The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

KCa3.1 expression is increased in atherosclerotic blood vessels.

In aortas (aortic root) of Apoe^+/+ mice, no atherosclerotic plaques were observed on Sudan III staining for lipid accumulation (Figure 1A). Immunohistochemical analysis with a polyclonal Ab specific for KCa3.1 (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI30836DS1) revealed that KCa3.1 expression was restricted to the intimal layer, and no KCa3.1 was detected in the medial layer (Figure 1, B and C). The intimal layer was defined by the expression of vWF, a marker of ECs, where the channel is normally expressed (9) (Supplemental Figure 2, middle). The medial layer was defined by the expression of α-SMA, a marker for VSMCs (Supplemental Figure 2, right), and by the expression of the large-conductance calcium-activated potassium channel KCa1.1 (Figure 1D). In Apoe^–/– mouse aortas, large, yellow-orange atherosclerotic plaques were visible with Sudan III staining (Figure 1E). A serial section revealed robust KCa3.1 expression in the intimal and medial layers of the lesion area (Figure 1, F and G), while KCa1.1 expression in the vascular wall was faint (Figure 1H). Western blot analysis of EC-intact aortic trees (ascending aorta to abdominal aorta) showed increased expression of KCa3.1 in Apoe^–/– mice compared with Apoe^+/+ mice (Figure 1I). EC denudation abolished KCa3.1 expression in Apoe^+/+ but not in Apoe^–/– mouse aortas. EC-denuded aortas from Apoe^–/– mice showed a significant elevation of KCa3.1 protein levels and reduced KCa1.1 levels compared with EC-denuded aortas from control Apoe^+/+ mice. EC denudation by this gentle method (23) did not cause the detachment of atherosclerotic plaques in the aortic trees (Supplemental Figure 3). A comparison of EC-denuded aortas from control Apoe^+/+ mice and atherosclerotic Apoe^–/– mice revealed a significant reduction in KCa1.1 and a parallel elevation of KCa3.1 and l-caldesmon, a marker of dedifferentiated, immature VSMCs (Figure 1I) (24). These data show a change in KCa subtype expression from KCa1.1 to KCa3.1 in VSMCs, paralleling their phenotypic conversion from a normal contractile state to a proliferative, dedifferentiated one in atherosclerosis.

A similar switch in KCa subtype expression was observed in human small coronary arteries (HCAs) from subjects with coronary artery disease (CAD), even though no plaques were present in these vessels. In subjects with no CAD, KCa3.1 was only present in the vWF⁺ intimal layer (Supplemental Figure 3), while it was expressed in both the intimal and medial layers in vessels from patients with CAD (Figure 1J). Western blot analysis was done on protein lysates from HCAs from 5 patients with CAD (patients 4–6, 9, and 10) and 5 patients with no CAD (patients 1–3, 7, and 8). EC-intact (patients 4–6) and -denuded (patients 9 and 10) HCAs from patients with CAD showed marked elevation of KCa3.1 protein levels and reduced levels of KCa1.1 compared with EC-intact (patients 1–3) and -denuded (patients 7 and 8) HCAs from patients without CAD (Figure 1K). KCa3.1 expression was abolished by EC denudation in non-CAD subjects but not in CAD subjects. These results in humans confirm the data in mice indicate that a change in vascular KCa subtype expression from KCa1.1 to KCa3.1 is a feature of atherosclerosis.

KCa3.1 is localized in vascular and inflammatory cells in plaques.

Two-color immunofluorohistochemistry was performed in plaques of Apoe^–/– mouse aortic sinus using cell-specific Abs to determine which cell types contribute to the increased KCa3.1 expression in atherosclerosis. KCa3.1 staining overlapped with vWF on the surface of intimal plaques (Figure 2A), indicating its expression in ECs. KCa3.1 staining also overlapped with α-SMA in the medial layer and in intimal plaques (Figure 2B), indicating its presence in VSMCs that had proliferated, migrated, and/or infiltrated into the intima. Macrophages, visualized in lesions by immunostaining for Mac3, also expressed KCa3.1 (Figure 2C), and T cells stained for CD3 in the lesions expressed KCa3.1 (Figure 2D, arrowhead). KCa3.1 localization in the medial layer was also observed in a variety of peripheral arteries from Apoe^–/– mice, where plaques were not observed, as we had noted in HCAs. Taken together, these results demonstrate KCa3.1 expression in 3 of the major cell types (VSMCs, T cells, and macrophages) involved in the formation of atherosclerotic plaques.

