Development of the atherosclerotic lesion involves the interaction of monocyte/macrophages, endothelial cells, and smooth muscle cells. the T- lymphocyte may also have a role in the late lesion. Each of these cells, given the right stimulus, can secrete bioactive peptides, cytokines. These cytokines may influence both the initiation and/or progression of the lesion by their influence on the behavior of surrounding cells. We will determine the ability of normal and modified triglyceride and cholesterol ester rich lipoproteins to initiate cytokine synthesis and secretion by human monocyte/macrophages, and human venous/arterial endothelial cells. Lipoproteins to be studied include LDL, VLDL, HTG-VLDL, Ac-LDL, and both Fe3+ and Cu2+ oxidized version of these lipoproteins. Cytokines to be examined include interleukins 1, 4, 6, 8, tumor necrosis factor, interferon-gamma, and granulocyte/macrophage colony stimulating factor. Cytokine synthesis and secretion will be determined at the mRNA level by Northern blotting or RT-PCR/Southern blotting, and at the protein level by antibody capture ELISA. The ability of monocyte/macrophages and endothelial cells, after preincubation with normal and modified lipoproteins, to respond to cytokine stimuli by cytokine secretion and cell surface changes will be examined. Cell surface changes will be evaluated by measuring H2 antigen expression, surface procoagulant activity, and monocyte adhesion. We will define the role of lipoprotein metabolism, lipid loading, and oxidized lipid components in initiating cytokine synthesis and modifying cellular response to cytokine stimuli. Oxidized lipid components will be extracted from lipoproteins, and active compounds from HPLC separations identified by GC-mass spectrometry. We will also determine the ability of the above cytokines, alone and in combinations, to directly regulate the uptake and metabolism of normal and modified cholesterol ester and triglyceride-rich lipoproteins in monocyte/macrophages and endothelial cells, and the mechanisms involved. Finally, cytokine synthesis and response will be examined in cocultures of human macrophages, endothelial cells, and smooth muscle cells to determine if the presence of multiple cell types alters the patterns of cytokine synthesis and response observed in pure cultures of endothelial cells and monocyte/macrophages. The overall goals of the project are (a) to understand how triglyceride-rich and cholesterol ester-rich lipoproteins, especially in modified forms that could accumulate in the lesion, can regulate both the initiation of cytokine production and cytokine response by arterial cells and, (b) determine the role of cytokines as regulators of normal and modified lipoprotein metabolism by arterial cells.
