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Exposure to hypercholesterolemic serum modifies the expression of cytoskeletal proteins in cultured endothelia
E Constantinescu1, D Alexandru, M Raicu
1Institute of Cellular Biology and Pathology, Nicolae Simionescu, Bucharest, Rumania.
Arterial endothelial layer dysfunction is considered to be one of the most important events which initiate the development of the atherosclerotic plaque and the cell cytoskeleton plays an essential role in maintaining the integrity of the endothelium exposed continuously to haemodynamic forces. The aim of this work was to study the modifications of the cytoskeletal proteins in the vascular endothelium exposed to atherogenic conditions. A hamster aortic endothelial cell line (HAEC) grown on glass coverslips was exposed for 24 h to hypercholesterolemic or normal homologous serum. Upon staining with Oil Red O and examination by phase contrast and fluorescence microscopy, HAEC incubated with hypercholesterolemic serum appeared heavily loaded with lipid droplets that showed a yellow autofluorescence in UV light and the general aspect of a foam cell. HAEC were incubated with: a) anti-actin serum; b) anti-vinculin monoclonal antibody (MoAb); c) anti-alpha actinin MoAb, and d) anti-talin MoAb, followed by appropriate secondary antibodies coupled with FITC or rhodamine. As compared to normal HAEC, the cells exposed to hypercholesterolemic serum showed a modified pattern for actin and vinculin localization. Actin appeared as a weakly stained network around the nuclear zone whereas vinculin was distributed as small granules throughout the cell cytoplasm. These experimental data suggest that in advanced atherosclerosis, some of the endothelial cytoskeletal proteins undergo modifications which could represent one of the important factors involved in further development of the atheromatous plaque. In addition they indicate that HAEC exposed to hypercholesterolemic serum could represent an in vitro working model for studying the events occurring in the endothelium at advanced stages of atherosclerosis.
Arterial endothelial layer dysfunction is considered to be one of the most important events which initiate the development of the atherosclerotic plaque and the cell cytoskeleton plays an essential role in maintaining the integrity of the endothelium exposed continuously to haemodynamic forces. The aim of this work was to study the modifications of the cytoskeletal proteins in the vascular endothelium exposed to atherogenic conditions. A hamster aortic endothelial cell line (HAEC) grown on glass coverslips was exposed for 24 h to hypercholesterolemic or normal homologous serum. Upon staining with Oil Red O and examination by phase contrast and fluorescence microscopy, HAEC incubated with hypercholesterolemic serum appeared heavily loaded with lipid droplets that showed a yellow autofluorescence in UV light and the general aspect of a foam cell. HAEC were incubated with: a) anti-actin serum; b) anti-vinculin monoclonal antibody (MoAb); c) anti-alpha actinin MoAb, and d) anti-talin MoAb, followed by appropriate secondary antibodies coupled with FITC or rhodamine. As compared to normal HAEC, the cells exposed to hypercholesterolemic serum showed a modified pattern for actin and vinculin localization. Actin appeared as a weakly stained network around the nuclear zone whereas vinculin was distributed as small granules throughout the cell cytoplasm. These experimental data suggest that in advanced atherosclerosis, some of the endothelial cytoskeletal proteins undergo modifications which could represent one of the important factors involved in further development of the atheromatous plaque. In addition they indicate that HAEC exposed to hypercholesterolemic serum could represent an in vitro working model for studying the events occurring in the endothelium at advanced stages of atherosclerosis.