The mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. This project is focused on investigating how these interactions regulate corneal keratocyte mechanical behavior and its role in corneal transparency, through the development of novel 3-D culture models, and the application of quantitative 3-D and 4-D imaging techniques. Research conducted in the prior grant period has provided important insights into the regulation of corneal keratocyte spreading and migration within 3-D matrices. We demonstrated that corneal keratocytes differentiate into distinct mechanical phenotypes (dendritic vs. contractile) in response to specific growth factors expressed during wound healing, and that this process is regulated, in part, by the balance between Rho and Rac activation and the mechanical stiffness of the ECM. We also demonstrated for the first time that whereas corneal fibroblasts generally move independently within collagen matrices, fibrin induces a switch to an interconnected, collective mode of cell spreading and migration, which is associated with localized fibronectin secretion, cadherin expression and development of intracellular stress fibers. Using an in vivo HRT- RCM confocal microscope that we custom-modified to allow quantitative full-thickness corneal imaging, we also generated pilot data suggesting for the first time that dendritic migration can occur in vivo following mechanical scrape injury in the rabbit, in which healing occurs without significant loss of transparency. In contrast, a collective, fibroblastic morphology was observed following transcorneal freeze injury (which leads to increased cellular light scattering and fibrosis). Corneal myofibroblasts have also been shown to organize into an interconnected mesh following incisional surgery or photorefractive keratectomy (PRK). In the current application, we will further characterize the mechanisms regulating corneal keratocyte mechanical behavior in vitro (in 3-D culture), and correlate these with wound healing phenotypes observed in vivo.
Specific Aim 1 will establish how growth factors modulate cell contractility, matrix reorganization production of normal and fibrotic ECM components and ALDH1A1 expression levels in collagen and fibrin matrices of varying stiffness, and determine whether these expression profiles are modulated by inhibiting Rho kinase. These studies will be the first to determine how matrix composition and stiffness, growth factors and Rho kinase activation regulate expression of normal and fibrotic keratocyte markers and corneal crystallins in 3-D culture;factors that are directly associated with maintenance or loss of corneal transparency during in vivo wound healing.
Specific Aim 2 will determine the dependency of collective cell migration on growth factors, expression and localization of fibronectin, ?5?1 integrin and cadherin, ECM degradation and patterning, and Rho kinase-mediated cell contractility, using both quiescent corneal keratocytes and activated dermal and corneal fibroblasts. Corneal fibroblasts and myofibroblasts have previously been shown to form an interconnected network during healing after incisional surgery, lamellar keratectomy and PRK. Our unique model for studying collective cell migration in 3-D culture should provide important new insights into the mechanical and biochemical regulation of this fundamental process.
Specific Aim 3 will in vivo confocal imaging and immunocytochemistry to correlate cell morphology, connectivity and backscattering with expression of fibroblast and myofibroblast markers after mechanical scrape, transcorneal freeze and lamellar keratectomy in the rabbit, and determine whether these responses can be modulated by inhibiting Rho kinase (using Y-27632). Fibroblast and myofibroblast transformation of quiescent corneal keratocytes lead to corneal haze development following injury or surgery. Our research suggests that Y-27632 is a natural candidate for inhibiting this transformation in vivo (possible bench to beside translation of a new therapy). During the course of the grant, training will be provided to a total of two biomedical engineering graduate (PhD) students, two post-docs, as well as 4 summer medical students and 3 undergraduate research fellows.

Public Health Relevance

In this application, we investigate how corneal keratocyte mechanical behavior is regulated in vitro (using innovative 3-D culture models), and correlate these findings with wound healing phenotypes observed in vivo. This bench to bedside studies should provide new insights into the factors contributing to the loss of corneal transparency (and thus visual acuity) that can occur following injury or refractive surgery, and directly test an inhibitor which may restore normal function.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Mckie, George Ann
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University of Texas Sw Medical Center Dallas
Schools of Medicine
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Miron-Mendoza, Miguel; Graham, Eric; Manohar, Sujal et al. (2017) Fibroblast-fibronectin patterning and network formation in 3D fibrin matrices. Matrix Biol 64:69-80
Robertson, Danielle M; Rogers, Nathan A; Petroll, W Matthew et al. (2017) Second harmonic generation imaging of corneal stroma after infection by Pseudomonas aeruginosa. Sci Rep 7:46116
Kivanany, Pouriska B; Grose, Kyle C; Petroll, W Matthew (2016) Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy. Exp Eye Res 153:56-64
Petroll, W Matthew; Robertson, Danielle M (2015) In Vivo Confocal Microscopy of the Cornea: New Developments in Image Acquisition, Reconstruction, and Analysis Using the HRT-Rostock Corneal Module. Ocul Surf 13:187-203
Petroll, W Matthew; Miron-Mendoza, Miguel (2015) Mechanical interactions and crosstalk between corneal keratocytes and the extracellular matrix. Exp Eye Res 133:49-57
Petroll, W Matthew; Lakshman, Neema (2015) Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness. J Funct Biomater 6:222-40
Miron-Mendoza, Miguel; Graham, Eric; Kivanany, Pouriska et al. (2015) The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments. Invest Ophthalmol Vis Sci 56:2079-90
Koppaka, Vindhya; Lakshman, Neema; Petroll, W Matthew (2015) Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices. Mol Vis 21:502-14
Petroll, W Matthew; Kivanany, Pouriska B; Hagenasr, Daniela et al. (2015) Corneal Fibroblast Migration Patterns During Intrastromal Wound Healing Correlate With ECM Structure and Alignment. Invest Ophthalmol Vis Sci 56:7352-61
Zhou, Chengxin; Petroll, W Matthew (2014) MMP regulation of corneal keratocyte motility and mechanics in 3-D collagen matrices. Exp Eye Res 121:147-60

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