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Am J Physiol Heart Circ Physiol 291: H2493-H2503, 2006. First published June 23, 2006; doi:10.1152/ajpheart.01254.2005
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Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle

D. L. Tharp,1 B. R. Wamhoff,4 J. R. Turk,1,2 and D. K. Bowles1,3

Departments of 1Biomedical Sciences and 2Veterinary Pathobiology and 3Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and the 4Department of Molecular Physiology and Biological Physics and The Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia

Submitted 29 November 2005 ; accepted in final form 19 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca2+-activated K+ channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K+ current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA ~4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle {alpha}-actin (SM{alpha}A), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SM{alpha}A, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by ~50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

migration; atherosclerosis; activator protein-1; dedifferentaiation; KCNN4; KCa 3.1

VASCULAR SMOOTH MUSCLE CELLS (SMCs) in mature blood vessels express a unique repertoire of smooth muscle-specific genes that serve as markers of a differentiated phenotype, e.g., smooth muscle {alpha}-actin (SM{alpha}A), smooth muscle myosin heavy chain (SMMHC), and smoothelin. A unique characteristic of SMCs is their ability to undergo rapid alterations in phenotype in response to both physiological and pathophysiological environmental cues, a process termed phenotypic modulation (32). For example, in response to experimental acute vascular injury, SMCs transiently suppress differentiation marker genes required for contraction and express a repertoire of genes that allow for proliferation and migration, a natural response to repair the injured vessel wall (25, 34). Similarly, during the development of complex atherosclerotic lesions, SMCs that compose the fibrous cap of the lesion and regions prone to rupture, such as plaque shoulder regions, show altered expression of SMC differentiation marker genes (1, 43). Although multiple humoral factors have been identified as potential mediators of SMC phenotypic switching, including platelet-derived growth factor-BB (PDGF-BB) (4, 20, 23, 51) and transforming growth factor-beta (28, 39), the role of ion channels, i.e., Ca2+ and K+ channels, in SMC phenotypic regulation in vascular disease is relatively unexplored.

Ca2+, a universal regulator of cell function, is required for vascular SMC contraction, growth, and differentiation (16, 42, 44, 51). We have previously demonstrated that Ca2+ influx through L-type voltage-gated Ca2+ channels induced SMC differentiation marker gene expression through a Rho kinase/myocardin/serum response factor (SRF)-dependent mechanism (42). Myocardin is an SRF coactivator that is required for selective expression of the promoter element containing CC (A/T) GG (CArG)-dependent SMC differentiation markers, e.g., SM{alpha}A and SMMHC (50), but not smoothelin-B, a CArG-independent gene (36, 49). Ca2+ influx through L-type voltage-gated Ca2+ channels is limited by hyperpolarization secondary to K+ channel activation. This raises the interesting paradigm that K+ channel activation may have opposing effects on SMC phenotype by suppressing SMC differentiation marker expression. Indeed, studies by Neylon et al. (30) demonstrated that proliferating rat aortic SMCs had enhanced charybdotoxin-sensitive K+ channels, which they concluded to be intermediate-conductance Ca2+-activated K+ (IKCa1) channels (31). Furthermore, Kohler et al. (24) demonstrated that restenosis after rat carotid balloon injury was attenuated in animals treated with the specific IKCa1 channel blocker TRAM-34 (46, 47). Taken together, these studies provide evidence for a role of IKCa1 channels as a regulator of vascular SMC proliferation. However, it is unknown whether IKCa1 channel activity influences cell phenotype by regulating SMC differentiation marker gene expression and whether IKCa1 function and expression are regulated by humoral factors proposed to play a pivotal role in SMC phenotypic modulation in vascular disease. Thus the present study tested the hypothesis that IKCa1 channel upregulation mediates SMC phenotypic modulation in response to PDGF-BB stimulation in vitro and is associated with the development of early atherosclerosis in vivo. Our results demonstrate that IKCa1 expression and activity are increased in proliferating coronary SMCs from atherogenic swine and that blockade of IKCa1 channels prevents PDGF-BB-induced suppression of SMC differentiation marker gene expression and migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of coronary arteries. Miniature swine were anesthetized with ketamine (35 mg/kg), rompun (2.25 mg/kg), and pentothal sodium (10 mg/kg), followed by administration of heparin (1,000 U/kg). Swine were euthanized by removal of the heart, which was immediately placed in 4°C physiological saline solution (PSS). Animal protocols were approved by the University of Missouri Animal Care and Use Committee. The right coronary artery (RCA) was isolated and cleaned of fat and connective tissue. It was then placed in low-Ca2+ PSS containing 20 mM HEPES and stored at 4°C until use (0–1 day).

Porcine coronary SMC culture. Primary cultures of medial SMCs were isolated from the medial portion of porcine RCA, after removal of adventitia and mechanical denudation to remove endothelium, as previously described (42). Cells were plated at 1.5 x 104 cells/cm2 in DMEM/F-12 media (GIBCO 11320-033) containing 100 U/ml penicillin/streptomycin, 1.6 mM L-glutamine, and 10% FBS for 4–5 days until postconfluent, changing media every 2 days. Cells (passages 2–6) were then serum restricted for 6 days to maximize expression levels of smooth muscle differentiation marker genes (SM{alpha}A, SMMHC, and smoothelin-B) as shown in Fig. 1.


