Glaucoma is a leading cause of preventable blindness. Elevated intraocular pressure (IOP) is a causitive risk factor and is due to altered drainage through the trabecular meshwork (TM). Extracellular matrix (ECM) turnover is known to be one of the factors that influences IOP regulation in the TM, but little is known of how ECM turnover is controlled. Furthermore, abnormal accumulations of ECM in the TM are seen in both untreated and treated eyes with primary open-angle glaucoma (POAG) and is, thus, a primary disease mechanism. Transforming growth factor beta-2 (TGF?2) is elevated up to 3-fold in the aqueous humor of POAG patients, and in various experimental systems, increases IOP with resultant alterations of ECM in the TM. Matricellular proteins are non-structural secreted glycoproteins that facilitate cellular control over their surrounding ECM. Matricellular proteins, and in particular SPARC, are associated with diseases of aberrent fibrosis. Under the auspices of the prior grant, we demonstrated that SPARC regulates IOP and increases fibronectin, collagens ?I and ?IV in the juxtacanalicular TM concurrent with decreased matrix metalloproteinase-9 (MMP-9) activity. The molecular mechanisms causing the changes in ECM and MMP activity are unclear. Integrin-linked kinase has been shown in other tissues as a downstream receptor for SPARC and is known to affect MMP activity and the actin cytoskeleton. Furthermore, we and others have shown that SPARC is the most highly expressed protein by TM cells in response to TGF?2. We demonstrated that TGFb2 regulates SPARC via smad3, JNK, and p38 signaling pathways. In SPARC -/- mice, there is a near complete block of the IOP elevation normally seen with increased TGF?2 indicating that SPARC is a critical regulatory node in the pathophysiology of POAG. We hypothesize that suppressing SPARC or inhibiting SPARCs downstream effector, ILK, will lower IOP in normotensive and TGF?2-induced hypertensive models. As a chaperone, SPARC allows accumulation of structural ECM proteins, especially COL4 and COL1, and the chaperone effect is dependent on the pattern of glycosylation, a cell/tissue specific post-translational modification that could potentially rectify incongruous observations of SPARC?s activity in different cell/tissue types. In this proposal, we will determine the general chaperone capability of SPARC using established assays and ascertain the effect of different glycosylation using surface plasmon resonance imaging on collagen binding. We will suppress SPARC using shRNA and the metabolic breakdown product of SPARC by MMP-3, SZ-1, in perfused anterior segment tissue, live mice, and TM cell cultures. We will determine the effect of inhibiting ILK, using shRNA and a small molecule inhibitor of ILK, and inhibiting ILK after SPARC overexpression on the actin cytoskeleton in TM cells as well as IOP and JCT ECM in both perfused anterior segment tissue and TM cells.
Primary open-angle glaucoma is a major cause of blindness worldwide with all current medical and surgical treatments attempting to lower eye pressure without interrupting the underlying cause of the elevated eye pressure. Our prior work has shown that SPARC regulates eye pressure under normal circumstances and is a critical mediator in the underlying cause of elevated eye pressure. In this application, we will elucidate important molecular pathways of how SPARC exerts its effects and show that suppressing SPARC is effective in cell and tissue culture models as well as both effective and safe in mice as a possible new disease-modifying treatment for glaucoma.
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|Keller, Kate E; Vranka, Janice A; Haddadin, Ramez I et al. (2013) The effects of tenascin C knockdown on trabecular meshwork outflow resistance. Invest Ophthalmol Vis Sci 54:5613-23|
|Oh, Dong-Jin; Kang, Min Hyung; Ooi, Yen Hoong et al. (2013) Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix. Invest Ophthalmol Vis Sci 54:3309-19|
|Kang, Min Hyung; Oh, Dong-Jin; Kang, Ja-heon et al. (2013) Regulation of SPARC by transforming growth factor ýý2 in human trabecular meshwork. Invest Ophthalmol Vis Sci 54:2523-32|
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