Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma, which is the 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 suppresses fibrosis by regulating ECM component expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4 in TM, thereby attenuating TGFβ2 induction of ECM proteins in TM cells, elevated IOP and decreased outflow facility in TGFβ2-induced ocular hypertension in mice. 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 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 intravitrealy. Ad5-CMV-TGFβ2C226S/C288S was injected along with Ad5- null vector as negative control, while Ad5-null injected mice were included as a positive control. IOP was measured using a Tonolab tonometer and outflow facility was measured using the constant flow infusion method. In addition we also performed luciferase repoter 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 changes in TGFβ2 induced ocular hypertensive 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 decrease outflow facility induced by TGFβ2. Further increasing activity of NFκB, Notch and cAMP/PKA suggest that ID1 and ID3 may regulate TGFβ2 effects in TM via activation of MMP pathway, inhibition of PAI-1 and Rho kinase activity, resist cytoskeleton changes and increase TM cellularity. This makes ID1 and/or ID3 strong candidates for developing disease-modifying IOP lowering therapies.
|Advisor:||Clark, Abbot F.|
|Commitee:||Krishnamoorthy, Raghu, Mao, Weiming, Mathew, Stephen, Millar, Cameron|
|School:||University of North Texas Health Science Center at Fort Worth|
|School Location:||United States -- Texas|
|Source:||DAI-B 79/03(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Ophthalmology|
|Keywords:||Bmp4, Id1, Id3, Intra ocular pressure, Outflow facility, Tgfbeta 2|
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