Functional consequences of KCa3.1 expression in VSMCs.

To study the role of KCa3.1 in the activation of VSMCs, we stimulated human coronary SMCs with PDGF, a mitogen widely used in vitro and in vivo (25). PDGF stimulation resulted in a time-dependent increase in KCa3.1 mRNA expression, peaking at 6 hours (Figure 3A). There was a corresponding increase in KCa3.1 protein expression in total cell lysates (Figure 3B) and in membrane and cytosolic fractions, peaking at 24–48 hours (Figure 3C). The delay between peak mRNA and protein expression likely reflects the time required for the functional channel to be synthesized, coassembled as tetramers with calmodulin, and trafficked to the membrane (7). A concomitant increase in l-caldesmon protein expression, a marker of dedifferentiated, immature VSMCs, was noted (data not shown). KCa1.1 mRNA increased slightly 6 and 12 hours after PDGF stimulation and then decreased significantly at 48 hours (Figure 3A). The mRNA expression of small-conductance calcium-activated potassium channels (KCa2.1–2.3) remained unchanged or decreased following stimulation with PDGF (Figure 3A). Protein expression was not detectable for KCa2.1–2.3 in any of 4 experiments and for KCa1.1 in 7 of 8 experiments before or after stimulation with PDGF (20 μg whole cell lysates; data not shown).

Whole-cell patch-clamp experiments corroborated the mRNA and protein expression data. PDGF induced a substantial increase in functional KCa3.1 channels in VSMCs (Figure 4A). KCa3.1 currents were elicited in VSMCs using a ramp protocol from –120 mV to 40 mV and 3 μM free calcium in the pipette (Figure 4, B–E). While KCa3.1 currents were barely detectable in unstimulated cells (Figure 4B), the amplitude of the KCa3.1 current increased 24 and 48 hours after PDGF stimulation (Figure 4C). The current was blocked by TRAM-34 (IC₅₀, 18 nM) (Figure 4C), a specific blocker of KCa3.1 channels (15) (Supplemental Figure 4), and by charybdotoxin (Figure 4D) with an IC₅₀ value (5 nM) similar to that obtained for the cloned KCa3.1 channel (15). Apamin, a blocker of KCa2.1–2.3, had no effect on the current (Figure 4D). Iberiotoxin, a specific blocker of KCa1.1, blocked a minor fraction of the current (data not shown), confirming the small amount of KCa1.1 protein in these cells. 1-Ethyl-2-benzimidazolinone (1-EBIO), an activator of KCa3.1 channels (26), increased the amplitude of the current (Figure 4E). VSMCs in the quiescent state expressed an average of 15 KCa3.1 channels/cell, which increased after PDGF stimulation to 85 channels/cell at 24 hours and 256 channels/cell at 48 hours (Figure 4A).

Due to the increased KCa3.1 expression in the medial layer of atherosclerotic vessels, 1-EBIO elicited robust vasodilatory responses in EC-denuded carotid artery segments from atherosclerotic Apoe^–/– compared with Apoe^+/+ mice (Figure 4F; maximum dilation, 66% ± 4% versus 13% ± 12% at 100 μM; P < 0.05; n = 5–6). Similar results were obtained with EC-denuded HCAs from CAD patients versus non-CAD subjects (Figure 4H; 49% ± 8% versus 9% ± 2% at 100 μM; P < 0.05; n = 10–11). Conversely, because KCa1.1 is decreased in the medial layer of mouse and human atherosclerotic vessels, the KCa1.1 opener pimaric acid (27) evoked markedly reduced vasodilation in vessels from Apoe^–/– compared with Apoe^+/+ mice (Figure 4G; 9% ± 3% versus 55% ± 10% at 10 μM; P < 0.05; n = 5–7) and CAD patients compared with subjects without CAD (Figure 4I; 31% ± 3% versus 59% ± 6% at 3 μM; P < 0.05; n = 4–5). Taken together, the calcium dependence of the current, its characteristic pharmacology, the mRNA and protein data, and the vasomotor responses to 1-EBIO verify the presence of functional KCa3.1 channels in activated VSMCs. These results suggest that the enhanced levels of KCa3.1 in atherosclerotic vessels may in part be a consequence of the upregulation of this channel during VSMC activation.

