Abstract 19061: Kca3.1 Regulates Monocytosis and Monocyte Adherence in Atherosclerosis

Abstract

Introduction: Atherosclerosis is associated with a monocytosis of an inflammatory subset (Ly-6Chi in mice), which is preferentially recruited to lesions and gives rise to pro-atherosclerotic macrophages (MΦ). We have shown that the intermediate-conductance Ca2+-activated K+ channel KCa3.1 is predominantly expressed in blood Ly-6Chi monocytes (MONO) and lesion MΦ, and KCa3.1 blockade or knockout reduces the development of atherosclerosis in mice. To further study the function of KCa3.1 in atherogenesis, we investigated its role in monocytosis and in MONO adherence to the endothelium in vivo. Methods and Results: Flow cytometric analyses of peripheral blood and bone marrow from Apoe-/- and Apoe-/- KCa3.1-/- mice showed that KCa3.1 deficiency decreased circulating MONO (8.8±1.5% of leukocytes, p<0.05 vs. 15.4±2.1%; n=7-8) and Ly6Chi MONO (4.2±0.7% of leukocytes, p<0.05 vs. 9.8±1.5%, n=7-8), with unchanged Ly6Clo MONO (4.6±1.3% of leukocytes, p=ns vs. 5.6±0.7%) and polymorphonuclar (11.7±1.4% of cells, p=ns vs. 12.5±1.2%; n=7-8). Apoe-/- KCa3.1-/- mice had reduced hematopoietic stem and multipotential progenitor cells (HSPCs) (2.4±0.5% of lineage negative cells, p<0.05 vs. 3.0±0.5%, n=7). In vivo confocal endomicroscopic analysis of leukocyte adherence was performed in anesthetized mice 4 hours after topical application of 0.5 μg TNF-α and 0.125 μg IL-1β onto the right external carotid artery where an imaging probe (Cellvizio®, field of view=8x105 μm2) was placed. Blood and circulating leukocytes were stained by intravenous injection of 200 μg FITC-dextran and 0.02% acrydine orange, respectively. KCa3.1 deficiency markedly inhibited leukocyte rolling (4±2 vs. 52±15 cells/min, contact time: 1.6±0.5 vs. 2.7±0.2 sec, distance: 78±11 vs. 165±5 μm, and velocity: 145±24 vs. 119±5 μm/sec) and adhesion (0.5±0.3 vs. 2.0±0.6 cells/min, p<0.05 for all data, n=6). Immunohistochemistry of lesions showed that Apoe-/- KCa3.1-/- mice had less CD68-positive MONO/MΦ compared to Apoe-/- mice (616±101, p<0.05 vs. 937±90 cells/mm2, n=8-9). Conclusion: KCa3.1 modulates monocytosis and plays a major role in MONO adherence to the endothelium in vivo. Thus KCa3.1 constitutes a promising therapeutic target for the prevention of atherosclerosis.