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Swarup Sai Swaminathan, Dong-Jin Oh, Min Kang, Allan R Shepard, Iok-Hou Pang, Douglas J Rhee; In vivo study of the role of SPARC in TGFb2-mediated ocular hypertension. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5987.
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© ARVO (1962-2015); The Authors (2016-present)
Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein that modulates extracellular matrix (ECM) in the trabecular meshwork (TM). In SPARC knockout (KO) mice, intraocular pressure (IOP) is reduced by 15-20%. Transforming growth factor-b2 (TGFb2) has been shown to be elevated in glaucomatous eyes up to 3-fold. When injected into rodent eyes, TGFb2 stimulates ECM deposition in the TM, which leads to IOP elevation. In TGFb2-stimulated TM cells, SPARC is the most upregulated protein. We have also shown that SPARC is downstream from TGFb2. We attempted to elucidate whether SPARC is essential to TGFb2-mediated ocular hypertension.
Adenovirus containing hTGFb2 (Ad.TGFb2) and empty vector (Ad.empty) were synthesized. Ad.TGFb2 or Ad.empty was injected intravitreally into the eyes of age-matched wild-type (WT) and SPARC KO mice at a titer of 6 x 10^6 pfu using a 35-gauge needle. IOP was measured every other day for two weeks using Tonolab tonometry. After identifying the peak temporal point of IOP elevation, additional WT and KO mice were injected with Ad.TGFb2 and Ad.empty, and were sacrificed at this time point. Their eyes were processed and utilized for immunohistochemistry (IHC) probing for ECM proteins, including collagens I, IV, and VI, fibronectin, connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1 (PAI-1).
IOP was significantly elevated in Ad.TGFb2-injected (n=8) vs. Ad.empty-injected WT (n=8) mice during days 4-11 (p<0.03). IOP was not significantly elevated in Ad.TGFb2-injected vs. Ad.empty-injected SPARC KO mice. Peak point of IOP increase was noted to be 8 days after injection (day 8). At this time, IOPs were as follows: Ad.empty-injected WT =14.3 mmHg, Ad.TGFb2-injected WT =18.3 mmHg, Ad.empty-injected KO =12.1 mmHg, Ad.TGFb2-injected KO =13.0 mmHg. The difference in percentage of IOP increase between WT and KO eyes was significant between days 4-8 (WT= 28.5 ± 4.2% vs. KO= 14.4 ± 4.9% at day 8; p=0.0444). IHC demonstrated that TGFb2 stimulated increases in collagen IV, fibronectin, CTGF, and PAI-1 in WT tissue, but only PAI-1 and CTGF in KO mice (p<0.05, n=3).
These data suggest that SPARC is an essential node in TGFb2-mediated ocular hypertension. Modulation of collagen IV and fibronectin levels are significantly restricted by the lack of SPARC. SPARC may play a key role in IOP regulation and potentially glaucoma pathogenesis.
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