Thrombospondin-1 in the trabecular meshwork: localization in normal and glaucomatous eyes, and induction by TGF-b1 and dexamethasone in vitro Cassandra Flu ¨gel-Koch a , Andreas Ohlmann a , Rudolf Fuchshofer a , Ulrich Welge-Lu ¨ssen b , Ernst R. Tamm a, * a Department of Anatomy, Molecular Anatomy and Embryology, University of Erlangen–Nu ¨rnberg, Universita ¨tsstr. 19, D-91054 Erlangen, Germany b Eye Hospital of the University of Munich, Munich, Germany Received 27 February 2004; accepted in revised form 19 July 2004 Available online 24 August 2004 Abstract Transforming growth factor-b2 (TGF-b2) is elevated in the aqueous humor of patients with primary open-angle glaucoma (POAG), and high levels of TGF-b2 are thought to contribute to the pathogenesis of POAG. Most TGF-b2 in the eye is present in a latent, inactive form and the mechanisms of its in vivo activation are unclear. Since thrombospondin-1 (TSP-1) is one of the most potent in vivo activating molecules of TGF-bs, we investigated the localization and expression of TSP-1 in the aqueous humor outflow pathways. TSP-1 immunohistochemistry was performed in the eyes of human donors (8 normal and 17 with glaucoma). In addition, the eyes of Tsp-1 K/K -deficient mice and normal Tsp-1 C/C mice were investigated. TSP-1 mRNA expression was assessed by reverse transcription-polymerase chain reaction and Northern blotting of RNA from fresh trabecular meshwork (TM), and human and mouse TM cells in vitro. In addition, Northern and Western blot analyses of TM cells after incubation with TGF-b and dexamethasone were performed. In most of the eyes, TSP-1 immunolabeling was predominately observed in extracellular areas of the juxtacanalicular (cribriform) part of the TM. Some focal staining was observed in the corneoscleral and uveal parts of the TM. In the eyes of six glaucoma patients (including one with steroid-induced glaucoma), TSP-1 immunoreactivity was considerably more intense and all regions of the TM were positively labeled. In double labeling experiments, staining for TSP-1 did not overlap with that of fibronectin or type VI collagen. mRNA for TSP-1 was detected in both fresh and cultured TM cells. Incubation of TM cells with TGF-b1 and dexamethasone caused a marked increase in TSP-1 expression. TSP-1 in the TM might act as a potent local endogenous activator of TGF-bs in the aqueous humor and mediate any local effects of TGF-b and/or dexamethasone on the outflow of aqueous humor. q 2004 Elsevier Ltd. All rights reserved. Keywords: trabecular meshwork; primary open-angle glaucoma; TGF-b; dexamethasone; extracellular matrix 1. Introduction Intraocular pressure (IOP) at a level that is too high for the health of the optic nerve head is a major risk factor for glaucoma (The AGIS Investigators, 2000), a leading cause of blindness worldwide (Thylefors et al., 1995). IOP critically depends on the resistance to aqueous humor outflow in the trabecular meshwork (TM). In primary open-angle glaucoma (POAG), IOP is elevated because of an abnormally high outflow resistance in the TM (Grant, 1963). The precise molecular mechanisms that are responsible for the increase in outflow resistance in POAG are unclear, but there is some evidence that changes in the amount and quality of the TM extracellular matrix are involved. Eyes with POAG show a significant increase in extracellular ‘plaque material’ in the TM (Rohen and Witmer, 1972; Lu ¨tjen-Drecoll et al., 1986), which correlates with the degree of axonal damage in the optic nerve head (Gottanka et al., 1997). The molecular nature of plaque material is unclear, but there is some evidence that collagen type VI is associated with it (Lu ¨tjen-Drecoll et al., 1989). Other components of the extracellular matrix that have been 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.07.005 Experimental Eye Research 79 (2004) 649–663 www.elsevier.com/locate/yexer * Corresponding author. Dr Ernst R. Tamm, Department of Anatomy, Molecular Anatomy and Embryology, University of Erlangen–Nu ¨rnberg, Universita ¨tsstr. 19, D-91054 Erlangen, Germany. E-mail address: ernst.tamm@anatomie2.med.uni-erlangen.de (E. R. Tamm).