The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans
Kazuyoshi Toyama, Heike Wulff, K. George Chandy, Philippe Azam, Girija Raman, Takashi Saito, Yoshimasa Fujiwara, David L. Mattson, Satarupa Das, James E. Melvin, Phillip F. Pratt, Ossama A. Hatoum, David D. Gutterman, David R. Harder, Hiroto Miura
Kazuyoshi Toyama, Heike Wulff, K. George Chandy, Philippe Azam, Girija Raman, Takashi Saito, Yoshimasa Fujiwara, David L. Mattson, Satarupa Das, James E. Melvin, Phillip F. Pratt, Ossama A. Hatoum, David D. Gutterman, David R. Harder, Hiroto Miura
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Research Article Vascular biology

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.

Authors

Kazuyoshi Toyama, Heike Wulff, K. George Chandy, Philippe Azam, Girija Raman, Takashi Saito, Yoshimasa Fujiwara, David L. Mattson, Satarupa Das, James E. Melvin, Phillip F. Pratt, Ossama A. Hatoum, David D. Gutterman, David R. Harder, Hiroto Miura

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Figure 1

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

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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).