Figure 1
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  		Fig. 1. Porcine coronary smooth muscle cell (SMC) culture model. Porcine coronary SMCs were grown to postconfluency and exposed to serum-free media (SFM) for either 1 (n = 4; 1dSFM) or 6 (n = 6; 6dSFM) days. Immunocytofluorescence was used to show the expression of smooth muscle {alpha}-actin (SM{alpha}A; A) and smoothelin (B) protein after 1 (top) and 6 (bottom) days of serum restriction. Nuclei are labeled red. C: bright-field image displays spindle-shaped 1-day (top) and 6-day (bottom) serum-restricted cells. D: quantitative RT-PCR (qRT-PCR) was used to measure expression of smoothelin-B in 1dSFM cells, 6dSFM cells, and intact coronary arteries. Data are presented as means ± SE and graphed relative to intact coronary arteries. *P < 0.05 vs. coronary artery. Smoothelin was expressed similarly in intact arteries and 6dSFM cells. E: SMCs exposed to SFM for 6 days had increased SM{alpha}A, smooth muscle myosin heavy chain (SMMHC), smoothelin-B, and myocardin mRNA expression compared with 1dSFM. Data are presented as means ± SE and graphed relative to 6dSFM. *P < 0.05 vs. respective 1dSFM.

 
Coronary artery organ culture. Segments of RCA were placed in DMEM/F-12 media with and without PDGF-BB and were incubated overnight at 37°C and 5% CO2. For subsequent quantitative RT-PCR (qRT-PCR), arteries were frozen in liquid nitrogen and stored at –80°C until use. For whole cell voltage-clamp experiments, vessels were opened longitudinally and incubated at 37°C in enzyme solution containing collagenase and elastase, followed by isolation of SMCs by gentle trituration, as previously described (6).

Laser capture microdissection. Frozen sections of porcine RCA (8.0 µm) taken from four high fat/high cholesterol-fed Yucatan swine were immunostained with Ki-67, a marker of proliferating cells, and counterstained with hematoxylin as modified from a previous study (6). The entire staining procedure took ~1 h and was performed on a cold plate to minimize RNA degradation. Laser capture microdissection (LCM; Pixcell IIe, Arcturus) was used to selectively capture Ki-67-positive (proliferating) media cells on a film-coated cap, which was immediately placed in RNA extraction buffer (Picopure RNA isolation kit, Arcturus). A second cap was subsequently used to capture Ki-67-negative (nonproliferating) medial cells from the same sample. A third cap was used to capture neointimal cells. Medial cells were defined as residing between the internal and external elastic laminas, while neointimal cells were defined as residing on the luminal side of the internal elastic lamina. cDNA was synthesized from total RNA using the Superscript III RT kit (Invitrogen) for subsequent qRT-PCR.

qRT-PCR. qRT-PCR was performed as previously described (6, 8). Samples were quick frozen in liquid nitrogen and stored at –80°C until processing. RCA samples were powdered under liquid nitrogen and placed in TRIzol. Cultured cells were frozen in TRIzol solution. Total RNA was isolated according to the manufacturer’s published protocol for TRIzol. cDNA was transcribed from total RNA using Superscript III RT kit (Invitrogen) in a 20-µl reaction containing 200 U RT, 100 ng of random hexamers, 5 mM MgCl2, 1 mM dNTPs, and 20 mM DTT. A minus RT reaction was also performed to ensure no genomic DNA contamination. qRT-PCR was performed on a Cepheid Smart Cycler (model no. SC1000-1). Each 25-µl reaction contained 1x SYBER Green Master Mix (Qiagen), 0.8 µM forward and reverse primers, and 1 µg of cDNA. Each reaction was initiated by a 95°C hold for 10 min, to activate heat-stable Taq. The reaction conditions were optimized for each set of primers: IKCa1, SM{alpha}A, SMMHC, smoothelin-B, myocardin, and 18S (for the primer sequences, refer to Table 1). Target gene expression was normalized to 18S ribosomal RNA using the 2–{Delta}{Delta}Ct method (26). Linearity and efficiency of each PCR condition were verified by creating a standard curve plotting the critical threshold vs. log of the dilution of cDNA.


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  		Table 1. Primer sequences (5' to 3')

 
Nucleofection and siRNA application. Postconfluent 6-day serum-starved cells were trypsinized and placed in basic SMC nucleofection solution (Amaxa) with either a negative control siRNA or IKCa1 siRNA (Ambion). Each sample contained 1 x 106 cells, 100 µl of nucleofection solution, and 600 pmol of siRNA. Samples were placed in electroporation cuvettes (Amaxa) and nucleofected using program D-33 of the Amaxa nucleofector device. After nucleofection, cells were immediately placed in RPMI media and incubated at 37°C and 5% CO2 for 15 min. Cells were then plated in six-well plates containing growth media (DMEM/F-12 plus 10% FBS). On adherence (4 h), cells were serum deprived for 18 h and treated with PDGF-BB (30 ng/ml) for an additional 12 h. Cells were frozen in TRIzol, and qRT-PCR was performed as stated above. The IKCa1 siRNA sequence (sense, CCUGUUCCUGGUUAAAUGCtt; antisense, GCAUUUAACCAGGAACAGGta) was designed by Ambion from the Sus scrofa KCNN4 sequence (accession no. NM_214018). The negative control siRNA was purchased from Ambion (Silencer negative control siRNA no. 1) and was described as having no specific homology to gene sequences from human, rat, or mouse.

