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Medical AI Inquiry: You was designed to assist in medical decision-making based on the provided user questions. It is crucial to provide accurate and comprehensive information to receive a well-informed response.  Labels: BACKGROUND, METHODS, RESULTS. Meshes: Calcium, Capillary Leak Syndrome, Capillary Permeability, Cells, Cultured, Endothelium, Vascular, Humans, Lung, Muscle Contraction, Myosin Light Chains, Phosphorylation.
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Is thrombin-mediated permeability of human microvascular pulmonary endothelial cells calcium dependent? Context: 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. Human lung microvascular endothelial cells, grown on 0.4 micromol/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 micromol 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 micromol Fura-2/AM were assessed at OD340/380nm after thrombin exposure to detect free Ca2+i. 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.
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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. Final Decision: yes