 3-AM following manufacturer's instructions (Dojindo, Japan) for 30 min at 37 °C. After that, cells were exposed to laminar SS of 12 dyn/cm2. The change of intracellular [Ca2+]i within 4 min of shear was analyzed by Ca2+ imaging as previously reported [13, 18]. Briefly, the Fluo 3-AM-loaded cell-seeded slide was mounted on a parallel-plate chamber, placed on an inverted fluorescence microscope (Olympus IMT-2), and left it undisturbed for 30 min. After that, the slide was exposed to laminar SS of 12 dyn/cm2. Images were collected for 1 min in the absence of shear, to establish a steady baseline, and for another 4 min following the onset of shear at 3-s intervals. The fluorescence intensity of each cell was analyzed by ImageJ software. The baseline was defined as the mean fluorescence value for each cell observed for 1 min in the absence of shear. The change in [Ca2+]i for each cell was presented as a ratio normalized to the base line.

The change of intracellular [Ca2+]i beyond 4 min of shear was determined by flow cytometry as previously reported [41]. After shear exposure for 5, 10, 20 and 30 min, the fluo 3-AM-loaded cells were harvested and analyzed by a flow cytometer (FACSCalibur, BD) on FL-1 channel. Mean fluorescence intensity was analyzed by CellQuest software.

Membrane potential determination

Membrane potential was measured with anionic oxonal dye, DiBAC4(3), which serves as an indicator of membrane potential [39]. Briefly, VSMCs treated with or without IBTX were incubated with 200 nM DiBAC4(3) (Sigma-Aldrich) for 30 min at 37 °C. Subsequently, cells were subjected to 12 dyn/cm2 of SS. For short and long duration, cells were harvested at 5 min and 1 h, respectively and incubated with 50 μg/ml PI. After that, fluorescence determinations were made with a flow cytometer (FACSCalibur, BD). PI fluorescence was measured (on FL-2 channel) to exclude non-viable cells, and DiBAC4(3) fluorescence intensity was determined by gating on viable cells on the FL-1 channel. Mean fluorescence intensity was analyzed by Cellquest software.

Statistical analysis

Each test was performed at least in triplicates, and all values are expressed as mean ± SD. Data comparison between static controls and shear exposure cases was made by one-way ANOVA analysis. A P value of <0.05 was considered significant.

Results

Laminar SS decreases PCNA expression and induces a G0/G1 cell cycle arrest

The expression of “proliferation marker” PCNA was measured to determine the effects of SS on cell proliferation. As shown in Fig. 1a and b, SS significantly decreased PCNA expression compared to static controls. At 6 h, the mean fluorescence intensities of PCNA were 0.9-, 0.6- and 0.6-fold lower for three shear cases than that of control (Fig. 1a, left panel). The expression of PCNA continued to decline at 12. The corresponding values were 0.6-, 0.3- and 0.3-fold lower for three shear cases than that of control (Fig. 1a, middle panel). As SS duration was prolonged to 24 h, PCNA expression still maintained a declining trend. Mean fluorescence intensities were 0.7-, 0.5- and 0.3-fold lower for three shear cases than that of control (Fig. 1a, right panel). Furthermore, this decline did not exhibit a regular duration or magnitude dependence.
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Fig. 1

The effects of laminar SS on the PCNA expression and cell cycle distribution of VSMCs. a Representative flow cytometry graphs for PCNA of VSMCs subjected to laminar SS with described magnitude and duration. NC negative control, SS shear stress, SC static control, SS-6 6 dyn/cm2 shear stress, SS-12 12 dyn/cm2 shear stress, SS-24 24 dyn/cm2 shear stress. b The value of PCNA fluorescence (minus negative control) of VSMCs. c Representative flow cytometry graphs for cell cycle distribution of VSMCs exposed to the described laminar SS for 12 and 24 h. d Mean values of cell distribution percentages in each phase for shear and static cases. *P < 0.05 and **P < 0.001, shear case vs. static case (n = 4–6)