Quantitative chromatin immunoprecipitation assay. The chromatin immunoprecipitation (ChIP) assay was performed as previously described (42, 43). Cultured cells were grown to the desired density in T-75s and treated with 1% formaldehyde for 10 min at 37°C to cross-link protein-DNA and protein-protein interactions within intact chromatin. The cross-linked chromatin was sonicated to shear chromatin fragments to 200–600 bp. The sonicated chromatin was immunoprecipitated with antibodies to c-jun (sc-45x, Santa Cruz Biotechnology) or acetylated H4 (H4Ac, Upstate Biotechnology), whereas negative control/input DNA was immunoprecipitated with no antibody, and immune complexes were recovered with agarose beads. Cross-links were reversed, chromatin was subjected to proteinase K digestion to remove protein from the DNA, and the DNA was purified via phenol-chloroform extraction. Recovered DNA was quantitated by fluorescence with picogreen reagent (Molecular Probes) according to the manufacturer's recommendations. For qRT-PCR, 1 ng of unknown and 1 ng of input DNA were amplified, and threshold cycle (Ct) number was determined in the linear amplification range. Dilutions of input DNA served as the standard curve. Ct of the unknown immunoprecipitated activator protein-1 (AP-1) samples (IP, 1 ng) were subtracted from the Ct of the same amount of reference input sample (Ref, 1 ng), and then 2 was raised to the power of that value. This provides fold enrichment of the target sequence relative to the Ref, which has a homogenous distribution of target sequence. Amplification can be described by the formula X = Xo(1 + E)n, where Xo is the initial DNA concentration of a target sequence, X is the final DNA concentration of a target sequence, E is the efficiency (a no. from 0 to 1), and n is the number of cycles. Therefore, if a threshold is set at which a specific final DNA concentration X is reached, X is a constant, and the number of cycles (Ct) required to reach X is inversely related to the initial target sequence concentration Xo, i.e., Xo(IP)/Xo(Ref) = [X(IP)/X(Ref)](1 + E)Ct(Ref) – Ct(IP). If X(Ref) = X(IP), which should be true for the same primer set, and E = 1, then Xo(IP)/Xo(Ref) = 2Ct(Ref) – Ct(IP). Therefore, for each primer set, the ratio of IP to Ref was calculated by subtracting the Ct determined for the target sequence of the IP sample from the Ct determined for the target sequence of the reference sample and taking the resulting power of 2. The data for the no antibody negative control were then subtracted from the other unknown samples to eliminate background

Formula
Data from two independent experiments were averaged for each ChIP antibody employed, and standard errors of the mean were calculated. qRT-PCR primers were designed to flank the 5'-AP-1-binding site of the IKCa1 promoter (accession no. AF305731). IKCa1 promoter primer sequences were 5'-ACC ATG TGT GTG GTG TCT GG-3' and 5'-GGC TTT GTC ACA CAC AAT GG-3', and 20 pmol of each primer with SYBER Green reagent were used in the reaction. PCR conditions were as follows: 15-s denaturation at 95°C, 60-s annealing at 65°C, 45-s extension at 72°C (50 cycles).

Immunocytofluorescence. Immunocytofluorescence was modified from a previous study (27). Porcine coronary SMCs were grown on glass coverslips in six-well plates until postconfluent and were serum restricted for 1 or 6 days. Cells were exposed to primary antibody, SM{alpha}A (1:200 dilution; Sigma), or smoothelin (undiluted; Santa Cruz Biotechnologies) overnight and Alexa 488-labeled secondary antibody for 4 h. Images were obtained at the Molecular Cytology Core (University of Missouri, Columbia) using confocal microscopy, as shown in Fig. 1.

Whole cell voltage clamp. Whole cell K+ current (IK) was measured as previously described (7, 19). SMCs were either trypsinized from cultured cells or enzymatically dispersed from RCA and suspended in low-Ca2+ PSS containing 20 mM HEPES and stored at 4°C until use (0–6 h). Cells were superfused with normal PSS containing (in mM) 2 CaCl2, 10 glucose, 10 HEPES, 5 KCl, 1 MgCl2, and 138 NaCl, pH 7.4. Pipettes (2–6 M{Omega}) were filled with solution containing (in mM) 0.15 CaCl2 (1 µM free Ca2+), 120 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.2 EGTA, with pH 7.1. Currents were elicited by 500-ms step depolarizations to potentials ranging from –60 mV to +60 mV (in 10-mV increments) from a holding potential of –80 mV. Current was recorded before and after addition of TRAM-34 (100 nM) to the bath.

Chemotaxis. Postconfluent 6-day serum-restricted porcine coronary SMCs were plated at 30,000–40,000 cells/well in the upper chamber of a 10-µm-pore, 96-well chemotaxis chamber (Millipore). The following solutions were placed in the lower chamber (diluted in serum-free media): vehicle, PDGF-BB (30 ng/ml), and PDGF-BB + TRAM-34 (100 nM). The chamber was placed at 37°C for 4 h. Cells from the upper chamber were removed, and the filters were stained using Diff-Quik staining kit (Fisher Scientific). The migrated cells in a x40 field were manually counted.

Statistics. All data are presented as means ± SE. One-way ANOVA was used for all group comparisons, with post hoc comparisons where appropriate. Significance was defined as P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Porcine coronary SMC culture model. Immunocytofluorescence demonstrated marked induction of SM{alpha}A (Fig. 1A, bottom) and smoothelin (Fig. 1B, bottom) protein in porcine coronary SMCs that were growth arrested in serum-free media for 6 days (6dSFM) compared with 1 day (1dSFM; Fig. 1, A and B, top), as previously described in rat aortic SMCs (42, 43). Porcine coronary SMCs in culture demonstrated a spindle-shaped morphology (Fig. 1C), as described previously (11, 18). 6dSFM porcine coronary SMCs demonstrated dramatic increases in SM{alpha}A, SMMHC, smoothelin, and myocardin mRNA compared with 1dSFM (Fig. 1E). Myocardin is an SRF coactivator that is selectively expressed in SMCs (50). Moreover, the late SMC-specific differentiation marker smoothelin was expressed similarly in 6dSFM cells and intact coronary arteries (Fig. 1D). Thus, under the appropriate conditions, porcine coronary SMCs in culture can recapitulate the expression profile of key late differentiation markers of native quiescent coronary SMCs.

