Wound healing in the cornea is incompletely understood;yet each year thousands of people elect corrective photorefractive surgery. Identification of mechanisms involved in wound healing will lead to the development of treatments for patients who do not heal properly. In this proposal, thrombin, a protease that generates fibrin and regulates cellular processes, will be studied. Scientific Description: The goal of this application is to test the hypothesis that thrombin is involved in corneal wound healing through cleavage of protease activated receptors and initiation of signaling pathways. Our preliminary data show the components required to convert prothrombin to thrombin and protease activated receptors mRNA in the human cornea and the ability of thrombin to alter corneal stromal cell gene expression and cell division. The thrombin inhibitor hirudin inhibits corneal epithelial wound healing.
The SPECIFIC AIMS of this proposal are: 1) TO DETERMINE WHETHER THROMBIN CAN REGULATE KNOWN CELLULAR STEPS IN CORNEAL WOUND HEALING. Cultured corneal epithelial cells, stromal keratocytes,fibroblasts and/or myofibroblasts treated with thrombin will be assayed for thrombin dependent changes in phenotype, apoptosis, total cell number, cell division, and migration. 2) TO DETERMINE WHETHER THROMBIN STIMULATES CORNEAL STROMAL CELL SYNTHESIS OF PROTEINS INVOLVED IN WOUND HEALING. The effect of thrombin on cytokine, chemokine, growth factor and plasminogen activator system component synthesis will be determined using real-time RT-PCR to characterize thrombin dependent mRNA changes and ELISA and/or western blots for changes in protein levels. 3) TO DETERMINE THE MECHANISM OF INDUCTION OF THROMBIN EFFECTS ON CORNEAL CELL FUNCTION AND GENE EXPRESSION. The mechanism of thrombin induced changes in cell division of stromal myofibroblasts and stimulation of PAI-1 synthesis will be determined using thrombin inhibitors, inactivated thrombin, thrombin peptides, agonist and antagonists to thrombin sensitive protease activated receptors and signaling pathway inhibitors. 4) TO DETERMINE WHETHER THROMBIN AND PAR-1 ARE IMPORTANT FOR CORNEAL WOUND HEALING IN VIVO AND IN ORGAN CULTURE. Rabbit and human organ culture models and an in vivo model using normal and PAR-1 deficient mice will be used for these studies.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012731-09
Application #
8013793
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
1999-08-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2012-12-31
Support Year
9
Fiscal Year
2011
Total Cost
$323,968
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Barnes, Jarrod; Warejcka, Debra; Simpliciano, Jennifer et al. (2012) Latency-associated peptide of transforming growth factor-β1 is not subject to physiological mannose phosphorylation. J Biol Chem 287:7526-34
Warejcka, Debra J; Narayan, Malathi; Twining, Sally S (2011) Maspin increases extracellular plasminogen activator activity associated with corneal fibroblasts and myofibroblasts. Exp Eye Res 93:618-27
Narayan, Malathi; Mirza, Shama P; Twining, Sally S (2011) Identification of phosphorylation sites on extracellular corneal epithelial cell maspin. Proteomics 11:1382-90
Endsley, Michael P; Hu, Yanqiu; Deng, Yong et al. (2011) Maspin, the molecular bridge between the plasminogen activator system and beta1 integrin that facilitates cell adhesion. J Biol Chem 286:24599-607
Narayan, Malathi; Twining, Sally (2010) Focus on molecules: maspin. Exp Eye Res 90:2-3
White, Mark J; He, Hongjun; Penoske, Renee M et al. (2010) PepD participates in the mycobacterial stress response mediated through MprAB and SigE. J Bacteriol 192:1498-510
Bohnsack, Richard N; Patel, Manish; Olson, Linda J et al. (2010) Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor. Biochemistry 49:635-44
Horswill, Mark A; Narayan, Malathi; Warejcka, Debra J et al. (2008) Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts. Exp Eye Res 86:586-600
Warejcka, Debra J; Twining, Sally S (2005) Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins. Biochem J 392:703-12
Warejcka, Debra J; Vaughan, Kimberly A; Bernstein, Audrey M et al. (2005) Differential conversion of plasminogen to angiostatin by human corneal cell populations. Mol Vis 11:859-68

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