The Functional Roles of Rho-Kinase and Matrix Metalloproteinases in Regulating Corneal Stromal Cell Mechanics in 3-D Collagen Matrices

The main focus of my research has been on understanding the biomechanical and biochemical mechanisms of cell-extracellular matrix (ECM) interactions during corneal wound healing, which may allow the development of new therapeutic strategies to promote corneal regeneration. Previous studies have established that the Rho GTPases play a central role in regulating the cytoskeletal changes associated with cell mechanical activity. A novel force monitoring system was successfully developed to investigate the role of Rho in corneal cell force generation in 3-D collagen matrices. Maximum tractional force generated by 9 million corneal fibroblasts in serum culture was around 265 Dynes. Inhibition of Rho kinase by Y-27632 induced a 69% force reduction. These results demonstrated that Rho/Rho kinase play a key role in mediating contractile force generation of corneal stromal fibroblasts in serum culture. I also investigated the functions of Rho GTPase signaling in corneal stromal fibroblast migration and cell-ECM interactions using a 3-D nested matrix construct. The experimental results showed that both the amount and the speed of corneal fibroblast migration and local collagen matrix reorganization were significantly inhibited by Y-27632. Following the inhibition, cells extended thinner dendritic processes into the outer matrix, and generated tractional forces at their leading edge. However, cells were unable to generate contractile forces needed to retract their tail and pull the cell body forward through the collagen matrix. I also studied the role of Matrix metalloproteinases (MMPs) in corneal cell mechanics, since these have been recognized as an influential component in extracellular matrix turnover and corneal repair. I first assessed the expression and collagenolytic activities of MMPs by primary corneal keratocyte in response to different signaling factors. I then studied the functions of MMPs in regulating keratocyte migration, cell-induced matrix contraction, and cell protrusive activity in 3-D collagen matrices. This study suggested that, in serum free PDGF culture, although collagenolysis was limited to a pericellular scale, primary corneal keratocytes utilized MMPs to facilitate cell migration, ECM contraction, cell spreading in 3-D collagen matrices. Thus MMPs may play a key role in facilitating cell-collagen matrix interactions by corneal keratocytes, without producing widespread disruption of corneal ECM structure.