PDGF-BB increases IKCa1 mRNA expression. We used both cultured porcine coronary SMCs and intact sections of porcine RCA to measure IKCa1 expression in the presence and absence of PDGF-BB. PDGF-BB has been demonstrated to be a potent stimulator of SMC phenotypic modulation, migration, and proliferation in vivo, as well as in vitro (20, 23, 51), and has been shown to increase in human atherosclerotic plaques (4). Treatment with PDGF-BB (+BB) for 24 h increased IKCa1 expression approximately fourfold in cultured coronary SMCs and approximately threefold in intact porcine RCA rings (Fig. 2A). Furthermore, the specific IKCa1 channel blocker TRAM-34 (46, 47) abolished the effect of PDGF-BB on IKCa1 expression in cultured coronary SMCs. Interestingly, TRAM-34 alone decreased expression of IKCa1 compared with control, suggesting a positive-feedback mechanism whereby IKCa1 activity stimulates IKCa1 expression in coronary SMCs.


Figure 2
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  		Fig. 2. TRAM-34 (TRAM) prevented platelet-derived growth factor-BB (PDGF-BB)-induced intermediate-conductance Ca2+-activated K+ channel (IKCa1) gene activation. Cultured coronary SMCs and segments of right coronary artery (RCA) were treated with PDGF-BB (30 ng/ml) or vehicle for 24 h. A: PDGF-BB (+BB; n = 6) increased IKCa1 mRNA in both SMCs and RCA compared with respective controls (–BB; n = 7). TRAM-34 (100 nM; n = 7) abolished PDGF-BB-induced increases in IKCa1, and alone decreased IKCa1 in SMCs relative to control (–BB, n = 6). *P < 0.05 vs. –BB. **P < 0.05 vs. –BB and +BB. B: quantitative chromatin immunoprecipitation (ChIP) assay was used to measure the enrichment of c-jun and acetylated histone-4 (H4Ac) to the 5'-activator protein-1 (AP-1)-binding site of the IKCa1 promoter. TRAM-34 blocked enrichment of both c-jun and H4Ac by PDGF-BB. n = 2 for each group.

 
TRAM-34 prevented PDGF-BB-induced AP-1 and H4Ac enrichment of the IKCa1 promoter. Quantitative ChIP was used to determine 1) whether PDGF-BB increased AP-1 and H4Ac enrichment of the IKCa1 promoter and 2) whether this enrichment was dependent on IKCa1 channel activation. PDGF-BB increased c-jun enrichment of the 5'-AP-1 (c-jun/c-fos)-binding site in the endogenous IKCa1 promoter approximately fivefold (Fig. 2B), as determined by ChIP. This enrichment was completely abolished by TRAM-34. In addition, PDGF-BB increased IKCa1 promoter enrichment of H4Ac, demonstrating PDGF-BB regulation of IKCa1 by histone acetylation. Consistent with c-jun binding, TRAM-34 prevented PDGF-BB H4Ac enrichment of the IKCa1 promoter. However, contrary to our finding with IKCa1 mRNA levels, TRAM-34 alone had no effect on either c-jun or H4Ac enrichment of the IKCa1 promoter. This finding indicates that the singular effect of TRAM-34 on IKCa1 mRNA levels may not be due entirely to inhibition of IKCa1 transcription. Together, these data are consistent with PDGF-BB-induced IKCa1 expression being enhanced by a positive-feedback mechanism involving AP-1 (c-jun/c-fos) and histone acetylation.

PDGF-BB increased IKCa1 activity. Porcine cultured coronary SMCs, as well as intact porcine RCA rings, were treated with PDGF-BB (30 ng/ml) or vehicle for 24 h to induce IKCa1 expression. Whole cell voltage clamp was used to measure IK before and after administration of the specific IKCa1 channel blocker, TRAM-34 (100 nM). Figure 3A displays representative traces obtained from a single PDGF-BB-treated cultured SMC (+BB), as well as a control SMC (–BB), demonstrating inhibition of whole cell IK with TRAM-34 (TRAM) in cells treated with PDGF-BB. Both cultured porcine coronary SMCs and freshly dispersed cells from RCA demonstrate an approximate fourfold increase in TRAM-34-sensitive current in cells exposed to PDGF-BB compared with control (Fig. 3B). These data are consistent with increased mRNA expression of IKCa1 by PDGF-BB, resulting in increased IKCa1 channel activity at the plasma membrane.


Figure 3
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  		Fig. 3. PDGF-BB increased IKCa1 channel activity. A: ensemble K+ currents in PDGF-BB-treated and control cultured SMCs (–60 to +60 mV) in the presence (+) and absence (–) of TRAM-34 (100 nM). B: PDGF-BB (+BB; SMC, n = 12; RCA, n = 12) increased TRAM-34-sensitive current (+60 mV) compared with control (–BB; SMC, n = 13; RCA, n = 13). *P < 0.05 vs. respective –BB.

