Abstract Title

Role of Extracellular Matrix Crosslinking Enzymes in the Trabecular Meshwork

Presenter Name

Urmimala Raychaudhuri

Abstract

Purpose - Transforming Growth Factor - β2 (TGFβ2) increases deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), which could be responsible for increased aqueous humor (AH) outflow resistance in primary open angle glaucoma (POAG). TGFβ2 induces expression of extracellular matrix (ECM) crosslinking enzymes tissue transglutaminase (TGM2), Lysyl oxidase (LOX) and Lysyl-oxidase like 2 (LOXL2) in the TM. These enzymes covalently crosslink ECM proteins leading to resistance to ECM degradation and turnover. In POAG, there is increased expression of TGFβ2, which increases TGM2, LOX and LOXL2 expression. Increased expression and crosslinking activity of these enzymes may enhance ECM deposition in the outflow pathway. This could lead to increased AH outflow resistance and elevated IOP. To determine whether these crosslinking enzymes play a role in regulating IOP, we developed and validated TGM2, LOX and LOXL2 expression vectors in-vitro. Viral vectors expressing these enzymes will be used in ex-vivo and in-vivo models to study the effect of their overexpression on IOP and AH outflow resistance.

Materials and methods – Transformed glaucomatous TM (GTM-3) and primary TM cells were transfected with plasmids expressing TGM2, LOX and LOXL2. Conditioned medium was collected, and overexpression was determined using western immunoblots. TGM2 activity was assessed by exposing cells to biotin-cadaverine followed by incubation with AlexaFluor 488 streptavidin-conjugate followed by fluorescence microscopy. LOX and LOXL2 enzyme activity was evaluated by western blots of the substrate tropoelastin in transfected cells with or without the LOX inhibitor b -aminoproprionitrile (BAPN) for 48 hours.

Results - TM cells transfected with TGM2, LOX and LOXL2 significantly overexpressed the enzymes. The TGM2 activity assay demonstrated increased crosslinking activity in cells transfected with TGM2 expression plasmid. LOX and LOXL2 activity was increased in cells transfected with LOX and LOXL2 expression vectors as exhibited by increased expression of the LOX substrate tropoelastin in western blotting

Conclusions - Our results indicate that TGM2, LOX and LOXL2 expression vectors significantly over-express these proteins and also show increased enzyme activity. In conclusion, these plasmid constructs will be packaged into adenovirus expression vectors and tested for effects on AH outflow and IOP in ex-vivo and in-vivo models.

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Role of Extracellular Matrix Crosslinking Enzymes in the Trabecular Meshwork

Purpose - Transforming Growth Factor - β2 (TGFβ2) increases deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), which could be responsible for increased aqueous humor (AH) outflow resistance in primary open angle glaucoma (POAG). TGFβ2 induces expression of extracellular matrix (ECM) crosslinking enzymes tissue transglutaminase (TGM2), Lysyl oxidase (LOX) and Lysyl-oxidase like 2 (LOXL2) in the TM. These enzymes covalently crosslink ECM proteins leading to resistance to ECM degradation and turnover. In POAG, there is increased expression of TGFβ2, which increases TGM2, LOX and LOXL2 expression. Increased expression and crosslinking activity of these enzymes may enhance ECM deposition in the outflow pathway. This could lead to increased AH outflow resistance and elevated IOP. To determine whether these crosslinking enzymes play a role in regulating IOP, we developed and validated TGM2, LOX and LOXL2 expression vectors in-vitro. Viral vectors expressing these enzymes will be used in ex-vivo and in-vivo models to study the effect of their overexpression on IOP and AH outflow resistance.

Materials and methods – Transformed glaucomatous TM (GTM-3) and primary TM cells were transfected with plasmids expressing TGM2, LOX and LOXL2. Conditioned medium was collected, and overexpression was determined using western immunoblots. TGM2 activity was assessed by exposing cells to biotin-cadaverine followed by incubation with AlexaFluor 488 streptavidin-conjugate followed by fluorescence microscopy. LOX and LOXL2 enzyme activity was evaluated by western blots of the substrate tropoelastin in transfected cells with or without the LOX inhibitor b -aminoproprionitrile (BAPN) for 48 hours.

Results - TM cells transfected with TGM2, LOX and LOXL2 significantly overexpressed the enzymes. The TGM2 activity assay demonstrated increased crosslinking activity in cells transfected with TGM2 expression plasmid. LOX and LOXL2 activity was increased in cells transfected with LOX and LOXL2 expression vectors as exhibited by increased expression of the LOX substrate tropoelastin in western blotting

Conclusions - Our results indicate that TGM2, LOX and LOXL2 expression vectors significantly over-express these proteins and also show increased enzyme activity. In conclusion, these plasmid constructs will be packaged into adenovirus expression vectors and tested for effects on AH outflow and IOP in ex-vivo and in-vivo models.