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Min Hyung Kang, Douglas J Rhee; Suppression of thrombospondin-1 (TSP1) in Human Trabecular Meshwork Alters Extracellular Matrix. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6024.
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Thrombospondins-1 (TSP1) is a matricellular protein which has been shown to regulate cytoskeleton, cell adhesion, and extracellular matrix remodeling. TSP-1 is found in the trabecular meshwork (TM) and is increased in one third of patients with primary open-angle glaucoma (POAG). As TSP1-null mice demonstrated a lower IOP resulting from increased outflow , we hypothesized that the suppression of TSP1would alter IOP by affecting extracellular matrix (ECM) synthesis and/or turnover in the trabecular meshwork (TM).
A lentivirus carrying the short hairpin RNA targeting human TSP1 (lenti-shTSP1) was constructed using the pLKO.1 vector system. Lenti-shTSP1 was then used to suppress TSP1 in human TM endothelial cells at the multiplicity of infections (MOI) 5. ECM proteins from cell lysates and conditioned media were analyzed by immunoblotting. Human anterior chamber perfusion culture (ex vivo) was treated with lenti-shTSP1 and IOP was recorded every 6 hours.
By infecting human TM endothelial cells in culture with lenti-shTSP1, we observed that TSP1 was suppressed (-24.6 ± 13.9 %, p=0.09, n=3), compared to lenti-control. Suppressed TSP1 attenuated the level of MMP2 (-42.93 ± 11.2 %, p=0.02, n=3) and collagen IV (-50.78 ± 8.6 %, p=0.009, n=3). The level of TIMP1 (-32.4 ± 4.8 %, p=0.067, n=2) showed the trend of suppression, but it was not significant.
As the suppression of TSP1 attenuated the level of ECM protiens, TIMPs and MMPs, TSP1 could play a regulatory role for IOP by altering ECM proteins, TIMPs and MMPs in the human TM.References1. Flugel-Koch C, Ohlmann A, Fuchshofer R, Welge-Lussen U, Tamm ER. Thrombospondin-1 in the trabecular meshwork: localization in normal and glaucomatous eyes, and induction by TGF-beta1 and dexamethasone in vitro. Exp Eye Res 2004;79:649-663.2. Haddadin RI1, Oh DJ, Kang MH, Villarreal G Jr, Kang JH, Jin R, Gong H, Rhee DJ. Thrombospondin-1 (TSP1)-null and TSP2-null mice exhibit lower intraocular pressures. Invest Ophthalmol Vis Sci. 2012 Sep 28;53(10):6708-17.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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