 
Proliferating cells have increased IKCa1 and decreased smooth muscle differentiation marker and myocardin mRNA expression. To determine whether similar changes occurred in vivo with early development of atherosclerosis, we used LCM on histological cross sections of RCA taken from swine exhibiting early-stage atherosclerosis induced by a 20-wk high-fat/high-cholesterol diet (6). Figure 4A demonstrates neointimal thickening in an RCA section taken from a high-fat/high-cholesterol-fed pig (black arrowheads designate the internal elastic lamina in Fig. 4, A and B). LCM was used to isolate proliferating and nonproliferating medial cells, as well as neointimal cells. Subsequent qRT-PCR was used to compare IKCa1 mRNA expression in proliferating (Ki-67 positive) and nonproliferating (Ki-67 negative) cells from the media (M), as well as cells from the neointima (NI). Figure 4B is a representative immunohistochemistry section illustrating LCM capture of Ki-67-positive medial cells (within black circles). After capture and removal of Ki-67-positive cells, Ki-67-negative cells were captured. Figure 4C demonstrates an ~22-fold greater IKCa1 expression in Ki-67-positive proliferating medial cells (M+) compared with Ki-67-negative nonproliferating medial cells (M–). A similar effect was seen comparing IKCa1 expression in neointimal cells (NI) with nonproliferating medial cells, as shown previously (24). These data support our hypothesis that there is increased IKCa1 expression in proliferating medial SMCs during early atherosclerosis. Figure 4 also demonstrates a 75–90% reduction in the expression of late SMC differentiation markers SMMHC (Fig. 4E) and smoothelin-B (Fig. 4F), as well as myocardin (Fig. 4G) in proliferating medial cells (M+) vs. nonproliferating medial cells (M–). Conversely, SM{alpha}A (Fig. 4D) expression was not significantly reduced in proliferating medial cells compared with nonproliferating medial cells. The loss of late differentiation markers SMMHC and smoothelin-B but not SM{alpha}A in proliferating medial SMCs is consistent with phenotypic modulation of medial coronary SMCs during early atherosclerosis. Similarly, SMC differentiation marker and myocardin gene expression are lower in the neointima (NI) compared with the media (M).


Figure 4
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  		Fig. 4. Proliferating coronary SMCs have increased IKCa1 and decreased smooth muscle differentiation marker and myocardin mRNA expression. A: RCA from high-fat/high-cholesterol-fed swine demonstrating neointimal (NI) thickening. B: proliferating, Ki-67-positive cells (dark nuclei) in the media (M) immediately after capture by laser capture microdissection (LCM; dark circles). C: qRT-PCR demonstrated that IKCa1 was ~22x higher in Ki-67-positive medial cells (M+; n = 4) compared with Ki-67-negative medial cells (M–; n = 4). IKCa1 was also higher in the neointima (~7x; n = 4). Inset: IKCa1 expression in Ki-67-positive (M+) and Ki-67-negative (M–) captured medial cells (–RT, no RT control). Late differentiation markers SMMHC (E; n = 4), smoothelin-B (F; n = 4), and myocardin (G; n = 4) but not SM{alpha}A (D; n = 5) were lower in proliferating medial cells (M+) and in neointimal cells compared with nonproliferating medial cells (M–). *P < 0.05 vs. M–. **P < 0.05 vs. M– and M+.

 
TRAM-34 blocked PDGF-BB-induced coronary SMC dedifferentiation. To determine whether IKCa1 activity is required for phenotypic modulation by PDGF-BB, porcine coronary SMCs in culture were exposed to PDGF-BB (30 ng/ml) in the presence or absence of TRAM-34 (100 nM) for 24 h. As shown in Fig. 5, relative to control (–BB), SMCs treated with PDGF-BB (+BB) had an ~60–80% decrease in SM{alpha}A (Fig. 5A), SMMHC (Fig. 5B), smoothelin-B (Fig. 5C), and myocardin (Fig. 5D) mRNA expression. Importantly, TRAM-34 abolished PDGF-BB-induced decreases in all SMC differentiation marker genes and myocardin, indicating that IKCa1 activity is required for PDGF-BB-induced inhibition of smooth muscle differentiation marker expression. Similar results were obtained using the nonselective IKCa1 channel blocker charybdotoxin (100 nM; data not shown). Interestingly, while TRAM-34 alone had no effect on SM{alpha}A, SMMHC, or myocardin mRNA, it increased smoothelin-B expression approximately fivefold (Fig. 5C), indicating that basal IKCa1 activity in these cells is sufficient to limit smoothelin-B expression. These data demonstrate a requisite role for IKCa1 activity in the downregulation of smooth muscle differentiation marker gene expression by PDGF-BB.


Figure 5
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  		Fig. 5. TRAM-34 blocked PDGF-BB-induced coronary SMC dedifferentiation. PDGF-BB (30 ng/ml) treatment for 24 h (+BB) decreased SM{alpha}A (A), SMMHC (B), smoothelin-B (C), and myocardin (D) mRNA. Addition of TRAM-34 (100 nM) blocked PDGF-BB-induced dedifferentiation (TRAM + BB). TRAM-34 alone increased smoothelin-B mRNA. n = 5–8 for each group. *P < 0.05 vs. respective –BB. **P < 0.05 vs. –BB and +BB.

 
IKCa1 siRNA prevented PDGF-BB-induced phenotypic modulation. To confirm that the effects of TRAM-34 were mediated by IKCa1 inhibition, cells were transfected with either IKCa1 siRNA or negative control siRNA. IKCa1 siRNA reduced IKCa1 mRNA expression by ~80% (Fig. 6A), whereas the negative control siRNA had no effect. As a control for "off-target" effects of the IKCa1 siRNA on other K+ channels, we demonstrated that IKCa1 siRNA had no effect on large-conductance calcium-activated K+ channel (BK) mRNA expression (Fig. 6A). Similar to the effects of TRAM-34, IKCa1 siRNA prevented increases in IKCa1 expression (Fig. 6B) and decreases in SMMHC (Fig. 6C) by PDGF-BB. However, unlike nontransfected cells, in transfected cells neither PDGF-BB nor IKCa1 siRNA had any effect on SM{alpha}A mRNA expression (data not shown).


