Migration of activated corneal keratocytes (corneal fibroblasts) is involved in matrix patterning during developmental morphogenesis and is also required for repopulation of corneal tissue following wounding due to injury or surgery.7, 13
Following lacerating injury or incisional surgery, contractile force generation is needed to facilitate wound closure and prevent loss of the mechanical integrity of the cornea. However, following refractive surgical procedures such as PRK or LASIK, it is preferable to minimize cellular force generation and fibrosis during repopulation, since these alter corneal shape and transparency. Understanding how cell-matrix mechanical interactions are regulated during migration is the first step towards the development of strategies to modulate these aspects of corneal wound healing in vivo.
The Rho-family GTPases, such as Rho and Rac, play a central role in the regulation of cell morphology, cytoskeletal organization and global contraction of 3-D collagen matrices. Rho is known to promote increased phosphorylation of myosin light chain via Rho-kinase (ROCK) inhibition of myosin light chain phosphatase (MLCPase), resulting in increased actin-myosin II based cell contractility.1, 14
In time-lapse studies of corneal fibroblasts plated on top or within restrained 3-D collagen matrices, addition of LPA activates Rho, and induces retraction of cell processes and a corresponding pulling in of the surrounding ECM. 18, 20
In contrast, inhibiting ROCK induces rapid cell body elongation, formation and extension of dendritic cell processes, and a corresponding relaxation of cell-induced tension on the matrix. 23
Quantitative analysis of static confocal images has directly demonstrated that when Rho kinase is inhibited, cell-induced matrix reorganization (compaction and alignment of fibrils) is also significantly reduced.10, 11
However, despite an overall decrease in cellular force generation, both corneal and dermal fibroblasts can still displace the collagen surrounding them when Rho kinase is inhibited.17, 22
Furthermore, PDGF-induced elongation, ruffling and branching of pseudopodia still occurs in the presence of Y-27632 and/or blebbistatin (which inhibits myosin II), and small tractional forces are generated at the tips of extending processes.17
The mechanism underlying the generation of these small tractional forces and their role in cell migration is unclear.
Rho Kinase has been shown to facilitate 3-D cell invasion by aligning collagen and inhibiting cell branching thereby facilitating directional persistence.3, 19
However, the effect of Rho kinase on migration varies depending on cell type and the culture model used.5, 21
Furthermore, dynamic assessments of cell/matrix interactions in 3-D culture following Rho kinase inhibition have been limited. Thus despite its role in regulating cell contractility and matrix reorganization, the mechanisms through which Rho kinase regulates fibroblast migratory behavior within fibrillar collagen matrices remain unclear.
We recently developed a model for directly investigating cell-matrix mechanical interactions during migration in which cell-seeded compressed collagen matrices are nested within acellular uncompressed matrices.9
Compressed matrices can be generated rapidly, and have a geometry and mechanical properties similar to in vivo corneal tissue.2, 12
In this study, we modify this model to facilitate time-lapse imaging, thereby allowing direct visualization of the pattern of cell-matrix interactions during migration. We apply this novel experimental model to study the role of Rho kinase in regulating the mechanics of fibroblast migration in this 3-D model.