Abstract
Purpose: :
To explore the roles of Rho-kinase (ROCK) and myosin light chain kinase (MLCK) in migration, cell shape and organization of actin stress fibers in human optic nerve head (ONH) astrocytes from normal Caucasian Americans (CA) and Caucasian Americans with glaucoma (CAG).
Methods: :
Three human eyes from age-matched CA donors and three eyes from age-matched CAG donors were used to generate primary cultures of ONH astrocytes. To study migration, a 1mm pipette tip scratch was made on a 90% confluent monolayer of cells on coverslips treated with ML-7, an MLCK inhibitor, or Y-27632, a Rho-kinase inhibitor. Untreated cells were used as a control. The cell-free area of the wound was measured initially and after 24 h using Metamorph 7.5 image analysis software and percent area change was calculated. Experiments were carried out in triplicate for each CA and CAG donor. Data were analyzed for significance with ANOVA. To study the effect of Rho-kinase in astrocyte morphology, Y-27632 treated and untreated cells were stained with phalloidin and anti-focal adhesion kinase (FAK) and analyzed by confocal microscopy.
Results: :
The scratch assay showed that untreated CAG astrocytes migrated 24% faster compared to CA cells (p=0.006). After 24h the CA and CAG cells migrated 323 + 34µm and 425 + 42µm, respectively. ML-7 significantly slowed migration by 39% (p=0.0059) in CA astrocytes and 46% (p=0.0046) in CAG astrocytes. The MLCK inhibitor also led to disruption of the stress fibers in the center of the cell. Y-27632 had no effect in astrocyte migration, but caused disassembly of the actin cytoskeleton within 1 hour and induced astrocyte stellation and internalization of FAK in both CA and CAG cells. Stellate astrocytes exhibited long thin processes compared to polygonal untreated astrocytes.
Conclusions: :
The scratch assay demonstrates glaucomatous astrocytes migrate faster than normal astrocytes, consistent with the reactive phenotype. Our data shows that MLCK plays an important role in migration while Rho-kinase contributes to the maintenance of polygonal shape in quiescent astrocytes by strengthening the actin cytoskeleton and maintaining the focal adhesion plaques.
Keywords: astrocytes: optic nerve head