Abstract
Purpose: :
Biochemical and morphological changes within the trabecular meshwork (TM) and its extracellular matrix (ECM) are associated with impaired aqueous humour outflow resistance in primary open angle glaucoma (POAG). There is an increase in the ECM components, including collagen IV, laminin and fibronectin. This may represent a disruption in the normal turnover of ECM which is influenced by transforming growth factor-beta (TGFβ) and matrix metalloproteinases (MMP). Thrombospondins (TSP)-1 and -2 are expressed in the TM and modulate cell-matrix interactions. TSP-1 is up-regulated by TGFβ and TSP-2 mediates MMP-2 and -9 activities. We have previously identified that TSP-2 is expressed in the TM and is up-regulated by TGFβ and Dexamethasone, coinciding with a decrease in MMP-2 levels. This study aims to determine the role of TSP-2 on the expression of ECM proteins by human TM cells.
Methods: :
Non-glaucomatous (NTM5) and glaucomatous (GTM3) human TM cell lines were treated with 2μg/ml TSP-2 and -1 recombinant human protein for seven days. Expression of fibronectin and collagen IV protein and mRNA levels were evaluated by western blotting and qPCR techniques, respectively.
Results: :
GTM3 and NTM5 cells expressed fibronectin and collagen IV at the protein and mRNA level. GTM3 cells showed a 3.2 and 1.4 fold increase in fibronectin and collagen IV protein levels under untreated conditions, compared to the NTM5 cell line. Fibronectin protein levels were further increased in the GTM3 cells following treatment with 2μg/ml TSP-2, showing a 6.3 and 1.9 fold increase compared to NTM5 and GTM3 controls, respectively. TSP-1 treatment did not increase fibronectin levels in either the NTM5 or the GTM3 cell line. Collagen IV protein levels in GTM3 cells were increased in TSP-2 and TSP-1 treated samples showing a 1.6 and 1.2 fold increase respectively, compared to untreated NTM5 control samples. Collagen IV mRNA levels were increased in GTM3 TSP-2 treated samples, whilst fibronectin mRNA levels did not change, compared to untreated GTM3 controls.
Conclusions: :
These data suggest that the TSP-2 increases the expression of fibronectin and collagen IV levels in the ECM of glaucomatous TM cells, relative to TSP-1. Given that TSP-2 is expressed in the TM and is upregulated in POAG and by TGFβ it has a potential role in the pathogenesis of glaucoma and may serve as a future therapeutic target in the management of POAG.
Keywords: outflow: trabecular meshwork • extracellular matrix • intraocular pressure