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

Abstract

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Figure 1. KCa3.1 upregulation in vessels of mice and humans with atherosclerosis.

Representative images of Apoe^+/+ and Apoe^–/– mouse aortic roots stained for lipid accumulation with Sudan III (A and E), KCa3.1 (B and F, C and G), and KCa1.1 (D and H). Sudan III staining is shown in yellow-orange and positive immunostaining in brown. Scale bars: 200 μm (A, B, E, and F), 50 μm (C, D, G, and H). (I) Western blot analysis of membrane fractions from Apoe^+/+ and Apoe^–/– mouse aortic trees with or without ECs for KCa3.1 (MW, 46 kDa) and KCa1.1 (110 kDa) and of whole-cell lysates from those without ECs for l-caldesmon (~70 kDa). KCa3.1, 40 μg and KCa1.1, 30 μg membrane protein; l-caldesmon, 20 μg whole-cell lysates. Western blotting was repeated 3 times by pooling 3 aortas from each strain (9 aortas in each strain total) and showed similar results. (J) Representative images of KCa3.1 expression in HCAs from non-CAD (left) or CAD subject (right). Red arrowheads indicate EC layers that were positively stained for vWF (Supplemental Figure 3B). Scale bars: 20 μm. (K) Western blot analysis of KCa3.1 and KCa1.1 in EC-intact or -denuded HCAs with or without CAD (10 patients total). The 5 patients with no CAD are numbered 1–3, 7, and 8, and the 5 patients with CAD are numbered 4–6, 9, and 10. ECs were denuded in vessels of patients 7–10 (Supplemental Figure 3B).

Figure 2. KCa3.1 localization in plaques of Apoe^–/– mouse aortic roots.

Double immunofluorohistochemical analysis of atherosclerotic plaques stained for vWF (A), α-SMA (B), Mac3 (C), and CD3 (D) with KCa3.1 staining. Representative images for each staining are in the far-left column (in red); KCa3.1 staining (in green) in the second column; overlays of the two (in yellow) in the third column; and bright-field images in the far-right column. Autofluorescence was not detectable in the plaques. Nuclei were stained blue with DAPI. White arrowhead indicates a T cell. Scale bars: 20 μm (A and D), 50 μm (B), 30 μm (C).

Figure 3. KCa3.1 upregulation in activated VSMCs.

(A) Stimulation with 20 ng/ml PDGF increased KCa3.1 mRNA in human coronary SMCs. mRNA expression of KCa1.1, -2.1, -2.2, and -2.3 was unchanged or decreased. n = 5–9. (B) Total KCa3.1 protein expression (20 μg whole-cell lysates) was increased in VSMCs in a time-dependent fashion. n = 9. (C) KCa3.1 protein expression in membrane and cytosolic fractions (50 μg) was increased during PDGF stimulation. PC, positive controls. ^#P < 0.05 versus control.

Figure 5. KCa3.1 blockade prevents VSMC activation.

KCa3.1 blockers suppressed PDGF-stimulated proliferation (A, cell count assay; n = 6–9), DNA synthesis (B, BrdU incorporation; n = 6–17), migration (C; n = 6–7), and ROS production (D) of human coronary SMCs. TRAM-7 (an inactive analog of TRAM-34), iberiotoxin (KCa1.1 blocker), and glibenclamide (ATP-sensitive potassium channel [K_ATP] blocker) had no effect. (A–C). ^#P < 0.05 versus PDGF alone; ^†P < 0.0005 versus quiescent cells; *P < 0.0005 versus PDGF-stimulated cells. Scale bars: 20 μm.

Figure 4. Increased KCa3.1 channel activity in activated VSMCs and in arteries from subjects with atherosclerosis.

