Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. The overall goal of this research is to determine the underlying cellular and molecular mechanisms that regulate these critical biophysical processes in corneal fibroblasts, which should ultimately lead to more effective approaches to directing their biomechanical behavior in vivo and in vitro. 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 regulating sub-cellular force generation, matrix reorganization, as well as the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) The balance between Rac and RhoA activity plays a central role in regulating the mechanical behavior of corneal fibroblasts inside 3-D matrices at the subcellular level. These effects are mediated by dynamic changes in cytoskeletal and focal adhesion organization, as well as differential regulation of myosin light chain (MLC) phosphorylation, 2) Corneal fibroblasts actively respond to increases or decreases in local matrix stress (including that produced by neighboring cells); these responses are mediated by compensatory and reciprocal changes in Rho and Rac activation, and 3) The pattern and amount of collagen matrix reorganization can be modulated by altering the balance between Rho and Rac activity, and is enhanced by cell-cell mechanical interactions. 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) Measure the dynamic mechanical response of corneal fibroblasts to changes in ECM stress; and, 3) Directly assess the process of cell-induced 3-D collagen matrix reorganization (alignment of collagen fibrils), and the roles of Rho and Rac in regulating this process. These studies will be the first to assess the roles of Rho and Rac on the subcellular pattern of force generation and ECM reorganization within 3-D collagen matrices. Overall, this research should provide unique insights into the mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization, critical processes in the fields of developmental biology, wound healing and tissue engineering. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY013322-05
Application #
7001201
Study Section
Special Emphasis Panel (ZRG1-BDCN-H (03))
Program Officer
Shen, Grace L
Project Start
2001-02-01
Project End
2007-11-30
Budget Start
2005-12-01
Budget End
2006-11-30
Support Year
5
Fiscal Year
2006
Total Cost
$342,752
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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