We used pharmacological blockade, gene silencing with siRNAs, and gene disruption to define the functional consequences of KCa3.1 upregulation in VSMCs. In the pharmacological experiments, TRAM-34, at concentrations that block the KCa3.1 channel, suppressed PDGF-induced proliferation (cell count assay; Figure 5A), de novo DNA synthesis (BrdU incorporation assay; Figure 5B), and migration (Figure 5C) of VSMCs in a dose-dependent fashion. Other KCa3.1 blockers (TRAM-3, clotrimazole, and charybdotoxin) had similar effects. TRAM-7 (an inactive analog of TRAM-34) (15), iberiotoxin, and glibenclamide (a blocker of ATP-sensitive potassium [K_ATP] channels) had no discernible effect on these processes. FBS also consistently induced strong cell cycle progression of VSMCs following 24-hour stimulation, with cell numbers decreasing in G₀/G₁ phase (FBS: 63% ± 1% of cells, P < 0.05 versus quiescent: 89% ± 1%; n = 4) and increasing in S phase. TRAM-34 suppressed FBS-induced cell cycle progression at the transition from G₀/G₁ to S phase (FBS plus TRAM-34: 72% ± 0%, P < 0.05 versus FBS). Since VSMC activation is associated with increased superoxide production (28), we also measured superoxide levels in VSMCs (Figure 5D) following 48-hour stimulation with PDGF. TRAM-34 reduced PDGF-stimulated superoxide production to a level comparable to that in quiescent cells. None of the KCa3.1 blockers (TRAM-34, TRAM-3, clotrimazole, charybdotoxin) altered the viability of VSMCs as judged by trypan blue exclusion (Supplemental Figure 5), and in a previous report, TRAM-34 was shown not to exhibit cytotoxicity against a panel of 9 cell lines and primary T cells (15). Similar results were obtained with gene silencing. KCa3.1-specific siRNA inhibited PDGF-stimulated increases in KCa3.1 mRNA and protein levels (Supplemental Figure 6) and suppressed the proliferation (Figure 6A), de novo DNA synthesis (Figure 6B), and migration (Figure 6C) of VSMCs, whereas control siRNA had no effect. Gene disruption studies also support a role for KCa3.1 in regulating VSMC proliferation and migratory responses. VSMCs from KCa3.1^–/– mouse aortas exhibited reduced proliferative and migratory responses to PDGF compared with VSMCs from KCa3.1^+/+ mice (Figure 6, D and E).

Functional consequences of KCa3.1 expression in macrophages.

Our in vitro studies showed that mouse macrophages but not mono­cytes express KCa3.1. Expression of KCa3.1 was detectable by immunofluorescence in thioglycollate-elicited peritoneal macrophages of Apoe^–/– mice but not in monocytes isolated from PBMCs of these mice (Figure 7A). In Transwell experiments, TRAM-34 and clotrimazole suppressed monocyte chemoattractant protein–1–induced (MCP-1–induced) migration of macrophages from Apoe^–/– mice in a dose-dependent manner (Figure 7B). Moreover, the migratory response of peritoneal macrophages from KCa3.1^–/– mice was significantly smaller than in KCa3.1^+/+ mice (Figure 7C). These data together with the results of VSMCs presented in Figures 5 and 6 and our previously published work on the role of KCa3.1 in T cells (7, 15) highlight the important role of KCa3.1 channels in the activation of VSMCs, macrophages, and T cells. A therapeutic strategy based on the selective targeting of KCa3.1 channels in these 3 cell types may therefore be beneficial in treatment of atherosclerosis.

