Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and remodeling of bioengineered tissues. The overall goal of this research is to determine the underlying biochemical and biophysical mechanisms which regulate these critical processes in corneal fibroblasts. In the first funding period, we developed a new experimental model for directly investigating cell-matrix mechanical interactions inside 3-D fibrillar collagen matrices. Data obtained using this innovative approach has provided new insights into potential mechanisms of sub-cellular force generation, matrix remodeling, and the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) Rac induces spreading of corneal fibroblasts via localized tractional force generation by extending pseudopodia, whereas Rho induces contractile force generati0n along the cell body; these effects ? are mediated by differences in sub-cellular regulation of myosin light chain phosphorylation, 2) corneal ? fibroblasts actively respond to increases or decreases in local matrix stress in order to maintain tensional homeostasis (constant tension); these responses are mediated by compensatory and reciprocal changes in Rho and Rac; and 3) the pattern and amount of permanent collagen matrix remodeling is maximal in stationary, contractile cells (high Rho and low Rac activity), and is enhanced by cell-cell mechanical communication at higher densities. To test these hypotheses, we propose the following Specific Aims: 1) determine the role of Rho and Rac in regulating cytoskeletal organization, mechanical behavior, and sub-cellular force generation by corneal fibroblasts inside 3-D matrices using our time-lapse imaging system, 2) investigate the mechanical response of corneal fibroblasts to changes in ECM stress by inserting microneedles into the ECM next to isolated cells, in order to modulate matrix stiffness; and, 3) compare the pattern and amount of local collagen matrix remodeling by corneal fibroblasts at different cell densities and culture conditions. Accomplishing these Aims should provide a better understanding of how the fundamental processes of cell spreading, contraction and matrix remodeling are regulated. This is an important step towards our long-term objective of controlling the organization and mechanical behavior of corneal fibroblasts inside artificial matrices ? through the design of """"""""smart"""""""" 3-D ECM scaffolds that incorporate both biochemical and mechanical cues. ? ?

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
3R01EY013322-04A1S1
Application #
7123593
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Fisher, Richard S
Project Start
2001-02-01
Project End
2007-11-30
Budget Start
2004-12-01
Budget End
2005-11-30
Support Year
4
Fiscal Year
2005
Total Cost
$71,387
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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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