The decrease in PCNA expression indicated that SS suppressed VSMC proliferation. To further confirm that, cell cycle was analyzed. In three shear cases, the cell number in S phase was significantly reduced while the number of cells in G0/G1 phase was increased (Fig. 1c and d). At 12 h, the percentages of cells in G0/G1 were about 1-fold higher for three shear cases than that of control. This increase was accompanied by a decrease in S phase percentage. The percentages in S phase were 2.1- to 3.4-fold less for three shear cases than that of control. The same trend was observed at 24 h of SS. The percentages of cells in S phase were 2.4- to 4.3-fold less for three shear cases than that of control. At the same time, the cell percentage in G0/G1 phase in the three corresponding shear cases was significantly increased compared with control. The percentages in G0/G1 phase were about 1.3-fold higher for three shear cases than that of control. At both 12 and 24 h, all the P values for control-shear pairs were less than 0.0001 (n = 3–5). These results further supported the contention that laminar SS restrained VSMC proliferation.

Laminar SS increases protein and mRNA expression of BKCa channel

BKCa channels are predominantly expressed in contractile rather than proliferative VSMC [5]. Since SS has an inhibitory effect on VSMC proliferation as described above, we speculated that laminar SS may be up-regulating BKCa expression. Hence, the protein expression of BKCa channel was detected by flow cytometry. Interestingly, the expression of both α- and β1-subunits was altered dramatically due to shear exposure, but each subunit responded to SS in a different manner. There was an increase in α-subunit expression which exhibited an inverse correlation with shear duration (left panel of Fig. 2a and left panel of Fig. 2b). At 6 h, α-subunit protein expression was enhanced by 1.6- to 1.9-fold for the three described shear cases, respectively. Subsequently this enhancement decreased to 1.3- to 1.5-fold at 12 h. Ultimately, up-regulation ceased and there was no significant difference between the shear cases and the control. In contrast, SS up-regulated β1-subunit protein in a time-dependent manner as shown in the right panels of Fig. 2a and b. At 6 h, the expression of β1-subunit in the three shear cases was increased by 2.2- to 2.4-fold compared with control. At 12 h, the increase was sustained, and the β1-subunit expression levels were 4.3- to 4.8-fold higher than that of control. Elevated β1-subunit expression was still maintained at 24 h. The levels were 4.7- to 10.2-fold compared to that of control.
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Fig. 2

The effects of laminar SS on the protein and mRNA expression of the two subunits of BKCa channel in VSMCs. a Representative flow cytometry graphs for α-subunit (left panel) and β1-subunit (right panel) of BKCa channel in VSMCs subjected to laminar SS with described magnitude and duration. b The value of α- and β1-subunit protein expression (normalized to static control). c Representative agarose electrophoretic gel for α- and β1-subunit mRNA of BKCa channel in VSMCs. d Mean values of α- and β1-subunit mRNA expression of BKCa channel in VSMCs (normalized to GAPDH). *P < 0.05 and **P < 0.001 vs. control (n = 4–6)

To further confirm these changes in gene expression levels, RT-PCR was performed. As shown in Fig. 2c and d, SS enhanced α-subunit transcription similarly to its effect on protein level. At 6 h, α-subunit mRNA levels were significantly elevated for the three described shear cases (0.97 ± 0.14, 0.98 ± 0.17 and 1.07 ± 0.24, respectively) compared with control (0.33 ± 0.09). Subsequently, the enhancement became weaker at 12 h and dropped to 0.86 ± 0.13, 0.87 ± 0.09 and 0.82 ± 0.04 for the three shear cases, respectively. This decline was even more pronounced at 24 h of SS. At this time point the α-subunit expression level under SS of 6 dyn/cm2 (0.32 ± 0.05) was almost equal to that in the control (0.31 ± 0.03, P = 0.68, n = 6). In the cases where SS was applied at 12 and 24 dyn/cm2 (0.50 ± 0.07 and 0.51 ± 0.02) the expression was still significantly higher than that of control (P ≤ 0.001, n = 6). In contrast, β1-subunit gene expression appeared significantly elevated in all shear cases (right panel of Fig. 2c and bottom panel of Fig. 2d). At 6 h, the β1-subunit mRNA reached levels 2.2- to 2.8-fold higher in the three shear cases compared to that of static control (P ≤ 0.005, n = 6). The values were 0.22 ± 0.06, 0.49 ± 0.09, 0.73 ± 0.17 and 0.62 ± 0.05 for static control and three shear cases, respectively. At 12 h the increase became 3.2- to 3.6-fold (P ≤ 0.005, n = 4), and the β1-subunit mRNA levels were 0.18 ± 0.08, 0.65 ± 0.06, 0.59 ± 0.03 and 0.65 ± 0.08 for static control and the three shear cases, respectively. This elevation was still maintained at 24 h and was 2.2- to 3.0-fold higher than the value for static control (P ≤ 0.005, n = 6).The β1-subunit mRNA levels were 0.21 ± 0.04, 0.48 ± 0.04, 0.65 ± 0.12 and 0.63 ± 0.09 for static control and the three described cases. These results were consistent with protein expression.

