Abstract 12861: Di-Phosphorylation of Myosin Light Chain and Actin fiber formation in the Submembranous Region at Inter-endothelial Junction Play a Crucial Role in Thrombin-induced Endothelial Barrier Disruption
Background: Endothelial barrier dysfunction plays a fundamental role in the early phase of development of vascular diseases. The sequential phosphorylation of myosin light chain (MLC) at S19 and T18 and the resultant actin stress fiber formation is thought to cause endothelial barrier disruption. However, it remains unknown whether mono- and di-phosphorylation of MLC (pMLC and ppMLC) play a distinct role in barrier disruption.
Objective: The mechanism of endothelial barrier disruption was investigated by using porcine aortic endothelial cells, in terms of MLC phosphorylation and actin fiber formation.
Methods and Results: Thrombin (1 u/mL) induced barrier disruption with a peak at 3-5 min, as evaluated by a decrease in transendothelial electrical resistance (TER). Phos-tag SDS-PAGE analysis revealed that the cells at confluence contained 25% pMLC and 2% ppMLC before thrombin stimulation. Thrombin slightly increased pMLC at 3 min in the perinuclear region. In contrast, ppMLC transiently increased to 35% in the submembranous region at the inter-endothelial junction, and it was associated with the actin fiber formation. The stress fiber formation was obscure. Blebbistatin, a myosin ATPase inhibitor, inhibited the thrombin-induced decrease in TER. ROCK inhibition abolished the thrombin-induced ppMLC and actin fiber formation in the submembranous region and the decrease in TER. Adenoviral expression of a phosphorylation defect T18A+S19A mutant of MLC abolished MLC phosphorylation and actin fiber formation in the submembranous region, and prevented the thrombin-induced decrease in TER. In the cell expressing T18A or S19A mutant, pMLC, but not ppMLC, was observed along with the actin fiber formation in the submembranous region, and thrombin decreased TER as in the cells expressing wild type MLC.
Conclusions: pMLC and ppMLC play a distinct role in endothelial barrier disruption. ROCK-mediated ppMLC and actin fiber formation seen in the submembranous regions and the circumferential, but not radial, contraction plays a crucial role in the thrombin-induced barrier disruption. However, pMLC is sufficient to induce barrier disruption. Inhibition of ppMLC in the submembranous regions is crucial for restoring normal endothelial barrier function.
- © 2013 by American Heart Association, Inc.