Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21683
Browse
Browsing Eye / Vision by Author "Hernandez, Humberto"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Crosstalk Between TGFβ2 and TLR-4 Signaling Pathways in the Glaucomatous Trabecular Meshwork(2017-03-14) Hernandez, Humberto; Curry, Stacy; McDowell, Colleen; Roberts, AmandaPurpose: Glaucoma is a heterogeneous group of optic neuropathies that increases extracellular matrix (ECM) proteins in the trabecular meshwork (TM) and leads to thinning of the retinal nerve fiber layer and vision loss. TM regulates aqueous humor outflow and intraocular pressure (IOP). In this study, we employ experimental cell culture methods to determine whether the crosstalk between transforming growth factor beta 2 (TGFβ2) and toll-like receptor 4 (TLR4) signaling pathways regulate ECM production. We hypothesize that TGFβ2-TLR4 crosstalk is a pathway that assist TLR4 ligands to augment TGFβ2 signaling and regulate ECM production in the TM. Methods: Transformed human TM (GTM3) and primary human TM (HTM) cells were grown to confluency. Cells were pre-treated with TAK-242 for 2 hours followed by treatment of TGFβ2 for 24 hours (RNA) or 48 hours (protein). To activate TLR4, HTM cells were plated on dishes pre-coated with cellular fibronectin (cFN) containing the FN-EDA isoform in the presence or absence of TGFβ2. ECM expression was assessed by western immunoblotting and quantified by densitometry. Results: These experiments revealed a TGFβ2-TLR4 signaling crosstalk that regulates ECM production. TGFβ2 treatment in GTM3 cells enhanced fibronectin (FN) and collagen-1 mRNA expression and FN protein expression in the condition medium and cell lysate, while TAK-242 significantly blocked this effect. Activating TLR4 using cFN-EDA alone increased FN expression in HTM3 cells. TGFβ2 with cFN-EDA treatment significantly enhanced FN expression compared to TGFβ2 alone. TLR4 inhibitor TAK-242 significantly blocked TGFβ2 and cFN-EDA induced ECM changes. For future studies, I will use low molecular weight hyaluronan (LMWH) as a TLR4 activator. I expect the LMWH to enhance the effect of TGFβ2 induced ECM production. Conclusions: These studies identified TGFβ2-TLR-4 crosstalk as a novel pathway involved in ECM regulation in the TM. These data provide novel targets to further explore the molecular mechanism involved in glaucomatous TM development.Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(2017-03-14) Medina-Ortiz, Wanda E.; Curry, Stacy; Luan, Tomi; Clark, Abbot; McDowell, Colleen; Hernandez, HumbertoPurpose: The trabecular meshwork (TM) plays an important role in the regulation of aqueous humor outflow and intraocular pressure (IOP). Regulation of the ECM by TGFβ2 in the TM and toll-like receptor 4 (TLR4) in fibrogenesis has been extensively studied. Here, we investigate the role of TGFβ2-TLR4 signaling crosstalk and BMP/activin membrane-bound inhibitor (BAMBI) in the regulation of the TM ECM and ocular hypertension. Methods: TLR4 expression was evaluated in cross-sections of human donor eyes, primary human TM cells, and dissected mouse TM rings. TM cells were treated with TGFβ2 (5ng/ml), TLR4 inhibitor (TAK-242, 15mM), and/or TLR4 ligand (cFN-EDA, 10mg/mL). A/J (n=13), AKR/J (n=7), BALBc/J (n=8), C3H/HeJ (n=20), and C3H/HeOuJ (n=10) were injected intravitreally with Ad5.hTGFβ2. Further, B6;129S1-Bambitm1Jian/J mice were injected intravitreally with either Ad5.TGFβ2 (n=10), Ad5.Cre (n=9), or Ad5.TGFβ2 + Ad5.Cre (n=10). The uninjected contralateral eyes served as controls. Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J mice using magnetic beads and transduced with Ad5.TGFβ2 or Ad5.Cre in cell culture. Results: TLR4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFβ2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFβ2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFβ2 induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre each induced ocular hypertension significantly throughout the time course compared to uninjected control eyes. Bambi knockdown by Ad5.Cre leads to increased fibronectin expression in MTM cells. Conclusions: Here we show a TGFβ2-TLR4 crosstalk pathway that we hypothesize is regulated by TGFβ2 negative regulator BAMBI. Conditional knockdown of BAMBI in the TM with Ad5.Cre induces fibronectin expression, reduces aqueous humor outflow facility and causes ocular hypertension. These data provide a novel pathway involved in the development of glaucomatous TM damage and provide potential new targets to lower IOP.