Crosstalk of transforming growth factor beta-2 and toll-like receptor 4 in the trabecular meshwork

Purpose: Glaucoma is characterized by progressive optic neuropathy that is associated with elevated intraocular pressure (IOP) and extracellular matrix (ECM) remodeling. The trabecular meshwork (TM) is involved in the outflow of aqueous humor and IOP regulation. Glaucomatous eyes show elevated levels of transforming growth factor-β 2 (TGF-β2) and its signaling pathways in the ECM of the TM have been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in the liver, kidney, lung and skin. Based on the potential for shared signaling pathways, we hypothesize that endogenous TLR4 ligands activate TLR4 and augment TGF-β2 signaling sensitivity by downregulating BAMBI, leading to increase ECM production in the TM and increase IOP. Methods: Cross-sections of human donor eyes and dissected mouse TM rings were used to determine TLR4 expression in the TM. Primary human TM cells were used to test for the expression of BAMBI. To study the role of TGF-β2 and TLR4 crosstalk in the expression of ECM proteins, four primary human TM cell strains were treated with a selective TLR4 inhibitor (TAK-242, 15µM), TGFβ2 (5ng/ml), and a TLR4 ligand (Fibronectin-EDA isoform). In-vivo studies were carried out to examine the induction of ocular hypertension in wild-type (A/J, AKR/J, BALBc/J, and C3H/HeOuJ) or Tlr4 mutant strains of mice (C3H/HeJ) by intravitreally injecting Ad5.hTGFβ2226/228 (2.5x107 pfu) in one eye while the contralateral uninjected eye was used as negative controls. Results: Our studies reveal the expression of TLR4 in the human and mouse TM. BAMBI is expressed in human TM cells and its expression is significantly decreased in the presence of TGF-β2. Inhibition of TLR4 in the presence of TGF-β2 decreases fibronectin and collagen-1 expression. Activation of TLR4 in the presence of TGF-β2 increases fibronectin and collagen-1 expression and TLR4 inhibition blocks this effect. Our in-vivo studies show that Ad5.hTGF-β2 induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant mice. Conclusions: These studies identify TGFβ2 – TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data provide potential new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.