The cause of intimal smooth muscle cell hyperplasia in atherosclerosis is unknown. Preliminary results are presented which support a link between smooth muscle cell proliferation and lipid accumulation, where cholesteryl ester-enrichment of arterial smooth muscle cells, achieved by exposing smooth muscle cells to cationized low density lipoproteins (cLDL), increases mitogen release. Mitogenic activity was assessed as the incorporation of 3H-thymidine into quiescent smooth muscle cells. the overall aim of this grant proposal is to identify the growth factors released, determine if cholesteryl ester-enrichment is responsible for increased mitogen release from cLDL-treated smooth muscle cells and to characterize the mechanisms of mitogen release from cholesteryl ester- enriched smooth muscle cells.
In Specific Aim 1, the growth factors released by cholesteryl ester- enriched cells will be identified. Mitogenic activity in the conditioned media will be measured as 3H-thymidine incorporation and cell counting assays. Mitogens will be identified on the basis of thermal stability, heparin binding characteristics and antibody neutralization. Further characterization of the growth factors in conditioned media and cell lysates will be achieved by SDS-PAGE, isoelectric focussing and N- terminal amino acid sequence analysis. Preliminary results indicate that a major mitogen release from smooth muscle cells is basic fibroblast growth factor (bFGF). Experiments proposed in specific aim 2 will determine if cholesteryl ester-enrichment is responsible for increased bFGF synthesis and release. Mitogen release in response to oxidized derivatives of cholesterol and LDL as well as the constituents of cLDL will be assessed. bFGF content of the conditioned media and cell lysates will be measured by radioimmunoassay (RIA). The experiments proposed in specific Aim 3 will characterize the mechanisms of release of bFGF-like molecules following cholesteryl ester-enrichment. The mechanisms to be examined are alterations of cell viability, increased synthesis of bFGF- like proteins, measured by immunoprecipitation and steady state levels of mRNA, increased release of bFGF from the extracellular matrix, and increased activities of cell surface proteases or heparinases. The experiments proposed in this study will provide critical information for the role of the smooth muscle cell-derived foam cell intimal smooth muscle cell hyperplasia in atherosclerosis.
