Browsing by Author "Curry, Stacy"
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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.Item Pharmacokinetic and Physicochemical Evaluation of Novel Drug Candidates for Retinitis Pigmentosa(2023) Garrett, Meredith; Curry, Stacy; Feris, Sherri; Martin, Stephen; Clark, Abbot; Kastellorizios, MichailPurpose: Retinitis pigmentosa is a set of inherited ocular diseases that affect nearly 3 million people worldwide. The condition is inherited and causes the progressive deterioration of the retina. Retinitis pigmentosa begins with the loss of rod photoreceptors which cause night blindness and a decrease in peripheral vision. After significant loss of rod cells, cone cells also begin to die, decreasing central vision until complete blindness. More than 150 genetic mutations in 80 different genes have thus far been identified to contribute to progression pathways of the condition. Despite ongoing stem cell and gene therapy investigations, thus far there are no curative options. Most existing treatments focus on slowing the progression of retinal deterioration by reducing oxidative stress on the retina. Unfortunately, these treatments only achieve limited success and cannot halt progression. Recently, the sigma 2 receptor (σ2r) was identified to be endoplasmic reticulum membrane protein 97 (TMEM97). This protein (σ2r/TMEM97) has been shown to have neuroprotective effects on retinal cells and is thus of interest as a potential drug target for retinitis pigmentosa. Here we synthesized and tested a series of six compounds which have previously been found to modulate σ2r/TMEM97. To determine which of these compounds is a suitable drug candidate, each underwent in vivo and in vitro testing with the goal of selecting the best candidate for further clinical development. Methods: We tested the compounds in a rat model to determine retinal uptake following intravitreal injection. Each drug was dissolved in dimethyl sulfoxide (DMSO) and injected into the eye. At set time points, animals were sacrificed, and retinas were isolated from harvested eyes. The retina was separated and homogenized using sonication. A small portion was removed and underwent protein precipitation to purify the sample. The samples were then analyzed via liquid chromatography mass spectrometry (LCMS) to find the drug concentration remaining at each timepoint. In addition to obtaining a pharmacokinetic profile, the compounds were physiochemically characterized for chemical stability, solubility, in vitro drug release from vitreous humor, thermal analysis, and surface tension. Conclusion: Our goal is to select those drug candidates with the highest chance of clinical success. The pharmacokinetic profiles as well as physicochemical characteristics and stability of the compounds obtained in this study revealed important differences between the compounds that were used in selecting which to advance to in vivo efficacy testing. Ongoing studies include completion of physicochemical characterization and in vivo efficacy in a retinitis pigmentosa rat model that will be used to identify top candidates for further development.Item Phenotypic and transcriptomic comparison of genetically distinct mouse strains for susceptibility to glucocorticoid-induced ocular hypertension (GC-OHT)(2024-03-21) Patel, Pinkal D.; Millar, J. Cameron; Curry, Stacy; Feris, Sherri; Clark, Abbot F.Purpose: Anti-inflammatory and immunosuppressive glucocorticoids (GCs) are widely prescribed for a variety of conditions and diseases. Unfortunately, a significant number of people experience negative side-effects associated with long term GC therapy and develop GC-induced ocular hypertension (GC-OHT) leading to secondary glaucoma. GC-OHT shares clinical and molecular signatures with primary open angle glaucoma (POAG) making this an appropriate model to study POAG. However, not all humans develop GC-OHT when treated with GCs. The ones that develop GC-OHT are called ‘responders’ whereas the ones that do not respond to GCs are called ‘non-responders’. The purpose of our study is to: (1) determine whether there are mouse strain differences in the development of GC-OHT, (2) whether resistance to develop GC-OHT is correlated with endogenous TM tissue gene expression using transcriptomic analysis. Methods: After measurement of baseline IOP, various mouse strains (B6, D2.gpnmb⁺, BALB/cJ, 129P3/J, C3H/HeJ) were treated with weekly periocular injections of potent GC dexamethasone (DEX; n=5-10) or vehicle (n=5-10) in both eyes for 4-5 weeks. IOPs were measured weekly using a TonoLab rebound tonometer in isoflurane anesthetized mice. “TM ring” tissue and underlying sclera was carefully collected, and mRNA libraries were prepared for sequencing. Differential expression analysis was performed to identify DEX-induced changes within each strain. Furthermore, Ingenuity Pathway Analysis (IPA) was used to identify DEX-altered pathways in each strain and compare differences between responder and non-responder strains. Results: B6 and C3H/HeJ mice robustly and reproducibly develop DEX-OHT with ΔIOP of 5-8 mmHg (P<0.0001). In contrast, D2.gpnmb⁺, 129P3/J, and BALB/cJ mice were resistant to the development of DEX-OHT. Differential analysis of gene expression between mouse strains showed novel DEX-responsive genes in all strains. Moreover, comparison of mouse strains using IPA showed similarities