Blockade of BKCa channel counteracts SS-induced PCNA decrease and promotes cell cycle progression

Having shown that SS up-regulates the mRNA levels and the protein expression of BKCa channel while suppressing VSMC proliferation, we proceeded to investigate a possible correlation between the two phenomena. To test this possibility, BKCa channel was blocked by IBTX. Then, PCNA expression was compared between the cases with and without blockade. As shown in Fig. 3a and Fig. S4a, PCNA expression was markedly increased after IBTX treatment in shear cases, with a P value of less than 0.001 (n = 9). In contrast, there was no significant difference between untreated (−IBTX) and treated (+IBTX) pairs in static culture (P > 0.5, n = 9) (see Fig. 3a and Fig. S4b). This result suggested that BKCa channel was involved in the laminar SS-induced inhibition of VSMC growth. Since IBTX treatment had no impact on BKCa channel expression (see Fig. S5), the inhibitory functions of BKCa on VSMC proliferation should be SS-induced.
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Fig. 3

The effects of BKCa blockade on the PCNA expression and cell cycle distribution in VSMCs. a Blockade of BKCa channel increases the PCNA fluorescence intensity of VSMCs in shear cases, but has no effect in static cases. **P < 0.001, +IBTX vs. −IBTX (n = 9–17). b BKCa blockade promotes cell cycle progress of VSMCs in all shear cases

To further evaluate the contribution of BKCa channel to SS-induced inhibition of cell proliferation, we set on to determine the effect of BKCa blockade on cell cycle progression. As illustrated in Fig. 3b, the percentages of cells in S phase were remarkably increased in all shear cases following BKCa channel blockade at both 12 and 24 h. At 12 h, the cell percentages in S phase in non-treated and treated cases are listed below: 4.41 ± 0.77 vs. 13.25 ± 0.23 for 6 dyn/cm2, 5.41 ± 0.32 vs. 11.41 ± 1.27 for 12 dyn/cm2 and 4.74 ± 1.46 vs. 11.45 ± 0.94 for 24 dyn/cm2, respectively (see left panel of Fig. 3b and Fig. S4c). This increase in the percentage of S phase cells was retained at 24 h. The corresponding values in non-treated and treated cases are shown below: 16.71 ± 0.46 vs. 20.24 ± 0.79 for 6 dyn/cm2, 15.36 ± 0.48 vs. 20.24 ± 0.79 for 12 dyn/cm2 and 10.19 ± 0.95 vs. 16.08 ± 0.64 for 24 dyn/cm2, respectively (see right panel of Fig. 3b and Fig. S4d). The P value for each pair was less than 0.001 (n = 4–6). Parallel to the increase in S phase, there was a decrease in the G0/G1 phase. In contrast to shear cases, BKCa blockade did not lead to an S phase accumulation in static controls. These results further supported the contention that BKCa channel exerts a regulatory effect on the transition from G0/G1 to S phase in response to SS.

