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Ayan Chatterjee, Min Kang, Dong-Jin Oh, Douglas J Rhee; Incubation with SPARC mitigates MMP-2-mediated collagen 4 degradation in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5685.
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Intraocular pressure (IOP) is a modifiable glaucoma risk factor and is a function of the rates of aqueous humor synthesis as well as outflow. Secreted protein, acidic and rich in cysteine (SPARC) regulates extracellular matrix (ECM) homeostasis within the human trabecular meshwork (TM), contributing to the rate of aqueous outflow. ECM turnover is a complex and dynamic process, involving a balance between collagen deposition and degradation by various matrix metalloproteinases (MMPs). The primary mechanism by which SPARC regulates ECM homeostasis is unknown.
MMP-2-mediated collagen 4 (COL4) digestions were carried out in 0.2mL capped PCR tubes. Reagents were prepared fresh for each experiment, with fourteen reaction conditions total (n = 4 each): reactions were carried out under low (2mM) or high (10mM) calcium conditions, with or without the addition of SPARC, and under native, oxidative, or heat-denaturing conditions. Control reactions (no MMP-2) were included for both the low and high calcium panels. Final concentrations were 2.5ng/μL for COL4 protein, 300μM for H202, 50ng/μL for SPARC protein, and 1ng/μL for active MMP-2 protein. For heat denaturation, reagents were pre-incubated for 1 hour at 45°C prior to addition of the MMP-2. All tubes were subsequently incubated for 6 hours at 37°C.
Under high calcium (10mM) conditions, the presence of 50ng/μL purified SPARC protein led to increased recovery of COL4 following MMP-2-mediated degradation. The relative amount of COL4 protein recovered was 0.42 ± 0.075 versus 0.18 ± 0.044 under native conditions in the SPARC versus control groups, respectively (p =0.031). COL4 recovered under conditions of oxidative stress was 0.31 ± 0.019 versus 0.17 ± 0.037, respectively (p = 0.017). COL4 recovered from reactions with heat-denatured substrate was 0.35 ± 0.049 versus 0.13 ± 0.037, respectively (p = 0.011). No significant differences in COL4 recovery were found under any of the low calcium conditions tested.
Our findings suggest that SPARC may act as a chaperone to mitigate MMP-2-mediated COL4 degradation under high calcium conditions. Further investigation is required in order to clarify whether this is true in physiologic and disease states within human TM, which may elucidate one mechanism by which SPARC regulates ECM homeostasis and IOP.
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