Thrombin-mediated permeability of human microvascular pulmonary endothelial cells is calcium dependent

Background: In response to inflammation, endothelial cytoskeleton rearrangement, cell contraction, and intercellular gap formation contribute to a loss of capillary barrier integrity and resultant interstitial edema formation. The intracellular signals controlling these events are thought to be dependent on intracellular calcium concentration ([Ca²⁺]_i). We hypothesized that, in human pulmonary microvascular endothelial cells, a thrombin-induced increase in permeability to albumin would be dependent on [Ca²⁺]_i and subsequent actin cytoskeleton rearrangements. Methods: Human lung microvascular endothelial cells, grown on 0.4 μmol/L pore membranes, were activated with 10 nmol/L human thrombin in Hank's balanced salt solution/0.5% fetal bovine serum. Select cultures were pretreated (45 minutes) with 4 μmol Fura-2/AM to chelate Ca²⁺_i. Permeability was assessed as diffusion of bovine serum albumin/biotin across the monolayer. Similarly treated cells were stained with rhodamine-phalloidin to demonstrate actin cytoskeletal morphology. Separately, cells loaded 2 μmol Fura-2/AM were assessed at OD_340/380nm after thrombin exposure to detect free [Ca²⁺]_i. Results: Intracellular [Ca²⁺] levels increased 15-fold (2 seconds) and fell to baseline (10 minutes) after thrombin. Permeability increased 10-fold (30 minutes), and a shift from cortical to actin stress fiber morphology was observed. Chelation of Ca²⁺_i diminished permeability to baseline and reduced the percentage of cells exhibiting stress fiber formation. Conclusion: Thrombin stimulates pulmonary capillary leak by affecting the barrier function of activated pulmonary endothelial cells. These data demonstrate a thrombin-stimulated increase in monolayer permeability, and cytoskeletal F-actin stress fibers were, in part, regulated by endothelial [Ca²⁺]_i. This early, transient rise in [Ca²⁺]_i likely activates downstream pathways that more directly affect the intracellular endothelial structural changes that control vascular integrity.