Abstract
Purpose:
We have shown that overexpression of Secreted Protein Acidic and Rich in Cysteine (SPARC), a matricellular protein known to modulate extracellular matrix (ECM) content, leads to increased intraocular pressure in human tissue. We thus attempted to identify potential mechanisms by which SPARC may induce ocular hypertension. Proteoglycans were the main targets investigated due to their role in ECM homeostasis. Bone morphogenic protein-4 (BMP-4) and gremlin were also assessed due to their known modulation of TGF-β2 signaling.
Methods:
Primary human trabecular meshwork (TM) cultures were derived from separate donors aged 12, 42, 44, 47, 57, 68, and 70 using a previously published protocol. TM cultures were allowed to grow to confluence at 37C in 10% CO2 atmosphere and then allowed 2-3 days to differentiate. For adenovirus infection, human TM cells were incubated in 2% FBS media with multiplicity of infection (MOI) 25 of Ad5.control or Ad5.hSPARC for 18 hours, after which 10% FBS media of an equal volume with 2% media were added for another 24 hours. TM cultures were incubated in serum-free media for 24 hours. Cultured TM cells were lysed in 1x radioimmunoprecipitation assay (RIPA) buffer or 1x Lysis Buffer with protease inhibitors. The serum-free conditioned media and cell lysates were then analyzed by immunoblot assays using antibodies against versican V0, versican V1, collagen XVIII, chondroitin-6-sulfate, decorin, opticin, fibromodulin, lumican, gremlin, and BMP-4.
Results:
Immunoblot assays demonstrated that SPARC overexpression significantly upregulated opticin (25.87 ± 9.44%; n=4, p=0.038) and decorin (16.36 ± 3.43%; n=4, p=0.004). SPARC overexpression did not affect the levels of versican V0, versican V1, lumican (n=4), fibromodulin, collagen XVIII, and chondroitin-6-sulfate (n=3). Interestingly, SPARC overexpression significantly upregulated gremlin (32.32 ± 7.18%; n=3, p=0.012) but had no effect on BMP-4 level (n=4).
Conclusions:
This study suggests that SPARC may modulate intraocular pressure through the proteoglycans opticin and decorin, as well as gremlin. Decorin has been shown to be upregulated by TGF-β2, and has also been found within SD plaque material in glaucomatous eyes. Both decorin and opticin are known regulators of collagen fibrillogenesis, which may affect aqueous humor outflow. Thus, SPARC may serve as an essential node in the TGF-β2-mediated pathogenesis of primary open-angle glaucoma.
Keywords: 519 extracellular matrix •
661 proteoglycans/glycosaminoglycans •
568 intraocular pressure