in the pathway and networks of the responder strains (B6 and C3H/HeJ). Conclusions: As observed in humans, we find that there are differences in GC responsiveness and the ability to develop GC-OHT among mouse strains. Transcriptomics evidence suggests that responder strains share common pathways that contribute towards development of GC-OHT. These studies will reveal the molecular mechanisms responsible for GC-OHT as well as provide insights into the pathogenesis of POAG.Item TGFβ2-TLR4 Crosstalk Signaling in the Glaucomatous Trabecular Meshwork(2019-03-05) Curry, Stacy; Clark, Abbot F.; McDowell, Colleen; Roberts, AmandaPurpose: Glaucoma is a group of optic neuropathies and the leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the most prevalent type of glaucoma. Elevated intraocular pressure (IOP) is a major risk factor for the development of POAG. Elevated IOP is caused by aqueous humor fluid not draining properly through the drainage structures in the eye and leads to vision loss. Discovering potential new targets to lower IOP is necessary to develop novel and effective drug therapies. Here we explore a novel molecular mechanism involved in the development of glaucomatous trabecular meshwork (TM) damage. The TM regulates aqueous humor outflow and IOP. The effects of transforming growth factor beta (TGFβ)signaling pathways on the TM’s extracellular matrix (EÇM) have been extensively studied. Recently, we identified TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk regulates changes in the TM ECM and mutation in Tlr4 rescues TGFβ2-induced ocular hypertension in mice. Here, we investigated the role of an endogenous TLR4 ligand, FN-EDA, and a downstream signaling molecule of TLR4, NFκB, in TGFβ2-induced ocular hypertension in mice. Methods: B6.FN-EDA+/+, B6.TLR4-/-, B6.FN-EDA-/-, B6.FN-EDA+/+/TLR4-/-, B6.FN-EDA-/-/TLR4-/-, and C57BL/6J mice were intravitreally injected with 2.0μL Ad5.TGFβ2 (2.5x107pfu) in one eye and the contralateral uninjected eye was used as a negative control. Likewise, we tested mice lacking the p50 subunit of NFκB (B6.Cg-NFκB1tm1Bal/J) and C57BL/6J mice. IOP was measured once per week using a TonoLab rebound tonometer on isoflurane-anesthetized mice 42 or 49 days post-injection. Significance determined by one-way ANOVA at each time point. Eyes were harvested, fixed in 4% paraformaldehyde, and sectioned for immunohistochemistry to access total fibronectin and FN-EDA isoform expression. Results: Ad5.TGFβ2significantly induced ocular hypertension in C57BL/6J mice and enhanced ocular hypertension in B6.EDA+/+ mice. Mutations in Tlr4,FN-EDA, and NFκB blocked Ad5.TGFβ2induced ocular hypertension with no significant IOP elevation at any time point. Total FN and FN-EDA isoform expression increased in Ad5.TGFβ2 injected C57BL/6J mice. Data suggest the onset of ocular hypertension developed in uninjected B6.EDA+/+ mice at 14 weeks of age and Ad5.TGFβ2 enhanced elevated IOP levels. Conclusions: TLR4, FN-EDA, and NFkB are necessary for TGFβ2 induced ocular hypertension in mice. In the absence of Ad5.TGFb2 constitutively active EDA (FN-EDA+/+) mice develop ocular hypertension and in the presence of Ad5.TGFβ2 ocular hypertension is enhanced. These data demonstrate that the crosstalk between TGFβ2 and TLR4 is involved in the glaucomatous development within the trabecular meshwork. In addition, it provides potential new targets to lower IOP and to further explore mechanisms involved in the development of glaucomatous TM damage.Item Toll-like Receptor 4, FIbronectin Extra Domain A, and Nuclear Factor kappa B are Necessary for TGFβ2-induced Ocular Hypertension(2020) McDowell, Colleen; Hernandez, Humberto; Curry, Stacy; Harris, Sherri; Roberts, AmandaPurpose: Ocular hypertension is the major risk factor for developing primary open-angle glaucoma (POAG). Trabecular meshwork (TM) damage in POAG patients leads to impaired aqueous humor outflow and retinal ganglion cell damage. We explored a novel molecular mechanism involved in glaucomatous TM extracellular matrix (ECM) development through the TGFβ2-TLR4 signaling crosstalk; which regulates TM ECM changes. We discovered that mutations in Tlr4, FN-EDA, and NFkB rescues TGFβ2 -induced ocular hypertension. Methods: B6.FN-EDA+/+, B6.FN-EDA-/-, B6.TLR4-/-, B6.FN-EDA+/+/TLR4-/-, B6.FN-EDA-/-/TLR4-/-, and C57BL/6J mice were intravitreally injected with 2.0µL Ad5.TGFβ2 (2.5x10^7 pfu) in one eye and the contralateral uninjected eye was the control. Likewise, we tested mice lacking the p50 subunit of NFκB (B6.Cg-NFκB1tm1Bal/J) and C57BL/6J mice. IOP was measured weekly using a TonoLab rebound tonometer on isoflurane-anesthetized mice. Significance determined by one-way ANOVA. Eyes were harvested, 4%-paraformaldehyde-fixed, and sectioned for immunohistochemistry to access total fibronectin and FN-EDA isoform expressions. Results: Ad5.TGFβ2 significantly induced ocular hypertension C57BL/6J and B6.EDA+/+ mice. B6.EDA+/+ mice developed spontaneous ocular hypertension. Mutations in Tlr4, FN-EDA, and NFκB blocked Ad5.TGFβ2 -induced ocular hypertension. Total FN and FN-EDA expression increased in uninjected B6.FN-EDA+/+ mice and increased in response to TGFβ2 in wildtype and EDA mice. Conclusions: TLR4, FN-EDA, and NFκB are necessary for TGFβ2 induced ocular hypertension and ECM deposition in mice. These data provide new targets to lower IOP and inhibit glaucoma disease progression.