Toll-like Receptor 4, FIbronectin Extra Domain A, and Nuclear Factor kappa B are Necessary for TGFβ2-induced Ocular Hypertension

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2020

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McDowell, Colleen
Roberts, Amanda
Hernandez, Humberto
Curry, Stacy
Harris, Sherri

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Purpose: Ocular hypertension is the major risk factor for developing primary open-angle glaucoma (POAG). Trabecular meshwork (TM) damage in POAG patients leads to impaired aqueous humor outflow and retinal ganglion cell damage. We explored a novel molecular mechanism involved in glaucomatous TM extracellular matrix (ECM) development through the TGFβ2-TLR4 signaling crosstalk; which regulates TM ECM changes. We discovered that mutations in Tlr4, FN-EDA, and NFkB rescues TGFβ2 -induced ocular hypertension. Methods: B6.FN-EDA+/+, B6.FN-EDA-/-, B6.TLR4-/-, B6.FN-EDA+/+/TLR4-/-, B6.FN-EDA-/-/TLR4-/-, and C57BL/6J mice were intravitreally injected with 2.0µL Ad5.TGFβ2 (2.5x10^7 pfu) in one eye and the contralateral uninjected eye was the control. Likewise, we tested mice lacking the p50 subunit of NFκB (B6.Cg-NFκB1tm1Bal/J) and C57BL/6J mice. IOP was measured weekly using a TonoLab rebound tonometer on isoflurane-anesthetized mice. Significance determined by one-way ANOVA. Eyes were harvested, 4%-paraformaldehyde-fixed, and sectioned for immunohistochemistry to access total fibronectin and FN-EDA isoform expressions. Results: Ad5.TGFβ2 significantly induced ocular hypertension C57BL/6J and B6.EDA+/+ mice. B6.EDA+/+ mice developed spontaneous ocular hypertension. Mutations in Tlr4, FN-EDA, and NFκB blocked Ad5.TGFβ2 -induced ocular hypertension. Total FN and FN-EDA expression increased in uninjected B6.FN-EDA+/+ mice and increased in response to TGFβ2 in wildtype and EDA mice. Conclusions: TLR4, FN-EDA, and NFκB are necessary for TGFβ2 induced ocular hypertension and ECM deposition in mice. These data provide new targets to lower IOP and inhibit glaucoma disease progression.

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