(A) Stimulation with PDGF increased KCa3.1 channel numbers in VSMCs. (B) Quiescent VSMCs demonstrated minimal KCa3.1 current. (C) The KCa3.1 current in PDGF-stimulated cells is blocked by TRAM-34 with an IC₅₀ value (18 nM) similar to that obtained for the cloned channel. (D) The current was blocked by charybdotoxin (IC₅₀, 5 nM) but not by the small-conductance calcium-activated potassium channel (KCa2.1–2.3) blocker apamin. (E) The KCa3.1 activator 1-EBIO enhanced the amplitude of the current. (F–I) Vasodilations to KCa3.1 stimulation with 1-EBIO and KCa1.1 stimulation with pimaric acid in EC-denuded carotid artery segments of Apoe^–/– mice (F and G) and in EC-denuded HCAs from CAD subjects (H and I). ^#P < 0.05 compared with Apoe^+/+ mice or non-CAD subjects. Max, maximum; [–logM], negative logarithm of the molar concentration; Unstim, unstimulated.

Figure 6. KCa3.1 gene silencing or KO prevents VSMC activation.

KCa3.1 gene silencing by specific siRNA transfection reduced PDGF-stimulated proliferation (A, cell count assay; n = 6), DNA synthesis (B, BrdU incorporation; n = 8), and migration (C; n = 6) of human coronary SMCs. ^#P < 0.05 versus PDGF. Aortic VSMCs from KCa3.1^–/– mice exhibited reduced proliferative (D; n = 7–8) and migratory (E; n = 7–8) responses to PDGF compared with VSMCs from KCa3.1^+/+ mice.

Figure 7. KCa3.1 blockade or gene KO prevents macrophage activation.

(A) KCa3.1 is abundantly expressed in macrophages but little in monocytes. Autofluorescence was not detectable in macrophages. ^#P < 0.05 versus monocytes; n = 3 mice, 445 monocytes, 1,572 macrophages. (B) TRAM-34 and clotrimazole dose-dependently inhibited macrophage migration to MCP-1. ^†P < 0.05 versus MCP-1 alone; n = 4–11. (C) Macrophage migration to MCP-1 was significantly reduced in KCa3.1^–/– mice compared with KCa3.1^+/+ mice. n = 8–9. Scale bars: 20 μm.

Figure 8. KCa3.1 blockade therapy prevents the development of atherosclerosis in Apoe^–/– mice.

(A) Representative images of atherosclerotic lesions in aortic trees from the ascending aorta to the iliac bifurcation (left) and in carotid arteries from the common carotid artery to the cervical bifurcation (right; stained yellow-orange with Sudan III) of Apoe^+/+ and Apoe^–/– mice treated with vehicle, clotrimazole, or TRAM-34. (B) Quantitative measurements of atherosclerotic lesion area (atherosclerotic lesion area/whole vascular area). Apoe^–/– mice treated with vehicle: 33% ± 3%, n = 14, versus clotrimazole-treated Apoe^–/– mice: 10% ± 2%, n = 10, ^#P < 0.05; and TRAM-34–treated Apoe^–/– mice: 15% ± 1%, n = 12, ^#P < 0.05. Scale bars: 2 mm (aortic trees), 1 mm (carotid arteries).

Figure 9. Antiatherosclerotic effect of KCa3.1 blockade therapy with TRAM-34 in Apoe^–/– mice.

Typical atherosclerotic lesions in the aortic root of a mouse treated with vehicle (A–E) or TRAM-34 (F–J). Serial 5-μm sections were stained with Sudan III (A and F); Abs specific to VSMCs (α-SMA; B and G), macrophages (Mac3; C and H), or T cells (CD3; D and I); or H&E (E and J). (K) Left: Representative images comparing fluorescence intensity of dihydroethidine, representing the production of superoxide in isolated iliac arteries of Apoe^–/– mice treated with vehicle or TRAM-34. Vessels with no intimal plaques were isolated, incubated with dihydroethidine, and laid on glass slides, and images were taken perpendicularly to vessels. Each vessel is traced with white dotted lines. Right: Summary of fluorescence intensities (normalized to vehicle-treated Apoe^–/– mice) in 7 arteries of each group. Dihydroethidine fluorescence intensity was significantly decreased in TRAM-34–treated Apoe^–/– mouse arteries. ^#P < 0.05 versus vehicle. Scale bars: 200 μm (A and F), 100 μm (B, C, E, G, H, J, and K), 50 μm (D and I).