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

J. Cameron Millar

Third Advisor

Raghu R. Krishnamoorthy

Abstract

Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma, which is a major cause of irreversible vision loss. The major risk factor associated with POAG is increased intra ocular pressure (IOP). Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) increases the deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor I (PAI-1) and ECM cross-linking enzymes. These disruptions in ECM physiology in the TM elevate IOP and decrease aqueous humor outflow facility. Bone morphogenetic proteins (BMPs) regulate TGFβ2 induced profibrotic ECM production. The underlying mechanism for BMP4 inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 induces inhibitor of DNA binding proteins (ID1, ID3), which are negative transcription regulators that suppress fibrosis by regulating ECM component expression. Our study sought to determine whether ID1and ID3 proteins are downstream targets of BMP4 in TM and thereby attenuate TGFβ2 induction of ECM proteins in TM cells, elevated IOP and decreased outflow facility. Methods: Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. GTM3 cells were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression and promoter activities. IOP and AH outflow facility changes were studied in female BALB/cJ mice. Ad5-CMV-ID1 and Ad5-CMV-ID3 viral vectors along with Ad5-CMV-TGFβ2C226S/C288S were injected intravitreally. Ad5-CMV-TGFβ2C226S/C288S was injected along with Ad5- null vector as positive control, while Ad5-null injected mice were included as a negative control. IOP was measured using a Tonolab impact tonometer and AH outflow facility was measured using a constant flow infusion method. In addition we also performed a luciferase reporter activity array assay to study the effect of ID1, ID3 and TGFβ2 on various transcription factor activity in TM. Results: Basal expression of ID1-3 was detected in primary human TM cells. BMP4 significantly induced early expression of ID1 and ID3 mRNA (p< 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells (p< 0.01). Transduction of mouse eyes with ID1 or ID3 significantly blocked TGFβ2 induced ocular hypertension (P< 0.0001) and AH outflow facility changes in living mice. Further, ID1 (P< 0.01) and ID3 increase NFκB, Notch and cAMP/PKA activity, even in presence TGFβ2. Conclusions: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 profibrotic activity in human TM cells. In addition ID1 and ID3 suppresses elevated IOP and decreases AH outflow facility induced by TGFβ2. Further, increased activity of NFκB, Notch and cAMP/PKA suggest that ID1 and ID3 may regulate TGFβ2 effects in TM via activation of the MMP pathway, inhibition of PAI-1 and Rho kinase activity, resisting cytoskeleton changes and lead to increased TM cellularity. This strongly supports ID1 and/or ID3 as robust candidates as a basis for developing disease-modifying IOP lowering therapies in POAG.

Share

COinS