In vivo KCa3.1 blockade therapy prevents atherosclerosis.

We tested the antiatherosclerotic potential of KCa3.1 blockade in Apoe^–/– mice. Clotrimazole or TRAM-34 dissolved in peanut oil was administered once daily (120 mg/kg, s.c.) (6) for 12 weeks. We used 120 mg/kg of each blocker because the bioavailability following s.c. administration was poor (0.5% of the availability after i.v. administration) and because the circulating half-life of these compounds in mice was short (1 hour) (Supplemental Figure 7). We chose this s.c. route because it was more convenient and less stressful on the mice than i.p. administration for a 12-week study. After a single s.c. injection of 120 mg/kg, total plasma levels of TRAM-34 averaged 782, 720, 112, and 88 nM at 1, 2, 12, 24 hours (Supplemental Figure 7C), which would be sufficient to inhibit VSMCs, macrophages, and T cells. Due to their high lipophilicity, both clotrimazole and TRAM-34 accumulated in the body after repeated administration, and after 12 weeks of treatment, total plasma levels 24 hours after the last drug administration were 1,719 ± 354 nM for clotrimazole (n = 8) and 866 ± 48 nM for TRAM-34 (n = 6). Considering TRAM-34’s protein binding of 98% (data not shown), the free plasma concentration of TRAM-34 was around the IC₅₀ for channel blockade for most of our study.

Long-term KCa3.1 blockade therapy with clotrimazole or TRAM-34 strikingly reduced atherosclerosis in Apoe^–/– mouse aortas and carotid arteries. Representative images of atherosclerotic lesions stained in yellow-orange with Sudan III in aortic trees (left panels) and carotid arteries (right panels) are shown in Figure 8A, with quantitative measurements of atherosclerotic lesions summarized in Figure 8B (n = 10–14 mice). No lesions were observed in control Apoe^+/+ mice. Aortas of Apoe^–/– mice treated for 12 weeks with vehicle developed extensive atherosclerotic lesions in carotid arteries and in the aortic trees. Mice treated with clotrimazole or TRAM-34 for 12 weeks showed a statistically significant reduction in the atherosclerotic lesion area, both in the various segments of the aorta and in the carotid arteries (Figure 8B).

Plaques in aortic roots where more advanced lesions developed in Apoe^–/– mice (29) were examined histologically to determine the effect of KCa3.1 blockade therapy in Apoe^–/– mice treated with TRAM-34 versus vehicle (n = 9–10). In a vehicle-treated mouse, large plaques stained with Sudan III were visible in the cross section (Figure 9A). Atherosclerotic lesions contained α-SMA⁺ VSMCs, Mac3⁺ macrophages, CD3⁺ T cells, and acellular/necrotic areas (30) (Figure 2 and Figure 9, B–E, respectively). Representative images of an aortic root from a TRAM-34–treated mouse are shown in Figure 9, F–J. The plaques were much smaller than in vehicle-treated Apoe^–/– mice (Figure 9F), showing smaller areas stained for α-SMA, Mac3, and CD3 and smaller acellular/necrotic areas (Figure 9, G–J, respectively). Statistical analysis of the histological data showed that TRAM-34 significantly decreased total lesion area, α-SMA⁺ area, Mac3⁺ area, the number of infiltrating CD3⁺ T cells, and the size of the acellular/necrotic area (Table 1). Since excess superoxide production in the medial VSMCs plays a causal role in diminishing NO bioactivity and impairing EC-dependent vasodilation (31), we measured superoxide production in the vessels of vehicle- and TRAM-34–treated Apoe^–/– mice. The therapy significantly reduced superoxide production compared with vehicle treatment (Figure 9K), consistent with its inhibition of superoxide production in activated VSMCs (Figure 5D). Because ECs contribute only 10% of total superoxide generated in whole vascular walls in aortas of Apoe^–/– mice (32), the inhibitory effect is most likely due to suppression of superoxide production by VSMCs and macrophages (8, 28, 31). Thus, TRAM-34 and clotrimazole may decrease atherosclerosis and the necrotic area by inhibiting VSMCs, macrophages, and T cells.

