Visualization of flow-dependent concentration polarization of macromolecules at the surface of a cultured endothelial cell monolayer by means of fluorescence microscopy

Abstract

To substantiate the occurrence of flow-dependent concentration or depletion of atherogenic lipoproteins, which has been theoretically predicted to take place at a blood/endothelium boundary, we have studied the effects of perfusion pressure and wall shear rate on the accumulation and uptake of microspheres by cultured vascular endothelial cells in a monolayer. The study was carried out by flowing a cell culture medium containing fetal calf serum and fluorescent microspheres through a parallel-plate flow chamber having a cultured bovine aortic endothelial cell (BAEC) monolayer on one wall of the chamber. The microspheres had a nominal diameter of 19 nm, approximately the same as that of low-density lipoproteins, and thus served as models and tracers of plasma proteins and lipoproteins. Experiments were carried out in steady flow in the physiological range of wall shear rate and water filtration velocity at the monolayer, while monitoring the intensity of fluorescence of the spheres accumulated at and taken up by the endothelial cells. It was found that in a perfusate containing only fluorescent microspheres, due to increased phagocytic activity of the endothelial cells, the intensity of fluorescence which reflected the number of the microspheres taken up by the endothelial cells, increased almost linearly with time and independently of wall shear rate. However, with perfusates containing fetal calf serum, this abnormal phenomenon did not occur, and the intensity of fluorescence increased with increasing perfusion pressure and decreasing wall shear rate. It was also found that the number of fluorescent microspheres accumulated at and taken up by the BAEC monolayer was shear-dependent only at low wall shear rates, and increased sharply when the flow rate was reduced to zero. These results provided solid experimental evidence that flow-dependent concentration or depletion of macromolecules occurs at the luminal surface of the endothelium at physiological wall shear rates and water filtration velocities, and strongly supports the hypothesis that flow-dependent concentration polarization of lipoproteins plays an important role in the localization of atherosclerosis and intimal hyperplasia in man by facilitating the uptake of atherogenic lipoproteins by endothelial cells.