Atherosclerosis is a chronic inflammatory disease, characterized by neointimal plaque formation due to proliferation and migration of vascular smooth muscle cells (VSMCs) and infiltration of T lymphocytes (T cells) and macrophages. Mitogens activate these cells, causing the induction of immature VSMCs and activated T cells and macrophages that produce cytokines and abundantly express intermediate conductance Ca2+-activated K+ channels (IKCa). The activity of IKCa regulates membrane potential, Ca2+ signaling and cell volume in a variety of cells, and involves in pro-atherogenic functions of these activated cells including proliferation and cytokine production. However, mechanisms for IKCa in regulating VSMC proliferation/migration and in atherogenesis are unknown. Our central hypothesis is that IKCa up-regulation mediates atherosclerosis. We shall test this hypothesis in three ways. First, we shall determine if IKCa up-regulation is associated with VSMC proliferation and migration in atherosclerosis. Western blotting, immunohistochemistry, videomicroscopy and proliferation and migration assays will determine the role of IKCa activity in VSMC proliferation/migration in atherosclerosis. Second, we shall test the hypothesis that increased IKCa activity regulates the gene transcription and cell cycle progression by raising [Ca2+]i in VSMCs. This will be done using fluorescence microscopy, 5-bromo 2'- deoxy-uridine incorporation assay, flow cytometry, and Western blotting. Third, the role of IKCa in the development of atherosclerosis will be tested using apolipoprotein knockout mice, IKCa knockout mice, and double knockout (apoE and IKCa) mice. The effect of long-term blockade or knockdown of IKCa on development of atherosclerosis, VSMC accumulation, and inflammatory cell proliferation/migration and cytokine production will be determined. Collectively, these aims address a novel mechanistic approach to the development of atherosclerosis by examining the role of IKCa activity in VSMC duplication and immune activation. These studies should open new therapeutic strategies for the prevention of atherosclerosis. ? ? ?