BKCa transfection decreases PCNA expression in CHO cells subjected to SS

To further clarify the role of BKCa channel in SS-induced changes of cell proliferation, transfections were performed, and then PCNA expression was compared between the cases with and without transfection at laminar SS of 12 dyn/cm2. In this study CHO cells were used because these cells have been used in BKCa transfection and shear experiments [21, 29]. The transfection efficiencies of α or β1 subunit were both higher than 95 % (see Fig. S2). As illustrated in Fig. 4, there was no significant difference in terms of PCNA expression in static cases (P > 0.5, n = 9), however, BKCa transfection resulted in a marked decrease of PCNA expression in shear cases (P < 0.001, n = 9). Similar to the trend observed in VSMC, the decrease did not depend on the duration of SS applied. The PCNA decrease was effectively reversed by the IBTX treatment in shear cases, but had no effects in static cases (see Fig. S6). This data further supports the hypothesis that BKCa channel mediates the laminar SS-induced inhibition of cell proliferation.
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Fig. 4

The effect of BKCa transfection on PCNA expression in CHO cells. BKCa transfection decreases the PCNA fluorescence intensity of CHO cells in shear cases, but has no effect in static cases. a Representative laser confoco microscope graphs. b Values of the PCNA fluorescent intensity with and without BKCa transfection. **P < 0.001, No transfection (α/β1) vs. transfection (α+/β1+) (n = 9)

Laminar SS induces an abrupt large and a subsequent small increase in intracellular [Ca2+]i

It is well known that BKCa channel is activated by the enhancement of [Ca2+]i [43]. Therefore, to investigate the possible cause of BKCa channel activation, we analyzed the effect of laminar SS on the change of intracellular [Ca2+]i. As illustrated in Fig. 5a, SS induced an abrupt increase in intracellular [Ca2+]i within 30 s and a subsequent decrease. It was noticed that, although [Ca2+]i was reduced, it was still slightly higher than the baseline. To avoid fluorescence quenching due to prolonged exposure, the fluorescence of the Fluo-3 AM-loaded VSMCs were determined by flow cytometry. As displayed in Fig. 5b, compared to static control, SS still induced a small (about 1-fold) but significant [Ca2+]i increase beyond 4 min of SS. These results were consistent with another report, which found that SS-induced [Ca2+]i change followed an initial drastic leap and a subsequent steady decline but still kept a minor increase beyond 3 min of SS [21]. Furthermore, this subsequent steady decline in [Ca2+]i was reversed by BKCa blockade (see Fig. 5b).
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Fig. 5

The effect of laminar SS on the change of intracellular [Ca2+]i in VSMCs. a The change of five representative cells analyzed by Ca2+ imaging within 4 min of SS. The arrow indicates onset of shear stress. b The change of intracellular [Ca2+]i analyzed by flow cytometry beyond 4 min of SS. **P < 0.001, static control vs. shear stress of 12 dyn/cm2 (n = 4)

Laminar SS induces a BKCa–mediated membrane hyperpolarization

Ion channels exert their functions by expression and activity. Therefore, to further investigate the impact of laminar SS on BKCa channel, we focused on examining the channel activity. Membrane potential was analyzed using DiBAC4(3) stain because that opening of BKCa channel leads to hyperpolarization[34]. The decrease in fluorescence intensity suggests membrane hyperpolarization as a result of a decrease in the intracellular concentration of negatively charged bisoxonol [39]. As shown in Fig. 6, SS induced a decrease in fluorescence intensity at 30 min or 1 h, and IBTX treatment could effectively override this decrease (P < 0.001, n = 3). However, IBTX blocker has no significant effect in static control cases. This data indicated that laminar SS induces the opening of the BKCa channel and subsequently leads to membrane hyperpolarization.
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Fig. 6

The effect of laminar SS on the change of membrane potential in VSMCs. a Representative flow cyotometry graphs for membrane potential of static case, 12 dyn/cm2 shear case, static plus IBTX case and 12 dyn/cm2 shear plus IBTX case at 5 min and 1 h. b Values of DiBAC4(3) fluorescent intensity in case of static, 12 dyn/cm2, static plus IBTX case and 12 dyn/cm2 shear plus IBTX. **P < 0.00, static vs. shear or shear vs. shear plus IBTX (n = 4)

