Abstract
Purpose:
The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. TGF-β 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 liver, kidney, lung and skin. We investigated whether the TLR4 signaling pathway is involved in the regulation of the ECM in the TM and if mutation in TLR4 rescues TGFβ2 induced ocular hypertension in mice.
Methods:
Cross-sections of human donor eyes, primary TM cells in culture, and dissected mouse TM rings were used to determine TLR4 expression in the TM. Human TM cells were treated with a selective TLR4 inhibitor (TAK-242, 15µM) in the presence or absence of TGFβ2 (5ng/ml). A/J (n=13), AKR/J (n=7), BALBc/J (n=8), C3H/HeJ (n=5), and C3H/HeOuJ (n=5) mice were injected intravitreally with Ad5.hTGFβ2226/228 in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer.
Results:
TLR4 was expressed in mouse (n=3 mice/strain) and human TM (n=8). TGFβ2 treatment increased both fibronectin (FN) (4.46 +/- 0.07 fold) and collagen 1 (COL1) (11.53 +/- 0.41 fold) mRNA expression in human cultured TM cells. However, inhibition of TLR4 in the presence of TGFβ2 decreased expression of FN (1.15 +/- 0.18 fold) and COL1 (4.35 +/- 0.85 fold) (n=3/treatment, p<0.001). Inhibition of TLR4 in the presence of TGFβ2 also decreased FN protein expression (0.88 +/- 0.02 fold; n=3) compared to cells treated with TGFβ2 alone (4.05 +/- 0.59 fold; n=3 p<0.01). In vivo, Ad5.hTGFβ2 induced ocular hypertension in A/J, AKR/J, and BALBc/J mice throughout an eight week time course. IOPs increased to approximately 25 mm Hg in all 3 mouse strains (p<0.05). IOPs were stable (12-15 mm Hg) in uninjected control eyes. C3H/HeJ and C3H/HeOuJ mice are genetically similar except for the genotype of Tlr4. Ad5.hTGFβ2226/228 did not induce ocular hypertension in Tlr4 mutant C3H/HeJ mice, but Tlr4 wildtype C3H/HeOuJ mice developed ocular hypertension to approximately 20 mm Hg (p<0.01).
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.
Keywords: 735 trabecular meshwork •
519 extracellular matrix •
568 intraocular pressure