Safety profile of KCa3.1 blockers.

To determine the safety profile of clotrimazole and TRAM-34, we monitored clinical parameters (body weight, heart weight, BP, and heart rate), blood chemistry, blood counts, and histopathology in Apoe^+/+ mice and in Apoe^–/– mice treated with vehicle or with clotrimazole or TRAM-34 during the 12-week trial (Supplemental Tables 1 and 2). Clotrimazole and TRAM-34 ameliorated atherosclerosis in Apoe^–/– mice, even though the cholesterol levels remained elevated. In Apoe^–/– mice, TRAM-34 did not cause any discernible change in the clinical parameters, blood counts, or blood chemistry compared with vehicle, whereas a body weight reduction of 17% was seen in clotrimazole-treated mice, which was accompanied by a significant increase in the levels of total protein, globulin, calcium, and the anion gap. In parallel toxicological experiments, normal C57BL/6J (Apoe^+/+) mice treated with TRAM-34 for 4 weeks did not exhibit any signs of toxicity or show visible abnormalities in the aortas, macroscopic organ damage, or any histopathological changes in the organs analyzed (Supplemental Table 3). Because of its immunosuppressive effects on T cells and macrophages, KCa3.1 blockers might compromise the immune response to acute infections. However, in rats infected intranasally with influenza virus, the viral clearance was delayed by dexamethasone but not by TRAM-34 (Supplemental Figure 9), indicating that KCa3.1 blockade with TRAM-34 does not affect the immune response to infectious agents. Additional information on the safety profile of KCa3.1 blockers is provided in Supplemental Results.

Materials.

The information on reagents and Abs other than KCa3.1 Ab is provided in Supplemental Methods.

KCa3.1 Ab.

The polyclonal primary Ab against human and mouse KCa3.1 was obtained from sera of rabbits immunized using oligopeptides with the following amino acid sequence: H-LNASYRSIGALNQVRC-NH2 (S4–S5 of human and mouse KCa3.1). The specificity of this Ab for KCa3.1 was verified by Western blotting and immunofluorescence (Supplemental Figure 1). To generate immunoabsorbed serum, purified anti-KCa3.1 Ab was incubated with its corresponding peptide for 60 or 120 minutes at 37°C or room temperature. We titrated each preparation of affinity-purified Ab to the range of 0.05–5.0 μg/ml before performing immunofluorescence or Western blotting experiments.

Animals.

Male mice (Apoe^+/+ [n = 16] and Apoe^–/– [n = 78] C57BL/6J mice; The Jackson Laboratory) were used as a model of in vivo atherosclerosis. Apoe^–/– mice were weaned at 4 weeks of age onto a high-cholesterol diet (1.25% cholesterol, 0% cholate, and 21% fat; 42% kcal as fat; Harlan Teklad) and treated with daily s.c. injection of clotrimazole (120 mg/kg/d), TRAM-34 (120 mg/kg/d), or vehicle (peanut oil) for 12 weeks (6, 9). Littermate Apoe^+/+ mice were used as controls. In separate experiments, male mice (KCa3.1^+/+, n = 11 and KCa3.1^–/–, n = 12) (49) were used for in vitro experiments involving VSMCs and macrophages. Mice were provided food and water ad libitum and maintained on a 12-hour light/120-hour dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

Mouse vessel acquisition.

Mice anesthetized with sodium pentobarbital were sacrificed by cardiac puncture. Isolation of aortas was performed as previously described (52). Under a microscope, common carotid arteries, first and second branches of external carotid arteries (150~250 μm in internal diameter, 3–5 mm in length) and iliac arteries were removed and placed immediately into cold (4°C) HEPES buffer (53).

Human vessel acquisition.

