Mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as morphogenesis, wound healing, and organization of bioengineered tissues. Our research focuses on how these interactions regulate corneal keratocyte behavior, through development of culture models that mimic the 3-D tissue environment, and use of multi-dimensional imaging approaches in vitro, in situ and in vivo. Research in the prior period using 3-D culture models demonstrated that matrix composition, stiffness and structure can influence corneal keratocyte mechanical behavior and patterning in response to wound healing cytokines and changes in Rho/Rac activation. In addition, using our custom-modified in vivo HRT-RCM confocal microscope combined with ex vivo fluorescence and second harmonic generation (SHG) imaging, we demonstrated for the first time that following freeze injury (FI) or lamellar keratectomy (LK) in the rabbit, migrating fibroblasts within the wounded stroma form long interconnected streams that often run in parallel, and that alignment of these cell streams is highly correlated with that of the collagen lamellae. In contrast, cells migrating on top of the stroma following LK form a randomly arranged, interconnected, meshwork. The biochemical factors which induce myofibroblast transformation and fibrotic tissue generation on top of the stroma following injury or refractive surgery have been studied extensively. However, little is known about biochemical and biophysical signals that regulate intra-stromal keratocyte behavior. The lamellar structure of the cornea, combined with powerful in vivo and ex vivo imaging capabilities, provides us with a unique opportunity to assess biophysical factors that regulate cell differentiation, migration and patterning within this tissue.
Aim 1 will use in vivo confocal microscopy and in situ fluorescent/SHG imaging in the rabbit to: a) perform the first comprehensive comparison of intra-stromal and extra-stromal cell differentiation and patterning following photorefractive keratectomy (PRK), and b) investigate whether intra-stromal and extra- stromal phenotypes are differentially regulated.
Aim 2 will investigate whether changes in ECM structure and stiffness modulate cell patterning and mechanical phenotype during stromal repopulation by comparing migration mechanisms in two distinct in vivo injury models. ECM structure and mechanical properties have become increasingly recognized as key factors in determining cell growth, differentiation and activity in a variety of cell types; thus our findings should have broad scientific impact. In order to isolate the specific factors regulating these in vivo processes, Aim 3 will assess how cytokines and downstream Rho/Rac signaling impact corneal keratocyte patterning, mechanical differentiation, fibronectin deposition and ECM reorganization using multiple novel experimental models in vitro. With this approach we hope to identify the key biochemical and biophysical signaling pathways that differentiate disruptive and non-disruptive cell patterning behavior within 3-D matrices, which may lead to new strategies to modulate cell behavior in vivo.

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In this application, we investigate how corneal keratocyte behavior is regulated by extracellular matrix structure and mechanical properties, through the application of quantitative 3-D and 4-D imaging approaches in vitro, in situ and in vivo. The studies should provide new insights into the key biochemical and biophysical signaling pathways contributing to keratocyte-induced loss of corneal transparency following injury, surgery or disease.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology of the Visual System Study Section (BVS)
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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; 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
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
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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