Discussion

As mentioned some cases in the introduction, flow SS has a significant impact on VSMC proliferation and vascular function [10, 12, 19, 27, 38]. Therefore, unraveling that underlying mechanisms is a current focus. Different from other published work, which mainly investigated the activation of a complex cascade of kinases and transcription factors [10, 12, 19, 27, 38], our study focused on the regulatory role of BKCa channel in SS-impacted VSMC proliferation. As for laminar SS, it has a dual effect on VSMC proliferation. Subnormal SS (≤5 dyn/cm2) has a promoting impact [12, 27], whereas normal SS has inhibitory properties [10, 38]. In addition, the theoretical modeling study of Tarbell and Shi showed that in vivo interstitial flow-induced SS on SMC could be up to 24 dyn/cm2 [37]. Our study mainly focused on the role of BKCa channel in laminar SS-induced inhibition of VSMC proliferation, therefore, laminar SS of 6, 12 and 24 dyn/cm2 was used.

The Ca2+-activated K+ (KCa) channel is commonly designated as an important biomechanosensor in EC [17, 22, 25]. In case of KCa family, BKCa in our study and the intermediate-conductance Ca2+-activated K+ (IKCa) channel were reported to be activated by SS. Sun et al. [35] demonstrated that BKCa is involved in flow-induced arteriole dilation, and that the activation of endothelial BKCa channel is an obligatory step in the transduction of the signal initiated by SS. More recently, it has been demonstrated that IKCa channel in HUVECs was up-regulated by laminar SS and Ca2+ sensitivity was strongly increased after shear exposure [4]. Compared to EC, there are rare reports on SS-induced response of BKCa channel in VSMC. In our study, we demonstrated that similar to IKCa in EC, BKCa in VSMC is up-regulated by laminar SS (as shown in Fig. 3). Considering the contractile phenotype-dependent expression of BKCa channel, this result provides another explanation for the fluid SS induced contraction of VSMC as reference reported [1, 7]. In addition, we also demonstrated that BKCa in VSMC was opened by laminar SS (as shown in Fig. 6).

An interesting phenomenon in our study is the difference between the α- and β1-subunit of BKCa channel in response to SS. The enhancement of the α-subunit showed an inverse correlation with shear duration. Thus, its increased expression was subdued by extending the duration of SS exposure (left panel of Fig. 2b and upper panel of Fig. 2d). The β1-subunit, however, showed a time-dependent up-regulation on protein level and a sustained elevation on transcription level (right panel of Fig. 2b and bottom panel of Fig. 2d). In some vascular diseases, as mentioned in the introduction such as hypertension and diabetes, it is the β1-subunit that has decreased expression or activity whereas the α-subunit has remained steady [3, 20, 23]. Therefore, it seems that the β1-subunit is more sensitive to stimulation than the α-subunit. To explore the difference between the two subunits in response to laminar SS, the change of [Ca2+]i for transient or longer periods of time was determined. As shown in Fig. 5, laminar SS induced an initial leap of intracellular [Ca2+]i (Fig. 5a), and a subsequent steady decline (Fig. 5b). These results are consistent with other published work [31]. BKCa can be activated by [Ca2+]i increase [26]. Therefore, it is reasonable to speculate that the transient [Ca2+]i increase induced by laminar SS would lead to activation of the BKCa channel, followed by K+ efflux and ultimately membrane hyperpolarization. As shown in Fig. 6, this BKCa-mediated hyperpolarization was also confirmed in our study. Based results from previous studies, membrane hyperpolarization induced by BKCa activation close the depolarization-dependent voltage-operated calcium channel, then the intracellular [Ca2+]i is decreased. That is why, as observed in Fig. 5b, following a transient [Ca2+]i increase the [Ca2+]i was reduced and this decline was reversed by BKCa blockade. However, we noticed that [Ca2+]i was still slightly higher for a longer time than baseline or static control although it was decreased (Fig. 5b).The main role of the β1-subunit is to enhance the Ca2+ sensitivity of BKCa and help the channel maintain an opening state [24, 26, 40]. Therefore, we speculated that the continuous enhancement of β1-subunit may increase the sensitivity of the BKCa to the relative lower but still higher [Ca2+]i.