HCAs (150~1,000 μm internal diameter, 1–2 mm length; n = 26) were isolated as reported previously (53–55). The schedules and procedures of cardiac surgery (coronary artery bypass graft, valve replacement, and congenital repair) were obtained from the hospitals mentioned below. Right atrial appendages, removed as discarded tissues for cannulation during cardiopulmonary bypass, were obtained at the time of cardiac surgery and immediately placed in cold (4°C), well-oxygenated HEPES buffer consisting of (in mM/l) 138.0 NaCl, 4.0 KCl, 1.6 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 0.026 Na₂EDTA, 10.0 HEPES, and 6 glucose, pH 7.4, before being brought to the laboratory. Using forceps and scissors, HCAs were dissected from adjacent tissue and cleaned of cardiomyocytes, fat, and connective tissues in cold HEPES buffer under a dissecting microscope. All protocols were approved by the local institutional review boards at the Medical College of Wisconsin, the Department of Veterans Affairs Medical Center, St. Luke’s Hospital, St. Joseph’s Hospital, Sinai Samaritan Hospital, and St. Mary’s Hospital in Milwaukee, Wisconsin, and at Waukesha Memorial Hospital in Waukesha, Wisconsin, USA.

Quantification of atherosclerosis.

For en face analysis of atherosclerosis (30, 32, 39), excised aortic trees (from ascending aorta to iliac bifurcation) and right carotid arteries (common carotid artery to cervical bifurcation) were dissected free from surrounding tissues, opened longitudinally, and pinned onto a silicon-coated dish filled with HEPES buffer. They were fixed with 4% paraformaldehyde in PBS overnight and stained 3 hours in 1.0% (vol/wt) Sudan III solution. Images were collected using a digital camera (C-755; Olympus).

Histological analysis of aortic sinus and HCAs were examined in cross sections (30, 32, 39). Aortic roots including aortic valve and sinus or HCAs were isolated and frozen in OCT compound. Every other section (thickness, 6 μm) was mounted and fixed with 4% paraformaldehyde in PBS. The lipid accumulated in the plaques was stained with Sudan III. H&E-stained sections were used to determine the total acellular/necrotic area in the lesions (30). Immunostains were visualized using avidin-biotin HRP visualization systems (VECTASTAIN Elite ABC kits or M.O.M. immunodetection kit; Vector Laboratories) and Mayer’s hematoxylin solution (Sigma-Aldrich) for counterstaining or using FITC- or Texas red–conjugated secondary Abs (53). Controls for nonspecific immunostaining were performed in the absence of primary Abs. Each section was photographed using a Nikon E600 microscope with a Spot R/T cooled CCD digital camera (Diagnostic Instruments Inc.) or a Nikon E200 inverted fluorescence microscope. No immunostaining was detected in negative controls (no primary Abs), except for autofluorescence from extracellular elastin/collagen fibers in the sections.

For quantitative analysis of histological characteristics in the plaques, the stained area as a percentage of total area in each aortic tree and carotid artery and the immunostained area (μm²), the number of immunostained cells (nuclei), and acellular/necrotic area (μm²) in each section of aortic roots were determined using MetaMorph imaging software (Molecular Devices); and the mean lesion area or the cell number per section per mouse were calculated for each group of mice.

Videomicroscopy.

Vasodilator responses of HCAs and mouse carotid arteries were examined by videomicroscopy as previously described (53–55). Vessels pressurized at 60 mmHg for HCAs and at 40 mmHg for mouse arteries were preconstricted with acetylcholine (HCAs) or U46619 (mouse arteries), and vasodilations to 1-EBIO and pimaric acid were studied.

EC denudation.

Mouse aortas were dissected free of adventitial fat and connective tissue and cut open longitudinally. ECs were removed without detachment of plaques by gently rubbing the intimal surface with a moist cotton swab under a microscope, which was followed by rinsing, as previously reported (23). ECs in HCAs and mouse arteries were also mechanically denuded using a modification of a method used previously by us (53–55). Removal of ECs was confirmed by lack of immunostaining for vWF and by abolishment of EC-dependent vasodilation and is described in Supplemental Methods and Supplemental Figure 3.

VSMC culture.

Human coronary artery SMCs were purchased and cultured according to the manufacturer’s instructions (Cambrex Inc.). Patient information is not released by the company. All experiments were performed between passages 5 and 7. Mouse aortic SMCs were isolated as previously reported (56) and described in Supplemental Methods. After the quiescent state of cells was achieved by 48-hour incubation in serum-free medium (SmBM), cells were stimulated with 20 ng/ml PDGF in each assay.

