Dry eye and associated ocular surface diseases affect over 40 million Americans and are the primary reason for visits to ophthalmologists. Unfortunately, treatments for dry eye are either palliative or treat inflammation and ocular surface apoptosis, but they are not cures and do not target the main cause of the disease, abnormal tear production. The present proposal focuses on the lacrimal gland secretion of the aqueous layer of tears and its dysfunction that causes aqueous deficiency dry eye (ADDE). The overall goal of this research is to understand the underlying cause of ADDE, the dysfunction of the lacrimal gland and to provide a broader base for the development of dry eye therapeutics beyond targeting inflammation that amplifies the original cause. In the present proposal the neurally-activated signaling mechanisms that regulate lacrimal gland secretion of proteins, electrolytes, and water will be compared in healthy and diseased lacrimal glands. The disease model to be used is the thrombospondin-1 (TSP-1)-/- mouse that mimics the chronic development of human ADDE. TSP-1 is a master regulator of cellular signaling and is best known for activation of TGF-b to prevent inflammation. TSP-1 additionally has multiple signaling domains and can function both extra- and intra-cellularly. Unlike other murine models of dry eye where inflammation remains indistinguishable as a cause or effect, in TSP-1-/- mouse lacrimal glands distinct secretory dysfunction is followed by destructive inflammatory responses. To date aberrant Ca2+ signaling and protein secretion induced by neurotransmitter activation of a1D- adrenergic and M3-muscarinic receptors has been documented in TSP-1 null mouse lacrimal glands. These abnormalities are consistent with abnormal tear secretion and impaired lacrimal gland cell proliferation in TSP-1-/- mice. Together these functional changes lead to dry eye disease. The goal of the present proposal is to identify the signaling mechanisms responsible for the alterations in TSP-1-/- mouse lacrimal glands thereby generating new targets for treatment of ADDE. The following specific aims are proposed: 1. Determine the signaling pathway components targeted in TSP-1-/- compared to wild type (WT) mouse lacrimal glands that cause the differential changes in a1D-adrenergic and M3-muscarinic receptor-induced changes in protein secretion that lead to secretion of abnormal tears, 2. Investigate the cellular mechanisms that lead to decreased cell proliferation and alteration of the progenitor cells in TSP-1-/- mouse lacrimal glands preventing repair, and 3. Investigate if replacement of TSP-1 in the lacrimal gland can restore lacrimal gland function and improve the signs of ADDE in TSP-1-/- mice. TSP-1-/- mouse lacrimal glands will be compared with WT glands at three ages: 1. no inflammation or secretory deficit, 2. secretory deficit and no inflammation, and 3. secretory deficit and inflammation. For in vitro studies peroxidase activity, FACS analysis, western blotting, ELISA, immunoprecipitation, single cell Ca2+, WST-8, and immunofluorescence microscopy will be used. For in vivo studies, corneal fluorescein staining and tear volume will be evaluated after treatment.

Public Health Relevance

Millions of Americans suffer from dry eye disease. The causes are varied and include aging, refractive surgery, menopause, autoimmune disease, and trauma causing damage to the ocular surface resulting in pain, inflammation, and irritation. There is no cure for dry eye and as the lacrimal gland is the primary contributor to the aqueous layer of the tear film, identification of the defective cellular mechanisms of a diseased lacrimal gland could lead to treatments for dry eye disease.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY006177-27A1
Application #
8372104
Study Section
Special Emphasis Panel (ZRG1-BDCN-H (02))
Program Officer
Mckie, George Ann
Project Start
1985-06-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
27
Fiscal Year
2012
Total Cost
$485,000
Indirect Cost
$235,000
Name
Schepens Eye Research Institute
Department
Type
DUNS #
073826000
City
Boston
State
MA
Country
United States
Zip Code
02114
Shatos, Marie A; Hodges, Robin R; Morinaga, Masahiro et al. (2016) Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1(-/-) mice, a model of dry eye. Exp Eye Res 153:27-41
Sanderson, Julie; Dartt, Darlene A; Trinkaus-Randall, Vickery et al. (2014) Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland. Exp Eye Res 127:270-9
Contreras-Ruiz, Laura; Ryan, Denise S; Sia, Rose K et al. (2014) Polymorphism in THBS1 gene is associated with post-refractive surgery chronic ocular surface inflammation. Ophthalmology 121:1389-97
Dartt, D A; Willcox, M D P (2013) Complexity of the tear film: importance in homeostasis and dysfunction during disease. Exp Eye Res 117:1-3
Shatos, Marie A; Haugaard-Kedstrom, Linda; Hodges, Robin R et al. (2012) Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland. Invest Ophthalmol Vis Sci 53:2749-59
Hodges, Robin R; Guilbert, Erin; Shatos, Marie A et al. (2011) Phospholipase D1, but not D2, regulates protein secretion via Rho/ROCK in a Ras/Raf-independent, MEK-dependent manner in rat lacrimal gland. Invest Ophthalmol Vis Sci 52:2199-210
Hodges, Robin R; Vrouvlianis, Joanna; Scott, Rachel et al. (2011) Identification of P2X₃ and P2X₇ purinergic receptors activated by ATP in rat lacrimal gland. Invest Ophthalmol Vis Sci 52:3254-63
Dartt, Darlene A; Hodges, Robin R (2011) Cholinergic agonists activate P2X7 receptors to stimulate protein secretion by the rat lacrimal gland. Invest Ophthalmol Vis Sci 52:3381-90
Dartt, Darlene A; Hodges, Robin R (2011) Interaction of alpha1D-adrenergic and P2X(7) receptors in the rat lacrimal gland and the effect on intracellular [Ca2+] and protein secretion. Invest Ophthalmol Vis Sci 52:5720-9
Ohtomo, Kaori; Shatos, Marie A; Vrouvlianis, Joanna et al. (2011) Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells. Invest Ophthalmol Vis Sci 52:9503-15

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