Apolipoprotein E expression in aortic smooth muscle cells: the effect of PVLDL

The expression of apolipoprotein E in cultured neonatal rabbit aortic smooth muscle cells was examined. Northern blot analysis determined that there was a single RNA transcript of approximately 1.2 kb. Moreover, a polyclonal antibody against rabbit apolipoprotein E was prepared in a goat and used in immunoprecipitations to demonstrate that the cultured cells secreted apolipoprotein E into the media. A double band typical of apolipoprotein E migrated at apparent molecular masses of 37 and 39 kDa. Analysis of steady-state levels of apolipoprotein E mRNA demonstrated that expression increased as cell seeding density increased. When examined as a function of time in culture, there were two peaks of expression evident l day and 8 days after seeding. The addition of PVLDL (@-very low density lipoprotein) to smooth muscle cells increased both [3H]thymidine incorporation into DNA as well as cell number and these increases were accompanied by a decrease in the levels of apolipoprotein E mRNA in cells treated with the lipoprotein for 1 and 7 days. After incubation of the cultures with PVLDL for 1 week, the cells were radiolabeled with [35S]methionine and the media was subjected to immunoprecipitation with antiapolipoprotein E. The data revealed that the amount of apolipoprotein E secreted into the media decreased in the presence of PVLDL. In summary, these results show that apolipoprotein E expression in aortic smooth muscle cells is regulated by cell density, time in culture, cell proliferative state, and PVLDL addition. These observations may have relevance to the conditions that are known to accompany the development of the atherosclerotic lesion.Schreiber, B. M., H. V. Jones, and C. Franzblau. Apolipoprotein E expression in aortic smooth muscle cells: the effect of PVLDL. J Lipid Res. 1994. 35: 1177-1186. Supplementary key words atherosclerosis apoE mRNA cell seeding density ELISA smooth muscle cell differentiation Apolipoprotein E is a major constituent of plasma lipoproteins that classically functions in lipid transport and redistribution (reverse cholesterol transport), although other roles have been postulated (1-4). Apolipoprotein E mediates the binding of lipoproteins to the apoB,E (LDL) receptor (5, 6). I n its capacity to remove cholesterol from peripheral tissues to the liver, apolipoprotein E probably plays an important role in inhibiting the development and/or progression of atherosclerosis. Synthesis of apolipoprotein E by aortic smooth muscle cells has been shown (7-9) and is likely to contribute to local arterial pools of the apolipoprotein. These cells are known to be intimately involved in atherogenesis as they migrate from the medial layer of the blood vessel to its intima where differentiation results in proliferation and connective tissue synthesis (10). Although Majack et al. (9) have suggested that the proliferative state of the smooth muscle cell influences apolipoprotein E expression, little else is known about the parameters that affect the synthesis of apolipoprotein E by these cells. These studies were undertaken to examine apolipoprotein E expression in cultured aortic smooth muscle cells. Studies from our laboratory have established that cultured neonatal rat cells synthesize and secrete apolipoprotein E (7). Moreover, cells from both rat and rabbit undergo phenotypic changes that result in a multilayered culture with an extensive insoluble extracellular matrix (11-14). Treatment with the atherogenic lipoprotein, PVLDL, causes intracytoplasmic lipid droplet formation reminiscent of the smooth muscle-derived foam cells characteristic of atherosclerosis (15). The experiments reported here demonstrate that the expression of apolipoprotein E mRNA in cultured neonatal rabbit aortic smooth muscle cells is a function of cell density and time in culture. Moreover, apolipoprotein E levels decrease in cell cultures