Date of Award

12-1-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Graduate School of Biomedical Sciences

First Advisor

Abbot F. Clark

Second Advisor

Raghu R. Krishnamoorthy

Third Advisor

Weiming Mao

Abstract

Increased intraocular pressure (IOP) is one of the major known risk factors for primary open angle glaucoma (POAG). The main cause of IOP elevation is obstruction to aqueous humor (AH) outflow at the trabecular meshwork (TM) in the eye. Cellularity and ECM turnover rates affect the normal physiology of the TM tissue. Various factors including transforming growth factor (TGFβ2) have been found responsible for a large number of glaucomatous changes. Increased expression and activity of tissue transglutaminase (TGM2), an enzyme induced by TGFβ2, has been seen in the glaucomatous TM. TGM2 can covalently crosslink ECM proteins including collagens, fibronectin and elastin. The purpose of this study was to investigate the role of TGM2 in ocular hypertension. For this, we overexpressed TGM2 using adenovirus serotype 5 (Ad5), which has tropism for TM cells, in TM cells in vitro and in mouse eyes in vivo. In vitro, we validated overexpression of TGM2 using Ad5.TGM2 and also found increased crosslinking following overexpression. For our animal studies we used BALB/cJ and C57BL/6J mice. In our mouse models, following intravitreal injection of Ad5.TGM2, we saw a significant increase in IOP and decrease in AH outflow facility in TGM2 overexpressed eyes compared to contralateral eyes. Immunohistochemical staining showed that there was increased expression of TGM2 and increased crosslinking in the TM region. There also appeared to be increased fibronectin at the TM region. We followed this with a knockout (KO) study to determine whether TGM2 KO could affect IOP. For this we used TGM2 floxed mice. Following intravitreal injection of Ad5.Cre, we find a significant reduction in IOP. We also found that KO of TGM2 significantly reduced TGFβ2 induced ocular hypertension. We further tested a small molecule TGM2 inhibitor ZM 449829. This inhibitor binds to TGM2 and inhibits crosslinking activity by locking it in an inactive state. In our findings, we observed that when treated with 5nM ZM 449829, at 48 hours it inhibited TGM2 crosslinking in 3 primary human glaucomatous cell strains. It also appears to reduce fibronectin deposition. We performed some preliminary tests of this drug in vivo. 5μM of the drug dissolved in PBS was administered as eye drops as once daily dosing. At 3 weeks, gross morphology of the eye and cornea looked normal. IOP measurements taken once a week till 3 weeks also did not show any aberrant changes. Overall, our findings suggest that TGM2 plays a significant role in inducing ocular hypertension. This makes TGM2 a potential therapeutic target; therefore inhibition or amelioration of TGM2 crosslinking activity such as with potent inhibitors like ZM 449829 should be further studied as a novel therapeutic strategy for glaucoma.

Share

COinS