Morphological study of the cytoskeleton of endothelial cells in vitro. Effects of heparin and protamine.

Abstract

The relation between the cytoskeleton and multiplication of cultured endothelial cells was observed immunocytochemically and electron microscopically. Heparin and protamine were used to control the multiplication of the cells. Cytochalasin D, a microfilament synthesis inhibitor, was also used, and its effect on cytoskeletal morphology was compared with the effect of heparin or protamine. In order to quantify changes in microfilaments, the ratio of the length of microfilaments to that of intermediate filaments (M/I ratio) was measured by transmission electron microscopy of the whole mounted cells. The addition of heparin to the culture medium not only enhanced multiplication of the endothelial cells, but also induced the outgrowth of many pseudopodia. In association with these changes, microfilaments became sparse, and stress fibers became short. The addition of protamine to the culture medium suppressed multiplication of endothelial cells and made them spherical. The thickening of the cytoplasmic layer increased the apparent density of the cytoskeleton, but the M/I ratio remained unchanged. Cytochalasin D made the endothelial cells spherical, and at the same time stress fibers disappeared, and microfilaments became sparse. The M/I ratio was smaller than that of the others. Protamine may inactivate protein which forms bridges to connect microfilaments or which binds microfilaments and the cell membrane.