Although the IKCa channel is activated by SS as mentioned above, it is not yet known whether this channel regulates EC proliferation. The major finding in our study is that BKCa channel in VSMC is not only activated by SS but also mediates the SS-induced inhibition of cell proliferation. To execute its inhibitory role, laminar SS initiates many complex signal pathways as reference reported [10, 12, 17, 19, 27, 38]. As for ion channel, the fast response is the change of membrane potential due to opening or closure induced by SS. Steady SS can activate K+ channel in EC and lead to a KCa-dependent hyperpolarization [4, 22, 25, 35]. Similar to that, laminar SS also induced hyperpolarization in VSMC, and IBTX blockade demonstrated that this hyperpolarization is BKCa-involved. It is well known that both changes in membrane potential and cell volume are necessary for progression of the cell cycle, and both require the action of a K+ channel [9, 36]. Membrane potential and cell proliferation share a close relationship. It has been demonstrated that membrane potential levels are tightly correlated with cell proliferation-related events, such as mitosis, DNA synthesis, and overall cell cycle progression. Furthermore, cells that have a high degree of membrane potential (hyperpolarization) tend to be quiescent and do not typically undergo mitosis [36]. Hence, it is reasonable to ascribe the inhibition of VSMC proliferation partly to the increased chance of membrane hyperpolarization induced by laminar SS.

In conclusion, our study demonstrated that (1) laminar SS induces an up-regulation of BKCa expression, but the two subunits of this channel respond in a different manner; (2) laminar SS induces the BKCa channel to open and leads to membrane hyperpolarization; (3) BKCa channel plays a regulatory role in laminar SS-induced inhibition of cell proliferation. More importantly, our findings may be helpful in exploring strategies to prevent flow-initiated vascular disease formation. Our study also gives rise to some additional questions that remain to be further investigated, for example, what is the electrophysiological characteristics of BKCa channel in response to SS, how does [Ca2+]i changes impact on BKCa activity in response to SS, and what is the mechanism of BKCa channel regulation of cell proliferation in response to SS?

Acknowledgments

The authors wish to thank Dr. Zhong-Dong Shi from Developmental Biology Program of Sloan-Kettering Institute for his helpful suggestions. We would also like to express our deep gratitude to Daniela C. Georgieva from Developmental Biology Program of Sloan-Kettering Institute for her help in polishing this paper. This work was supported by the National Natural Science Foundation of China (Nos. 10972024, 11120101001, 10925208 and 10802006). National Basic Research Program of China (973 program, 2011CB710901)

Conflict of interest

None.

Supplementary material

424_2012_1182_Fig7_ESM.jpg (17 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM1_ESM.tif (7.3 mb)
High Resolution Image(TIFF 7512 kb)
424_2012_1182_Fig8_ESM.jpg (32 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM2_ESM.tif (14 mb)
High Resolution Image(TIFF 14308 kb)
424_2012_1182_Fig9_ESM.jpg (16 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM3_ESM.tif (2.5 mb)
High Resolution Image(TIFF 2572 kb)
424_2012_1182_Fig10_ESM.jpg (189 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM4_ESM.tif (16.1 mb)
High Resolution Image(TIFF 16518 kb)
424_2012_1182_Fig11_ESM.jpg (123 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM5_ESM.tif (15.6 mb)
High Resolution Image(TIFF 16007 kb)
424_2012_1182_Fig12_ESM.jpg (16 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM6_ESM.tif (1.8 mb)
High Resolution Image(TIFF 1832 kb)
424_2012_1182_Fig13_ESM.jpg (102 kb)
Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

424_2012_1182_MOESM7_ESM.tif (11.5 mb)
High Resolution Image(TIFF 11760 kb)
424_2012_1182_MOESM8_ESM.docx (28 kb)
ESM 8(DOCX 27 kb)

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