ABSTRACT
Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGFß2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGFß2 BALB/cJ mouse model of ocular hypertension. BALB/cJ mice that had been intravitreally injected with an adenovirus (Ad5) expressing a bioactive TGFß2226/228 showed a significant increase in IOP after 2 weeks. When 1µM FUD was injected intracamerally into mice 2 weeks post Ad5-TGFß2 injection, FUD significantly reduced IOP after 2 days. Neither mutated FUD (mFUD) nor PBS had any effect on IOP. Four days after FUD was injected, IOP returned to pre-FUD injection levels. In the absence of TGFß2, intracameral injection of FUD had no effect on IOP. Western blotting of mouse anterior segments expressing TGFß2 showed that FUD decreased fibronectin levels 2 days after intracameral injection (p<0.05) but not 7 days compared to eyes injected with PBS. mFUD injection had no significant effect on fibronectin levels at any time point. Immunofluorescence microscopy studies in human TM (HTM) cells showed that treatment with 2ng/ml TGFß2 increased the amount of EDA+ and EDB+ fibronectin incorporated into fibrils and 2µM FUD decreased both EDA+ and EDB+ fibronectin in fibrils. An on-cell western assay validated this and showed that FUD caused a 67% reduction in deoxycholate insoluble fibronectin fibrils in the presence of TGFß2. FUD also caused a 43% reduction in fibronectin fibrillogenesis in the absence of TGFß2 while mFUD had no effect. These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP.
Subject(s)
Fibronectins/metabolism , Intraocular Pressure/drug effects , Ocular Hypertension/metabolism , Peptides/therapeutic use , Trabecular Meshwork/drug effects , Adolescent , Adult , Animals , Cells, Cultured , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Fibronectins/chemistry , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ocular Hypertension/chemically induced , Ocular Hypertension/drug therapy , Peptides/metabolism , Peptides/pharmacology , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolism , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta2/toxicityABSTRACT
Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.
Subject(s)
GTP-Binding Proteins/genetics , Intraocular Pressure , Ocular Hypertension/prevention & control , Trabecular Meshwork/enzymology , Transglutaminases/genetics , Adenoviridae/genetics , Animals , Gene Expression Regulation, Enzymologic/physiology , Gene Knockout Techniques , Intraocular Pressure/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ocular Hypertension/chemically induced , Ocular Hypertension/enzymology , Protein Glutamine gamma Glutamyltransferase 2 , Real-Time Polymerase Chain Reaction , Tonometry, Ocular , Transfection , Transforming Growth Factor beta2/toxicityABSTRACT
PURPOSE: To validate a new culture model of primary human trabecular meshwork cells (p-hTMCs) using Matrigel®, in order to mimic in vitro 3D-TM organization, and to investigate the proinflammatory effect of benzalkonium chloride (BAK) in 3D p-hTMC cultures. METHODS: p-hTMCs, seeded onto Matrigel®-coated inserts were stimulated with BAK (10-4%), dexamethasone (DEX) (10-6M) or transforming growth factor-beta 2 (TGF-ß2) (5ng/ml) for 48h and observed with confocal microscopy. The BAK effect at 10-4% or 5.10-3% on the gene expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and matrix metalloproteinase (MMP-9) was investigated using qRT-PCR in 2D and 3D p-hTMC cultures. RESULTS: p-hTMCs seeded in Matrigel® were able to organize themselves in a 3D-spatial conformation in the different conditions tested with cross-linked actin network (CLAN) formation in presence of DEX or TGF-ß2 and intercellular space contraction with TGF-ß2. IL-6 and IL-8 gene expressions increased in presence of BAK in 2D and in 3D p-hTMC cultures. BAK 10-4% only showed a tendency to stimulate MMP-9 expression in p-hTMCs after 24h-recovery. CONCLUSIONS: We investigated this new 3D-TM in vitro model in Matrigel® matrix for pathophysiological and toxicological purposes. It appears as a new promising tool for a better understanding of TM behavior in physiological and stress conditions, as well as toxicological evaluations of antiglaucoma eyedrops and preservatives.
