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Colleen M McDowell, Humberto Hernandez, Abbot F Clark; Crosstalk between TGFβ2 and TLR4 in the trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
The trabecular meshwork (TM) regulates aqueous humor outflow and intraocular pressure (IOP). The effects of TGF-β signaling pathways on the TM extracellular matrix (EÇM) have been extensively studied. Recent evidence implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung and skin. We investigated whether TGFβ2-TLR4 signaling crosstalk regulates changes in the TM ECM and if mutation in Tlr4 rescues TGFβ2-induced ocular hypertension in mice.
Primary human TM cells were treated with combinations of a selective TLR4 inhibitor (TAK-242, 15µM), cFN containing FN-EDA (10ug/ml), and TGFβ2 (5ng/ml). Gene and protein expression were measured by qRT-PCR and western immunobots. C3H/HeJ (n=15) and C3H/HeOuJ (n=11) mice were injected intravitreally with Ad5.hTGFβ2226/228 in one eye. The uninjected contralateral eye served as a control. IOP measurements were taken using a TonoLab rebound tonometer.
In confluent HTM cells, TGFβ2 treatment increased both fibronectin (FN) (4.46 +/- 0.07 fold) and collagen 1 (COL1) (11.53 +/- 0.41 fold) mRNA expression. 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, p<0.001). To activate TLR4, cells were plated on dishes pre-coated with cFN in the presence of TGFβ2 and FN expression was increased (4.86 +/- 1.06 fold), while treatment with TGFβ2 + TLR4 inhibitor blocked this effect (2.36 +/- 0.45 fold) (n=4, p<0.05). Activation of TLR4 by cFN increased NF-κB activity (12.1 +/- 1.8 RLU, n=6) while treatment of cFN + TLR4 inhibitor reduced NF-κB activity (6.3 +/- 1.7 RLU, n=6, p<0.001). Similarly, treatment with TGFβ2 increased NF-κB activity (15.3 +/- 2.2 RLU n=6) while treatment of TGFβ2 + TLR4 inhibitor reduced NF-κB activity (4.2 +/- 0.96 RLU, n=6, p<0.001). pNF-κB and pSMAD3 expression also increased in cFN treated cells and reduced with addition of the TLR4 inhibitor. Intraocular injection of 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 mmHg (p<0.01).
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 to further explore the molecular mechanisms involved in the development of glaucomatous TM damage.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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