Browsing by Author "Medina-Ortiz, Wanda E."
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Item CELLULAR FIBRONECTIN SUPRESSES NORMAL HUMAN TRABECULAR MESHWORK CELL METALLOPROTEINASE EXPRESSION AND ACTIVATES TRANSFORMING GROWTH FACTOR-BETA 2 SIGNALING PATHWAY(2014-03) Medina-Ortiz, Wanda E.; Wordinger, Robert J.; Clark, Abbot F.Glaucoma is one of the leading causes of blindness, set off generally by changes on eye pressure and tissue modifications. One of the common tissue modifications includes induction of extracellular proteins expression on the human eye trabecular meshwork. This study provides important information on one of the tissue changes caused by a factor involved in glaucoma, Transforming Growth Factor-beta 2, the induction of the expression of the extracellular protein fibronectin. Fibronectin is a protein that can be found in two types of forms, a plasma soluble form and a cellular insoluble form. This study showed how interaction with cellular fibronectin form can affect normal human trabecular meshwork cells. This type of tissue modification may be part of the trabecular meshwork changes that can lead to glaucoma. Understanding the tissue changes and factors affecting the eye conditions leading to glaucoma will help in the discovery of agents protecting the eye from glaucoma damage. Purpose (a): The expression of cellular fibronectin isoforms (cFN) are induced by transforming growth factor-beta 2 (TGF-β2) in cultured human trabecular meshwork (TM) cells, and TGF-β2 expression is elevated in glaucomatous TM tissues. Cellular interaction with cFN isoforms can affect extracellular matrix (ECM) homeostasis, as well as the cellular interaction and response to the surrounding microenvironment. Our purpose is to determine the impact of the interaction of normal HTM (NTM) cells with cFN isoforms on the metalloproteinase (MMP) expression and the TGF-β2 signaling pathway. Methods (b): NTM cell strains were cultured for up to 2 days on surfaces coated with cFN, and the responses were compared to control uncoated surfaces. In addition, to show that the EDA domain of cFN was involved, NTM cells were cultured in the presence of anti-EDA antibodies. Changes on gene and protein expression and cellular distribution of MMPs and TGF-β2 signaling pathway components were analyzed using qRT-PCR, Western immunoblots and immunocytochemistry. Results (c): NTM cell strains exposed to cFN isoforms significantly decreased MMP-1 and MMP-3 expression, and this effect was blocked by anti-EDA pre-incubation. cFN significantly altered the expression of TGF-β2 signaling pathway components, including regulatory and inhibitory SMADs. The phosphorylation and nuclear translocation of regulatory SMADs also was increased, indicating activation of the TGF-β signaling pathway. Conclusions (d): Our results demonstrate that NTM cell interactions with cFN isoforms decreases levels of critical components involved in ECM homeostasis. Furthermore, we show that interaction with cFN affects different TGF-β2 signaling components further activating this signaling pathway. In summary, our data suggest that interaction of NTM cells with a glaucoma-like ECM (i.e. cFN) further exacerbates TGF-β2 signaling leading to decreased ECM turnover and fibrosis.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.Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(ARVO Journals, 2022-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Luan, Tomi; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFbeta2 signaling pathways in the TM has 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. Here, we investigated the role of TGFbeta2-TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods: Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFbeta2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 muM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFbeta2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results: Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFbeta2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFbeta2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFbeta2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions: These studies identify TGFbeta2-TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.Item Crosstalk of transforming growth factor beta-2 and toll-like receptor 4 in the trabecular meshwork(2015-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Clark, Abbot F.; McDowell, Colleen M.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.Item EFFECT OF CELLULAR AND PLASMA FIBRONECTIN ISOFORMS ON NORMAL HUMAN TRABECULAR MESHWORK CELLS(2013-04-12) Medina-Ortiz, Wanda E.Purpose: The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression. Methods: Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays. Results: GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes. Conclusions: Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.