ChronicIn VitroFlow Promotes Ultrastructural Differentiation of Endothelial Cells

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Abstract
Endothelial cells in vivo are exposed to flowing blood and are therefore subject to continuous apical shear stress. This study explored the feasibility of endothelial cell culture with chronic shear stress. Bovine glomerular capillary and aortic endothelial cells were cultured under conventional conditions without flow, or in polypropylene hollow fibers perfused for 9 days with venous or arterial shear stress. Cells were then examined by scanning and transmission electron microscopy. For both cell types, ultrastructural differences between conventional culture and culture with chronic arterial flow were quantified morphometrically. In the hollow fibers, capillary and aortic endothelial cells formed adherent, confluent monolayers with chronic flow, but not without flow. With venous and arterial shear stress, aortic, but not glomerular capillary endothelial cells, aligned themselves in the direction of flow. In aortic endothelial cells, the density of Weibel Palade bodies was, on average, 38 fold greater with arterial flow than in conventional culture. Lysosomal content was also significantly greater in aortic endothelial cells under arterial shear stress compared to conventional culture. In glomerular capillary endothelial cells, the density of organized actin microfilaments was nearly 7 fold greater under arterial flow compared to conventional culture. In these microvascular endothelial cells, the density of clathrin coated pits and vesicles was 115 ± 15 compared to 40 ± 11 (n = 3, p < 0.01) per linear μm plasma membrane with arterial flow and conventional culture, respectively, and the density of endocytic vesicles was 2.7 fold higher with arterial flow. Thus, chronic in vitro culture of capillary and aortic endothelial cells with flow is associated with cytoskeletal reorganization and increased endocytic activity in the microvascular endothelial cells and, in aortic cells, with a much higher density of Weibel Palade bodies when compared to conventional, static culture conditions. The data are interpreted to indicate greater differentiation of endothelial cells grown with flow than with conventional culture.