Figure 6
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  		Fig. 6. IKCa1 siRNA prevented PDGF-BB-induced coronary SMC phenotypic modulation. A: IKCa1 siRNA (IK) reduced IKCa1 mRNA expression by ~80% compared with both nontransfected (control) cells and cells transfected with negative control (Neg. con.) siRNA. IKCa1 siRNA did not affect large-conductance calcium-activated K+ channel (BK) mRNA levels. B: PDGF-BB increased IKCa1 expression by ~2-fold, which was blocked by the IKCa1 siRNA. C: IKCa1 siRNA prevented PDGF-BB-induced reductions in SMMHC expression. Data are presented as means ± SE (n = 5–7 for each group) and expressed relative to the negative control siRNA. *P < 0.05 vs. negative control siRNA.

 
Time course of phenotypic modulation. To determine the time at which IKCa1, SMMHC, smoothelin-B, and myocardin expression is altered with PDGF-BB, cells were treated with PDGF-BB for 0, 0.5, 1, 2, 4, or 10 h as shown in Fig. 7. PDGF-BB increased IKCa1 and decreased myocardin mRNA expression at 0.5 h and decreased SMMHC and smoothelin-B mRNA expression at 10 h, consistent with IKCa1 and myocardin-regulating smooth muscle differentiation marker expression and cell phenotype.


Figure 7
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  		Fig. 7. Time course of PDGF-BB effect on mRNA. Cultured coronary SMCs were treated with PDGF-BB (30 ng/ml) for 0–10 h. IKCa1 was increased and myocardin was decreased at 30 min and remained altered throughout. SMMHC and smoothelin-B were not decreased until 10 h to a level similar to that seen at 24 h (Fig. 5). n = 4–5 for each time point.

 
TRAM-34 blunted PDGF-BB-induced coronary SMC migration. To determine the role of IKCa1 activity in coronary SMC migration, cells were exposed to either vehicle, PDGF-BB (30 ng/ml), or PDGF-BB + TRAM-34 (100 nM) for 4 h. Compared with vehicle (0.11 ± 0.11 cells/field, n = 4), PDGF-BB increased migration ~280-fold (31.5 ± 4.1 cells/field, n = 4; P < 0.05 vs. vehicle). TRAM-34 (100 nM) inhibited PDGF-BB-induced migration by ~50% (18.8 ± 2.4 cells/field, n = 4; P < 0.05 vs. PDGF-BB), demonstrating that blockade of IKCa1 channels inhibits PDGF-BB-stimulated migration of coronary SMCs.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Coronary smooth muscle plasticity permits stimuli-specific modulation of phenotype to both physiological and pathophysiological conditions, allowing vascular remodeling associated with hypertension, atherosclerosis, and postangioplasty lesion development. We recently demonstrated that ion channels in the plasma membrane, specifically L-type voltage-gated Ca2+ channels, regulate coronary SMC phenotype by altering smooth muscle differentiation marker gene expression (42). The present study expands this concept and provides novel insight into the role of IKCa1 in the regulation of coronary SMC phenotype. We tested the hypothesis that functional upregulation of IKCa1 regulates coronary SMC phenotype. Three key findings of the present study support this hypothesis: 1) PDGF-BB increased expression and activity of IKCa1; 2) blockade of IKCa1 prevented PDGF-BB-induced coronary SMC migration and phenotype modulation, i.e., TRAM-34 or IKCa1 siRNA prevented the loss of smooth muscle marker gene expression; and 3) we provide the first evidence for upregulation of IKCa1 during the development of coronary atherosclerosis in vivo, as demonstrated by increased IKCa1 and decreased smooth muscle late differentiation marker expression in proliferating coronary SMC during early atherosclerosis.

Previous studies have described a role for IKCa1 in the proliferation of cancer cells (22), endothelial cells (17), and SMCs of rat carotids after balloon injury (24). However, a role for IKCa1 in coronary SMC phenotypic modulation with coronary atherosclerosis had not been previously investigated. The present study is the first to show increased IKCa1 expression and activity in coronary SMCs treated with PDGF-BB. PDGF-BB is a potent stimulator of SMC phenotypic modulation (20, 23, 51). Previous studies demonstrate increased PDGF-BB in human atherosclerotic plaques in vivo and proliferating vascular SMCs in vitro (4, 20). In animal models of experimental vascular disease, PDGF-BB has been shown to stimulate migration and intimal thickening in injured rat carotid arteries (23). Moreover, in ApoE–/– mice fed a Western diet for 18–20 wk, blockade of the PDGF-beta receptor with a monoclonal antibody resulted in an 80% reduction in SMC investment of the neointima (38). Although SMC conditional knockout of the PDGF-beta receptor has not been performed to definitively show that PDGF-BB alters SMC phenotype in vivo, significant evidence suggests that PDGF-BB plays a role in this process. Thus the upregulation of IKCa1 by PDGF-BB is consistent with a role for IKCa1 in coronary SMC phenotypic modulation.

