Sinusoidal endothelial fenestrae organization regulated by myosin light chain kinase and Rho-kinase in cultured rat sinusoidal endothelial cells

Abstract

The presence of actin filaments in the neighborhood of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. We examined the roles of Rho-kinase and myosin light chain kinase (MLCK) in the organization of SEF. Cultured SEC were treated with MLCK inhibitor (ML-7) and Rho-kinase inhibitor (Y-27632). SEF morphology was observed by scanning electron microscopy. F-actin stress fibers were observed by confocal microscopy and heavy meromyosin-decorated reaction under transmission electron microscopy. Y-27632 caused disassembly of stress fibers in the center of the cell, while SEF clustered and dilated. However, stress fibers located in the periphery of the cell were not severely affected by Y-27632. ML-7 caused disruption and/or shortening of peripheral stress fibers, leaving the central stress fibers relatively intact. ML-7, but not Y-27632, caused cells to lose the spreading morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. Thus, there are at least two different stress fiber systems in SEC. The central stress fiber system and SEF microfilaments depend more on the activity of Rho-kinase, while the peripheral stress fiber system depends on MLCK. These results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.