Abstract
Abstract: :
Purpose: High blood pressure is associated with increased retinal vascular permeability in patients with diabetes. However, a sudden increase in transmural pressure gradient across retinal and aortic endothelial monolayers reduces hydraulic conductivity (Lp), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70 kDa dextran, Lp, and the solvent–drag reflection coefficients (σ) during the sealing process. Methods and Materials: The rate of flux of water across BAEC monolayers was measured by tracking the displacement of an air bubble in hydraulic tubing after application of a 10–cmH2O pressure gradient. Transport of fluorescent–labeled albumin and dextran were measured with and without pressure gradient. The BAEC monolayers were fixed and examined for ZO–1 by immunohistochemistry. Results: The diffusional permeability coefficients for albumin (Pdalb = 1.33 ± 0.18 x 10–6 cm/s) and dextran (Pddex = 0.60 ± 0.16 x 10–6 cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran (Pealb and Pedex) were measured after application of a 10–cmH2O pressure gradient. During the first 2 h of pressure application, Pealb, Pedex, and Lp were significantly reduced by 2.0 ± 0.3–fold, 2.1 ± 0.3–fold, and 3.7 ± 0.3–fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens–1 (ZO–1) was significantly increased at cell–cell contacts after application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered σ despite significantly elevating both Lp and Pe. Conclusion: Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO–1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system, which allows the transport of water but not dextran or albumin. Furthermore, the TJ determines transport rates but not the solute–selectivity of the monolayer.
Keywords: diabetic retinopathy • vascular cells • diabetes