A plausible mechanism for upregulation of IKCa1 by PDGF-BB is activation of immediate early-response genes encoding transcription factors, i.e., c-fos and c-jun, and posttranslational modification of chromatin promoter structure by histone modifications, e.g., increased histone acetylation. PDGF-BB increases expression of c-fos and c-jun (2, 12, 13), which are constituents of the AP-1 transcriptional complex. AP-1 is involved in the regulation of cell proliferation by serum, growth factors, and/or cytokines (2, 13). Importantly, the IKCa1 promoter region contains an AP-1-binding site, which is necessary for promoter activity (15, 37). Our findings demonstrate that PDGF-BB increased the enrichment of c-jun to the IKCa1 promoter region containing the 5'-AP-1-binding site, consistent with PDGF-BB activation of AP-1 (c-jun/c-fos) as a mechanism for increased IKCa1 expression. Aside from AP-1, it has also recently been demonstrated that downregulation of repressor element-1 silencing transcription factor (REST) can increase expression of IKCa1 (10), providing another pathway that could potentially mediate the upregulation of IKCa1 by PDGF-BB. Further studies are needed to determine the role of REST in PDGF-BB-induced regulation of IKCa1. In addition, we found that blockade of IKCa1 by TRAM-34 prevented both PDGF-BB-induced c-jun enrichment of the 5'-AP-1-binding site and histone H4 acetylation (H4Ac) of the IKCa1 promoter, consistent with regulation of IKCa1 expression by a positive-feedback mechanism involving AP-1 and increased histone acetylation. Indeed, a recent report by Owens and colleagues (29) showed in SMCs, in vitro and in vivo, that increased H4Ac within promoter regions is associated with increased transcription factor binding required for promoter activity, a mechanism that has been proposed to maintain chromatin in an open state for easy access of transcription factors. For example, treatment of cultured SMCs with PDGF-BB or acute balloon injury in vivo resulted in decreased H4Ac and SRF enrichment at CArG SMC differentiation marker promoter regions but increased H4Ac and SRF enrichment at the c-fos CArG promoter region, similar to PDGF-BB-induced H4Ac and c-jun enrichment of the IKCa1 AP-1 promoter region shown herein.

One potential cellular mechanism for increased AP-1 binding to the IKCa1 promoter involves store-operated calcium entry (SOCE) via transient receptor potential (TRP) channels. PDGF-BB stimulation of pulmonary arterial SMC proliferation involves enhanced SOCE (51) leading to nuclear AP-1 binding as determined by EMSA (14). TRP channels have been demonstrated to be the likely candidate responsible for SOCE in smooth muscle (5, 16, 48, 51). Furthermore, PDGF-BB has been shown to upregulate TRPC6 (51), and both TRPC1 and TRPC6 are upregulated with vascular balloon injury (5). Future studies will be required to determine whether upregulation of IKCa1 involves enhanced SOCE and AP-1 binding to the IKCa1 promoter and which histone acetyl transferases (HAT) are involved in posttranslation modification of histone H4 in the IKCa1 promoter.

Previously, Kohler et al. (24) demonstrated that blockade of IKCa1 using TRAM-34 prevented rat carotid restenosis after balloon injury, providing evidence that functional upregulation of IKCa1 is required for smooth muscle proliferation. In addition, a reciprocal expression between IKCa1 and the BK channel was shown in intimal vs. medial SMCs after injury (24). However, the molecular phenotype of the cells was not determined, and the role of IKCa1 in regulating expression of smooth muscle-specific marker genes remained unknown. The present study is the first to show a requisite role for IKCa1 in modulating coronary SMC phenotype by demonstrating that blockade of IKCa1 with TRAM-34 or IKCa1 siRNA prevented PDGF-BB-induced reductions in smooth muscle-specific markers. Furthermore, PDGF-BB increased IKCa1 and decreased myocardin within 30 min but did not decrease smoothelin-B or SMMHC until 10 h, demonstrating that PDGF-BB-induced phenotypic modulation is preceded by changes in IKCa1 and myocardin. These findings are especially intriguing, as myocardin has been shown to be a key regulator of SRF/CArG-dependent smooth muscle differentiation marker gene expression (e.g., SM{alpha}A and SMMHC) (9, 42, 45, 50). Myocardin selectively regulates smooth muscle-specific genes that contain at least one CArG cis regulatory element (e.g., SM{alpha}A, SMMHC, SM22{alpha}, and telokin) but not CArG-dependent growth response genes such as c-fos or the CArG-independent SMC-selective gene smoothelin-B (36, 42, 49, 50). Thus myocardin may serve as a transcriptional point of divergence whereby IKCa1 selectively regulates CArG-independent and CArG-dependent SMC differentiation markers. However, the ability for IKCa1 to regulate both CArG- and non-CArG-driven smooth muscle-specific genes in this study implicates another, as yet unidentified, pathway/factor modulated by IKCa1 that is responsible for regulating smoothelin-B expression. Further evidence that IKCa1 differentially regulates smoothelin-B was observed by treatment of SMCs with TRAM-34, which resulted in increased smoothelin-B expression; TRAM-34 alone had no effect on SM{alpha}A, SMMHC, or myocardin. The smoothelin gene contains two promoter regions that produce two isoforms: smoothelin-B, the long, vascular form our primers were designed against, and smoothelin-A, the short, visceral form (36). The promoter regions are differentially regulated (36); however, a detailed mechanism(s) regulating smoothelin-B gene expression is unclear. Together, these results provide the first evidence for the regulation of smooth muscle-specific gene expression by IKCa1 and provide novel evidence that coronary SMC phenotypic modulation by IKCa1 may occur, in part, via myocardin downregulation.

Whereas IKCa1 siRNA prevented PDGF-BB-induced reductions in the late differentiation marker SMMHC, neither PDGF-BB nor IKCa1 siRNA had any effect on SM{alpha}A expression (data not shown). Because of the technical requirements of the transfection procedure, transfected cells are no longer in a postconfluent, serum-starved state and, as such, begin to lose differentiation marker expression compared with postconfluent, adherent, serum-starved cells. The inability of PDGF-BB to reduce SM{alpha}A levels in transfected cells compared with nontransfected cells may indicate that there is a low, basal level of SM{alpha}A expression in SMCs that is not suppressed by dedifferentiation, nor dependent on IKCa. This is supported by the fact that low levels of SM{alpha}A expression are found in dedifferentiated SMCs and myofibroblasts.

