Rationale Acute lung injury (ALI) is a frequent complication of sepsis in which disruption of the pulmonary endothelial cell (EC) monolayer leads to increased vascular permeability and life-threatening pulmonary edema. Activated protein C (APC) reduces mortality in sepsis; however, the mechanism through which it exerts its protective effect remains undefined. We hypothesized that APC, via interaction with the endothelial protein C receptor (EPCR), and the EC cytoskeletal protein phosphorylated myosin light chain (phosphoMLC), enhances EC barrier function.
Methods Transendothelial electrical resistance (TER) was measured in confluent human pulmonary artery EC monolayers cells treated with APC alone and with the addition of thrombin(0.2 U/mL). Activation of MLC via phosphorylation was assessed using Western blot analysis.
Results APC dose-dependently attenuated the thrombin-induced reductions in TER at 1 hr and Western blot analysis of APC (1 μg/mL)-challenged EC demonstrated a 3 fold increase in phosphoMLC (5 min) compared to control. Addition of an EPCR blocking antibody abolished APC-induced barrier-protection and increases in phosphoMLC, while incubation with a control antibody did not alter APC-mediated changes in TER or phosphoMLC levels. Incubation with an antibody against the thrombin receptor, PAR1, had no effect on APC-mediated increases in phosphoMLC.
Conclusion APC significantly reduces thrombin-induced barrier dysfunction in ECs via an EPCR-dependent mechanism. APC-mediated alterations in the cytoskeletal protein phosphoMLC was EPCR-dependent but independent of an interaction of APC with PAR1. Modulation of EC barrier function may play a crucial role in the improved survival in patients with sepsis and ALI treated with APC.
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