The corneal endothelium is responsible for maintaining corneal clarity, but these cells do not divide throughout life. Our long-term goal is to develop methods to stimulate proliferation and increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Human corneal endothelial cells CAN divide in organ culture, suggesting that multiple factors together exert a strong anti-proliferative pressure in vivo. Contact-dependent inhibition of proliferation appears to be an important anti-proliferative factor, which is regulated, in part, by phosphotyrosine phosphatases (PTPs). Activation of receptor tyrosine kinases, such as the EGF receptor, leads to tyrosine phosphorylation (Tyr-PO4)-induced cell cycle entry. Cell-cell contacts can be disrupted by Tyr-PO4 of cadherin-associated proteins. PTPs inhibit both Tyr-PO4 events, suggesting that PTP activity helps maintain contact-dependent inhibition of proliferation. Release of cell-cell contacts in confluent endothelium does NOT stimulate cell cycle entry in the absence of mitogens. Sodium orthovanadate (SOV), a PTP inhibitor, releases cell contacts and promotes cell cycle entry without mitogens, suggesting that inhibition of PTP activity might augment the proliferative effect of mitogens, such as EGF, in confluent endothelium. We hypothesize that, by down-regulating the protein expression or inhibiting the activity of specific PTPs in the endothelium, we will be able to make the cells more responsive to mitogenic stimulation, thus providing a method to increase cell density in individuals at risk for vision loss due to low endothelial cell counts.
Specific Aims proposed to test this hypothesis include: I. Identify specific PTPs expressed in corneal endothelial cells; II. Determine the importance of each PTP in regulating EGF-driven mitogenic signaling; III. Determine whether the cadherin-catenin complex is a target for PTP regulation in corneal endothelium; and IV. Test if down-regulating the expression or inhibiting the activity of specific PTPs will promote proliferation and increase cell density in human corneal endothelial cells in situ. Immunoprecipitation, Western blotting, RT-PCR, immunocytochemistry, flow cytometry, antisense methods, and expression of dominant-negative mutants will be used to accomplish the objectives of this proposal.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY005767-18
Application #
6769511
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Fisher, Richard S
Project Start
1985-07-01
Project End
2006-06-30
Budget Start
2004-09-01
Budget End
2005-06-30
Support Year
18
Fiscal Year
2004
Total Cost
$343,000
Indirect Cost
Name
Schepens Eye Research Institute
Department
Type
DUNS #
073826000
City
Boston
State
MA
Country
United States
Zip Code
02114
Joyce, Nancy C (2012) Proliferative capacity of corneal endothelial cells. Exp Eye Res 95:16-23
Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L et al. (2008) Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells. Mol Vis 14:61-70
Harris, Deshea L; Joyce, Nancy C (2007) Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells. Mol Vis 13:785-96
Konomi, Kenji; Joyce, Nancy C (2007) Age and topographical comparison of telomere lengths in human corneal endothelial cells. Mol Vis 13:1251-8
Konomi, Kenji; Zhu, Cheng; Harris, Deshea et al. (2005) Comparison of the proliferative capacity of human corneal endothelial cells from the central and peripheral areas. Invest Ophthalmol Vis Sci 46:4086-91
Joyce, Nancy C; Zhu, Cheng Chris (2004) Human corneal endothelial cell proliferation: potential for use in regenerative medicine. Cornea 23:S8-S19
Zhu, Cheng; Joyce, Nancy C (2004) Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci 45:1743-51
Joyce, Nancy C; Harris, Deshea L; Mello, David M (2002) Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2. Invest Ophthalmol Vis Sci 43:2152-9
Zieske, J D; Hutcheon, A E; Guo, X et al. (2001) TGF-beta receptor types I and II are differentially expressed during corneal epithelial wound repair. Invest Ophthalmol Vis Sci 42:1465-71
Senoo, T; Obara, Y; Joyce, N C (2000) EDTA: a promoter of proliferation in human corneal endothelium. Invest Ophthalmol Vis Sci 41:2930-5

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