Abstract
Purpose: :
To determine how substrate stiffness underlying human trabecular meshwork (HTM) cells alters their response to the cytoskeleton disrupting agent Latrunculin B (Lat B). Changes in the inherent stiffness of the cells as well as their migration rates were measured
Methods: :
Substrates used in this study were glass, tissue culture plastic and hydrogels with Young’s moduli of 4 kPa and 92 kPa stiffness. The 4 kPa hydrogels mimic the modulus of normal HTM while the 92 kPa hydrogels mimic glaucomatous HTM. After adhering to the surfaces, cells were treated for 30 minutes with 0.2µM Lat B in phosphate buffered saline. Using atomic force microscopy, the modulus of the cells was taken prior to treatment, and 90 minutes and 270 minutes after removal of the Lat B. For migration analysis, the cells were followed for 6 hours before treatment and for 7 hours after Lat B.
Results: :
On stiffer substrates, HTM cells were intrinsically stiffer and had more numerous actin stress fibers. When Lat B was added to the medium, the cells rounded up, and modulus was difficult to measure accurately. After removal of Lat B at 90 minutes, the cells had Young’s modulus values greater than the predose levels. At this time point, cell moduli were increased 1.4, 2.2 and 4.1 fold for the 4 kPa gels, 92 kPa and the glass substrates, respectively. On all substrates, the cells returned to their pre-dose moduli at 270 minutes. The migration rates for the HTM cells decreased by 2 fold on the 92 kPa substrate, but changed very little on the 3 kPa hydrogels.
Conclusions: :
With HTM cells, more stress fibers were visible on stiffer substrates. Latrunculin B decreased the modulus of the cells. After removal of latrunculin B, there was a rebound of cell modulus to values greater than their pre-dose levels. The amount of rebound was dependent of the substrate modulus. The migration rate was lower on all substrates 7 hours after Lat B. The data show biophysical properties significantly influence cellular behaviors.
Keywords: trabecular meshwork • cytoskeleton