Abstract Title

Protective effect of ID protein on TGFβ2 induced fibrosis in human trabecular meshwork cells: implication for developing a glaucoma therapy.

Presenter Name

Avani A Mody

RAD Assignment Number

1010

Abstract

Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma. Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) causes increased deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor-I (PAI-1), leading to elevated intraocular pressure (IOP) in POAG patients. In fibrotic pulmonary diseases, bone morphogenetic proteins (BMPs) induce inhibitor of DNA binding proteins (ID1, ID3), which are transcription regulators known to suppress bHLH promoter activity, thereby inhibiting TGFβ induced ECM production. However, in TM cells the underlying mechanism for BMP4 inhibition of TGFβ2-induced fibrosis remains undetermined. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGFβ2 induction of ECM proteins in cultured human TM cells.

Methods: Primary human TM (HTM) cells were treated with BMP4 for 0-48hr, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. HTM cells were treated with a BMPR inhibitor to confirm that BMP4 signaling is necessary for induction of ID1 and ID3 protein expression. GTM3 cells were transfected with ID1 or ID3 vectors to determine their inhibitory effects on TGFβ2 induced fibronectin and PAI-1 protein expression. Ad5-CMV-hId1 and Ad5-CMV-hID3 viral vectors along with Ad5-CMV-hTGFb2C226S/C288S were injected intravitreally to observe IOP changes in female BALB/cJ mice.

Results: BMP4 significantly induced early expression of ID1 and ID3 mRNA (p<0.05) and protein in HTM cells, while the BMPR 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). We currently are determining whether overexpression of ID1 and ID3 can suppress TGFb2-induced ocular hypertension in mice.

Conclusions: BMP4 induced ID1 and ID3 suppresses TGFβ2 induced fibronectin and PAI-1. This makes ID1 and/or ID3 strong candidates for developing disease-modifying IOP lowering therapies.

Research Area

Eye/Vision

Presentation Type

Poster

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Protective effect of ID protein on TGFβ2 induced fibrosis in human trabecular meshwork cells: implication for developing a glaucoma therapy.

Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma. Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) causes increased deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor-I (PAI-1), leading to elevated intraocular pressure (IOP) in POAG patients. In fibrotic pulmonary diseases, bone morphogenetic proteins (BMPs) induce inhibitor of DNA binding proteins (ID1, ID3), which are transcription regulators known to suppress bHLH promoter activity, thereby inhibiting TGFβ induced ECM production. However, in TM cells the underlying mechanism for BMP4 inhibition of TGFβ2-induced fibrosis remains undetermined. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGFβ2 induction of ECM proteins in cultured human TM cells.

Methods: Primary human TM (HTM) cells were treated with BMP4 for 0-48hr, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. HTM cells were treated with a BMPR inhibitor to confirm that BMP4 signaling is necessary for induction of ID1 and ID3 protein expression. GTM3 cells were transfected with ID1 or ID3 vectors to determine their inhibitory effects on TGFβ2 induced fibronectin and PAI-1 protein expression. Ad5-CMV-hId1 and Ad5-CMV-hID3 viral vectors along with Ad5-CMV-hTGFb2C226S/C288S were injected intravitreally to observe IOP changes in female BALB/cJ mice.

Results: BMP4 significantly induced early expression of ID1 and ID3 mRNA (p<0.05) and protein in HTM cells, while the BMPR 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). We currently are determining whether overexpression of ID1 and ID3 can suppress TGFb2-induced ocular hypertension in mice.

Conclusions: BMP4 induced ID1 and ID3 suppresses TGFβ2 induced fibronectin and PAI-1. This makes ID1 and/or ID3 strong candidates for developing disease-modifying IOP lowering therapies.