Crosstalk of TGF-beta and TLR4 pathways in the trabecular meshwork

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Elevated intraocular pressure (IOP) is one of the major risk factors for the development and progression of glaucoma and is due to damage to the trabecular meshwork (TM). We have identified a novel molecular pathway of TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk involved in the development of ocular hypertension and glaucomatous TM damage. We identified that the TLR4 signaling pathway is involved in the regulation of the extracellular matrix (ECM) in the TM and activation of TLR4 can enhance the effect of TGFβ2- induced glaucomatous phenotypes, while inhibition of TLR4 blocks the effect. We have identified both BMP and activin membrane bound inhibitor (BAMBI) and fibronectin extra domain A (FN-EDA) as important regulators of pathogenic TLR4 and TGFβ2 signaling in the TM. FN-EDA contains both integrin α4β1 and α9β1 binding sites which are known to regulate FN-EDA function. Recently integrin α4β1 was identified as a TLR4 co-receptor necessary for EDA-initiated activation of TLR4 in dermal fibroblasts. A20, an endogenous regulator of downstream TLR4 signaling, has been identified as an important regulatory molecule in other fibrotic diseases. Here we propose to determine the molecular mechanism of FN-EDA regulation of TLR4, determine the role of endogenous regulators BAMBI and A20 on pathological TGFβ2-TLR4 signaling, and identify novel therapeutic targets to prevent glaucomatous TM damage and elevated IOP. Our hypothesis is TGFβ2-TLR4 signaling crosstalk leads to pathological glaucomatous TM damage and elevated IOP, which can be controlled by both therapeutic and endogenous regulators. We will address this hypothesis with 4 specific aims: Specific Aim #1 will determine whether FN-EDA alters the TM morphology and function through TLR4 signaling. We will utilize B6.EDA+/+ mice which constitutively express only FN containing EDA and will analyze the IOP profiles, TM morphology, and ECM makeup. Specific Aim #2 will determine whether the EDA-binding integrins a4b1 and a9b1 are necessary for EDA activation of TLR4 signaling and ocular hypertension utilizing primary human TM cells as well as a4 and a9 integrin floxed mice. Specific Aim #3 will determine whether the endogenous negative regulators of TGFβ2-TLR4 signaling, BAMBI or A20, can block IOP elevation in mice. We will overexpress Bambi (using Ad5.Bambi virus) or A20 (using Ad5.A20 virus) in the TM and determine whether they can block Ad5.TGFβ2-induced ocular hypertension in C57BL/6J mice. Specific Aim #4 will determine whether inhibiting FN-EDA expression can block IOP elevation in mice and in a human anterior segment perfusion organ culture model (POC). Studies will use a fibronectin-binding peptide called FUD to block EDA+/+-induced and TGFβ2-induced ocular hypertension. These studies aim to explore a novel pathway involved in the development of glaucomatous TM damage. The data will be invaluable to the field of glaucoma research and could provide new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.