Monocyte and macrophage isolation.

PBMCs were isolated from peripheral blood of Apoe^–/– mice by modified density-gradient centrifugation using Nycoprep (Nycomed) (57) and by positive selection using CD11b magnetic cell separation system beads (Miltenyi Biotec) (58). Macrophages from the peritoneal cavity of thioglycollate-injected Apoe^–/–, KCa3.1^+/+, and KCa3.1^–/– mice were isolated 4 days after the injection (59).

Real-time PCR.

Total RNA from VSMCs was isolated with TRIzol Reagent (Invitrogen), and real-time PCR analysis (iCycler; Bio-Rad) was performed for quantification of transcripts for human KCa1.1, -2.1, -2.2, -2.3, and -3.1 (GenBank accession number NM002250) and for GAPDH (AF106860) as previously described (6). Primer sequences and the reaction conditions are available in Supplemental Methods.

Immunocytochemistry.

VSMCs, monocytes, and macrophages seeded onto a Lab-Tek Chamber Slide system (Nunc) were immunolabeled for KCa3.1. VSMCs adherent to the chamber and monocytes and macrophages spun and deposited onto the chamber by Cytospin were immediately fixed with 4% paraformaldehyde and dried. The remainder of the immunostaining methodology was performed as described previously (53). No immunostaining or autofluorescence was seen in negative controls (no primary Abs).

siRNA transfection.

siRNA transfection was performed as previously reported (60) and described in Supplemental Methods.

Western blotting.

Total cell lysates or membrane and cytosolic fractions (20–50 μg) of VSMCs, HCAs, or mouse aortic trees were analyzed by Western blotting. The gels were stained in Coomassie blue to confirm equal protein loading. Rat brain extract, PC-12 cell lysate, and COLO 320DM cell lysate (Santa Cruz Biotechnology Inc.) were used as positive controls.

Cell proliferation and migration.

Cell proliferation (cell count and BrdU incorporation assays) and migration assays were performed as previously reported (59). VSMCs were stimulated with 20 ng/ml PDGF for 8 hours in the migration assay and for 48 hours in the proliferation assay. Macrophages were stimulated with 1 nM MCP-1 for 90 minutes in the migration assay. All control experiments (PDGF or MCP-1 alone) were performed in the presence of vehicle for blockers.

Fluorescence detection of ROS.

Fluorescence microscopy was performed as previously described (55). Changes in superoxide generation in vessels and VSMCs were assessed by measuring dihydroethidine fluorescence intensity.

Electrophysiology.

VSMCs were patch clamped in the whole-cell mode of the patch-clamp technique. KCa3.1 currents were elicited by dialysis with 3 × 10^–6 M of free calcium as previously described (6, 15).

Cell cycle analysis by flow cytometry.

Flow cytometry was performed using VSMCs stimulated with 10% FBS as previously reported (33). Cells stained with 50 μg/ml propidium iodide were analyzed with a BD FACScan. Data were analyzed with CELLQuest software.

Statistics.

All data are expressed as mean ± SEM. Data other than those from the vasodilation studies were statistically analyzed by paired t test; ANOVA for 1-way, 2-way, and repeated measures; and nonparametric tests for data that were not distributed normally, where appropriate. Vasodilation data were analyzed as previously reported (53, 55). Agonist-induced dilations are expressed as a percentage, with 100% dilation representing the change from the constricted diameter to the maximal diameter obtained by addition of papaverine (10^–4 mol/l). Statistical comparisons of maximum percent vasodilation were performed using paired t test, whenever applicable. A 2-factor repeated-measures ANOVA was used to compare dose-response (factor 1) relationships between the treatment groups (factor 2). Interactions, if any, were noted between dose and treatments groups. Corollary dose-specific comparisons were tested using a Bonferroni-adjusted t test whenever the interactions were statistically significant. All procedures were done using “proc mixed” or “proc glm” programs of SAS for Windows version 8.2. Statistical significance was defined as a value of P < 0.05.