While PDGF-BB is a useful tool to investigate smooth muscle phenotypic modulation in vitro, we sought to verify IKCa1 upregulation during coronary atherosclerosis in vivo. We have previously demonstrated that swine placed on a high-fat/high-cholesterol diet develop early-stage atherosclerosis, as evidenced by type I–III Starry lesions, similar to humans (6). At this early stage of atherosclerosis, only ~5% of medial SMCs are proliferating (6); therefore, we used LCM to compare mRNA expression in nonproliferating and proliferating coronary medial SMCs. LCM has been used previously in human coronary arteries with neointimal thickening to show decreased smoothelin expression in the neointima vs. media (40), consistent with smoothelin being a late differentiation marker, similar to SMMHC (33, 35, 41). Similarly, Bar et al. (3) observed decreased smoothelin expression after rat carotid balloon injury, suggesting that smoothelin expression is downregulated in phenotypically modulated SMCs (33). Using LCM, we demonstrated increased IKCa1 expression in proliferating compared with nonproliferating coronary medial cells from swine exhibiting signs of early coronary atherosclerosis. In addition, proliferating cells had decreased expression of SMMHC, smoothelin-B, and myocardin; however, SM{alpha}A mRNA levels were not decreased. This latter finding is significant, as, developmentally, SMCs initially express SM{alpha}A, followed by expression of SMMHC and smoothelin as the cells enter the more mature/contractile phenotype (32, 33). Thus SMMHC and smoothelin are typically expressed in fully differentiated/contractile SMCs, whereas SM{alpha}A is also expressed in myofibroblasts or SMCs undergoing phenotypic modulation (3, 32, 33). The loss of late differentiation markers SMMHC and smoothelin-B but not SM{alpha}A in proliferating medial SMCs is consistent with increased IKCa1 expression and phenotypic modulation of medial coronary SMCs during early atherosclerosis. Although we cannot definitively exclude proliferating medial cells arising from adentitial fibroblast migration or infiltration and transdifferentiation of cells of blood origin, i.e., bone marrow-derived stem cells (21, 32), it is highly unlikely, as proliferating medial cells still express SMMHC, smoothelin, and myocardin, a molecular phenotype that has never been observed in either myofibroblasts or transformed blood-derived cells (32).

In summary, this study provides compelling, novel evidence that IKCa1 channels play a significant role in coronary SMC dedifferentiation and migration in response to PDGF-BB and during the development of early atherosclerosis. We propose a model for smooth muscle phenotypic modulation incorporating our findings with relevant findings from the literature (Fig. 8). This study and previous work by others (12) demonstrate that PDGF-BB and Ca2+ stimulate AP-1 (c-fos/c-jun), which regulates IKCa1 expression (15, 37, 51). IKCa1-induced hyperpolarization would increase Ca2+ influx through nonvoltage-dependent Ca2+ channels, such as TRP channels, which would, in turn, activate AP-1 and posttranslational histone acetylation to further drive the expression of IKCa1 in a positive-feedback manner. Conversely, hyperpolarization due to IKCa1 upregulation will inhibit calcium influx through L-type voltage-gated channels, which would reduce Rho kinase (ROK)/myocardin-induced expression of SM{alpha}A and SMMHC (42). Blockade of IKCa1 would prevent L-type voltage-gated Ca2+ channel inhibition and thus serve to maintain the quiescent, contractile phenotype of smooth muscle. Future studies will be necessary to fully characterize the molecular mechanisms that link IKCa1 channel activity with changes in smooth muscle marker gene expression and cell phenotype. Of major importance will be discerning how plasticity in specific ion channels is linked to specific changes in smooth muscle gene expression, e.g., how the cell discriminates between L-type voltage-gated Ca2+ channel and SOCE Ca2+ signals to produce disparate phenotype responses. These findings also reinforce the importance of membrane excitability in regulating gene expression, i.e., excitation-transcription coupling, in vascular smooth muscle (44).


Figure 8
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  		Fig. 8. Proposed mechanism for IKCa1-induced coronary SMC dedifferentiation. PDGF-BB (BB) binds to its receptor (BBR), initiating a signaling cascade in which AP-1 and histone (H) acetylation (Ac) upregulates IKCa1 (IK). Hyperpolarization due to activation of IKCa1 increases Ca2+ influx through nonvoltage-gated channels (e.g., transient receptor potential; TRP) while simultaneously inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels (Cav). Inhibition of Cav inhibits Rho-kinase (ROK)/myocardin (Myo)/serum response factor (SRF) smooth muscle differentiation marker gene expression, as detailed previously (42), producing a dedifferentiated, noncontractile phenotype.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants P01-HL-052490 (D. K. Bowles, J. R. Turk) and T32-HL-07094 (D. L. Tharp) and the American Heart Association (B. R. Wamhoff).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Heike Wulff for generously providing TRAM-34 and Jennifer Casati and Rebecca Shaw for technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Bowles, E102 Veterinary Medicine Bldg., Univ. of Missouri, Columbia, MO 65211 (e-mail: BowlesD{at}missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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Potassium Channels Keep Mobile Cells on the Go
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Sphingosine-1-Phosphate Receptor Subtypes Differentially Regulate Smooth Muscle Cell Phenotype
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Preventing Stenosis by Local Inhibition of KCa3.1: A Finger on the Phenotypic Switch
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V. Kaushal, P. D. Koeberle, Y. Wang, and L. C. Schlichter
The Ca2+-Activated K+ Channel KCNN4/KCa3.1 Contributes to Microglia Activation and Nitric Oxide-Dependent Neurodegeneration
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