Lisha Ma

Dynamic Assessment of Fibroblast Mechanical Activity During Rac-Induced Cell Spreading in 3-D Culture

The goal of this study was to determine the morphological and sub‐cellular mechanical effects of Rac activation on fibroblasts within 3‐D collagen matrices. Corneal fibroblasts were plated at low density inside 100 µm thick fibrillar collagen matrices and cultured for 1–2 days in serum‐free media. Time‐lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y‐27632 or blebbistatin were used to inhibit Rho‐kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac‐induced cell spreading within 3‐D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II‐based tractional forces during cell spreading within 3‐D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II‐independent. J. Cell. Physiol. 217: 162–171, 2008.

CHANGES IN CORNEAL ENDOTHELIAL APICAL JUNCTIONAL PROTEIN ORGANIZATION AFTER CORNEAL COLD STORAGE

PURPOSE Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium. METHODS Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media. RESULTS In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas. CONCLUSION These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.

Organization of junctional proteins in proliferating cat corneal endothelium during wound healing

Purpose. To evaluate for the first time cell junctional protein organization in proliferating corneal endothelial cells during in vivo wound healing. Methods. A total of 16 cats (32 eyes) were used in this study. A single 3-mm diameter (n = 24) or 1-to 2-mm diameter (n = 8) scrape injury was created in the central corneal endothelium of each eye. Twenty-four, 48, 72 hours or 5 days after scrape injury, eyes were collected for in situ double-or triple-labeling with phalloidin, anti-ZO-1, &agr;-catenin, &bgr;-catenin, and MIB-1 (monoclonal antibody to Ki67, a marker for actively cycling cells) and were imaged using confocal laser microscopy. Results. In 3-mm diameter injuries, endothelial cells completely resurfaced the wound 48 to 72 hours after scrape injury; smaller wounds resurfaced by 48 hours. Ki67 staining was negative 24 hours after scrape injury in all cases. Ki67-positive cells were observed in the central region of the wounds after 48 and 72 hours, and mitotic figures and pairs of postmitotic cells were observed. On day 5, Ki67-positive cells were rarely detected, and no mitotic figures were observed. In the wound area, a significant increase in cell area and a reduction in hexagonality were observed in cycling cells after 48 and 72 hours. Normal apical, pericellular staining of f-actin, ZO-1, &agr;-catenin, and &bgr;-catenin was partially maintained at all times during wound healing of small and large wounds. Double-labeling confirmed that these proteins were also present along the apical cell border in Ki67-positive cells. Conclusions. After in vivo scrape injury, proliferation is limited temporally and spatially to spreading endothelial cells within the wound. Cell junctional connections appear to be maintained in actively cycling cells during healing.

Analysis of the pattern of subcellular force generation by corneal fibroblasts after Rho activation

Purpose. To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. Methods. Human corneal fibroblasts were plated at low density in 100-&mgr;m thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 &mgr;mol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. Results. Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (−12.1% ± 7.0%; P<0.05) and cell area (−13.1% ± 13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. Conclusions. The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.

Experimental Models for Investigating Intra-Stromal Migration of Corneal Keratocytes, Fibroblasts and Myofibroblasts

Following laser vision correction, corneal keratocytes must repopulate areas of cell loss by migrating through the intact corneal stroma, and this can impact corneal shape and transparency. In this study, we evaluate 3D culture models for simulating this process in vitro. Buttons (8 mm diameter) were first punched out of keratocyte populated compressed collagen matrices, exposed to a 3 mm diameter freeze injury, and cultured in serum-free media (basal media) or media supplemented with 10% FBS, TGFβ1 or PDGF BB. Following freeze injury, a region of cell death was observed in the center of the constructs. Although cells readily migrated on top of the matrices to cover the wound area, a limited amount of cell migration was observed within the constructs. We next developed a novel “sandwich” model, which better mimics the native lamellar architecture of the cornea. Using this model, significant migration was observed under all conditions studied. In both models, cells in TGFβ and 10% FBS developed stress fibers; whereas cells in PDGF were more dendritic. PDGF stimulated the most inter-lamellar migration in the sandwich construct. Overall, these models provide insights into the complex interplay between growth factors, cell mechanical phenotypes and the structural properties of the ECM.

Localized application of mechanical and biochemical stimuli in 3-D culture.

The goal of this study was to investigate the responses of isolated cells in 3‐D culture to localized application of mechanical and biochemical signals. Corneal fibroblasts were plated inside collagen matrices for 24 hours, then imaged using time‐lapse DIC. For mechanical perturbation, a microinjection needle (Femtotip) was inserted axially into the ECM, then displaced laterally to alter local ECM stress. For biochemical stimulation, PDGF or vehicle control solution was microinjected into the matrix. Compressing the ECM perpendicular to the cell axis had no appreciable effect on cell behavior. However, pushing the ECM parallel to the cell axis induced rapid cellular contraction, followed by secondary cell spreading and tractional force generation. Injection of PDGF induced a similar cell spreading response. Cells in 3‐D matrices showed remarkable plasticity, and extension of pseudopodia could be induced at both the leading and trailing edges of migrating cells. Developmental Dynamics 237:2726–2736, 2008.