Corneal opacities, a leading cause of blindness worldwide, are commonly a result of stromal fibrosis or haze from dysregulated wound healing. Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical as well as biophysical cues associated with the wound space. Research from our laboratory and others have demonstrated biophysical attributes of the extracellular matrix profoundly modulate a host of fundamental cellular behaviors essential to the maintenance of homeostasis and wound repair including adhesion, migration, proliferation and differentiation. We have demonstrated that the intrinsic mechanical properties of the corneal stroma are significantly altered in a time dependent manner throughout wound healing. The matrix associated with the wound space was stiffest at 7 days with the greatest myofibroblast numbers and degree of stromal haze occurring at a later time point. This temporal relationship, combined with in vitro data documenting stiffer substrates to promote myofibroblast transformation, suggests a causal relationship.
In Aim 1 we propose to directly modulate matrix stiffness in vivo using clinically relevant approaches and establish the impact of stiffening and softening of the wound matrix on wound healing outcomes using a well-defined rabbit PTK model. The complex inter-relationship between cytoskeletal dynamics and matrix remodeling and stiffness is central to determining wound healing outcomes but remains little investigated and poorly understood and are the central focus of Aim 2. Here, the impact of altering cytoskeletal dynamics in vitro on cell derived extracellular matrix elaboration, remodeling and mechanics, and the impact of altering cell derived matrix stiffness on keratocytes to myofibroblast transformation will be determined. Preliminary data demonstrate that the mechanical properties of ECM derived from cell in vitro can be modulated by applying diverse clinically relevant crosslinking methods. Preliminary data also document that modulating the cellular stiffness using drugs that alter the cytoskeleton can dramatically influence myofibroblast transformation. Stiffening of the cell promoted myofibroblast transformation. While softening of the cell inhibited transformation even in the presence of TGF? signaling. Additionally, the complex signaling dynamic that exist between stromal and epithelial cells will be investigated using a novel microfluidic co-culture platform in Aim 3. This novel system allows for the simultaneous presentation of multiple yet distinct biophysical and biochemical stimuli in addition to controlled exchange of soluble signaling molecules between epithelial and stromal cells. We will determine if soluble signals from the epithelial cells have a differential effect on KFM transformation under various biophysical stimuli. In aggregate the experiments detailed in this proposal are aimed at testing our central concept that optimal outcomes in corneal wound repair can be facilitated by engineering the intrinsic biophysical attributes of cells and/or matrices.

Public Health Relevance

The cornea can be wounded by trauma, infection and from complications of common surgical procedures such as LASIK. If corneal wound healing is not optimal then corneal haze can develop which can significantly impair vision. The goal of this project is to better understand the mechanobiology of the cornea and to improve corneal wound healing outcomes for patients. Studies are aimed at altering the inherent mechanical properties of the tissue and/or cells.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY019970-06
Application #
9185333
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Mckie, George Ann
Project Start
2010-12-01
Project End
2020-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
6
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California Davis
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Thomasy, Sara M; Raghunathan, Vijay Krishna; Miyagi, Hidetaka et al. (2018) Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation. Exp Eye Res 170:101-107
Miyagi, Hidetaka; Jalilian, Iman; Murphy, Christopher J et al. (2018) Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance. Exp Eye Res 176:235-242
Raghunathan, VijayKrishna; Eaton, J Seth; Christian, Brian J et al. (2017) Biomechanical, ultrastructural, and electrophysiological characterization of the non-human primate experimental glaucoma model. Sci Rep 7:14329
Raghunathan, Vijay Krishna; Thomasy, Sara M; Strøm, Peter et al. (2017) Tissue and cellular biomechanics during corneal wound injury and repair. Acta Biomater 58:291-301
Ali, Maryam; Raghunathan, VijayKrishna; Li, Jennifer Y et al. (2016) Biomechanical relationships between the corneal endothelium and Descemet's membrane. Exp Eye Res 152:57-70
Thomasy, Sara M; Cortes, Dennis E; Hoehn, Alyssa L et al. (2016) In Vivo Imaging of Corneal Endothelial Dystrophy in Boston Terriers: A Spontaneous, Canine Model for Fuchs' Endothelial Corneal Dystrophy. Invest Ophthalmol Vis Sci 57:OCT495-503
Strom, Ann R; Cortés, Dennis E; Rasmussen, Carol A et al. (2016) In vivo evaluation of the cornea and conjunctiva of the normal laboratory beagle using time- and Fourier-domain optical coherence tomography and ultrasound pachymetry. Vet Ophthalmol 19:50-6
Strom, Ann R; Cortés, Dennis E; Thomasy, Sara M et al. (2016) In vivo ocular imaging of the cornea of the normal female laboratory beagle using confocal microscopy. Vet Ophthalmol 19:63-7
Horikawa, Taemi; Thomasy, Sara M; Stanley, Amelia A et al. (2016) Superficial Keratectomy and Conjunctival Advancement Hood Flap (SKCAHF) for the Management of Bullous Keratopathy: Validation in Dogs With Spontaneous Disease. Cornea 35:1295-304
Morgan, Joshua T; Kwon, Heung Sun; Wood, Joshua A et al. (2015) Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells. Exp Eye Res 135:127-33

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