Browsing by Author "Tovar-Vidales, Tara"
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Item ADAM19 and ADAMTS4 expression in Human Optic Nerve Head Astrocytes(2024-03-21) Easo, Tony; Rangan, Rajiv; Tovar-Vidales, TaraPurpose: Glaucoma is characterized by the degeneration and death of retinal ganglion cells and their axons causing irreversible blindness. Various risk factors contribute to glaucoma onset, including intraocular pressure (IOP), age, and family history. In glaucoma, the primary site of damage is the optic nerve head (ONH). ONH astrocytes, a major cell type in the LC, are believed to play a significant role in the pathological remodeling of extracellular matrix (ECM) during glaucoma. Glaucoma patients have increased levels of transforming growth factor beta 2 (TGFβ2) in their aqueous humor, trabecular meshwork and ONH. TGFβ2 is a profibrotic cytokine known to induce the synthesis and deposition of ECM. Previously, we performed RNA sequencing of ONH astrocytes in which disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTS) were significantly dysregulated with TGFβ2 treatment compared to controls. ADAMs and ADAMTS4 influence cell phenotype by affecting cell adhesion, migration, proteolysis, and signaling pathways. The purpose of the present study was to determine a) if ONH astrocytes express ADAM19 and ADAMTS4 in ONH astrocytes and b) determine if TGFβ2 regulates ADAM19 and ADAMTS4 expression in ONH astrocytes. Methods: Primary human ONH astrocyte cell strains (n=3) were treated with TGFβ2 (5ng/ml) or with a vehicle control for 48 hours. The effects of TGFβ2 on ADAM19 and ADAMTS4 were determined by qPCR and western blots using primary human ONH astrocyte cell cultures. Results: ADAM19 and ADAMTS4 were expressed in ONH astrocytes. Treatment with TGFβ2 significantly increased mRNA levels of ADAM19 and ADAMTS4. However, western blot analysis showed no significant change in ADAM19 protein expression compared to control, while ADAMTS4 was increased with TGFβ2 compared to control. Conclusions: TGFβ2 modulated the expression of ADAM19 and ADAMTS4 in ONH astrocytes. ADAM19 and ADAMTS4 may contribute to the pathogenic remodeling of the ONH in glaucoma. While further studies are needed, this research aims to shed light on the intricate mechanisms underlying glaucoma pathogenesis.Item In Vitro Effect of CNTF, FGF-9, IL-1α on Human Optic Nerve Head Astrocytes(2004-08-01) Tovar-Vidales, Tara; Wordinger, Robert J.; Alvarez-Gonzales, Rafael; Agarwal, NeerajTovar, Tara., In Vitro Effect of CNTF, FGF-9, and IL-1α on Human Optic Nerve Head Astrocytes. Master of Science (Biomedical Sciences), August 2004, 100 pp., 4 tables, 35 illustrations, bibliography, 163 titles. Glaucoma is a leading cause of blindness worldwide. A major risk factor for glaucoma is increased intraocular pressure that leads to pathological changes in the optic nerve head (ONH). Astrocytes within the ONH become activated in glaucoma and may create an environment detrimental to retinal ganglion cell axons. The factors that cause activation of the ONH astrocytes (ONA) are unknown, although there is evidence that CNTF, FGF-9, and IL-1α activate glial cells within the CNS. The purpose of this research was to determine if exogenous CNTF, FGF-9, and/or IL-1α activate human ONH astrocytes.Item Increased Fibronectin Serotonylation in Stretched Optic Nerve Head Astrocytes(2023) Rangan, Rajiv; Clark, Abbot; Tovar-Vidales, TaraPurpose: Elevated intraocular pressure contributes to glaucomatous optic nerve degeneration by inducing biomechanical stress at the optic nerve head (ONH), especially in the lamina cribrosa (LC). ONH astrocytes (ONHA) in the LC respond to biomechanical signals through extracellular matrix (ECM) remodeling activities, promoting tissue fibrosis and damage to retinal ganglion cell axons. The enzyme transglutaminase 2 (TG2) plays a role in ECM remodeling, in part, due to its ability to post-translationally modify and cross-link ECM proteins. A unique post-translational modification mediated by TG2 is "serotonylation” - the transamidation of the monoamine serotonin (5-hydroxytryptamine, 5HT) to glutamine residues on proteins. It is speculated that serotonylation contributes to fibrotic tissue remodeling, but this process has not been studied in ocular tissues or in primary glial cells. In this study, we examined changes in the serotonylation of fibronectin (FN; a major ECM glycoprotein) by ONHA after exposure to cyclic stretch. Methods: Primary human ONHA strains (n=3) were exposed to 0-12% cyclic stretch for 24h using a FlexCell FX-6000 system. Cell lysates and conditioned medium samples were collected from stretched and control cells. Serotonylation was assessed by probing for serotonin in samples of FN immunoprecipitated out of conditioned media. Protein levels for potential extra- and intra-cellular mediators of serotonylation were examined using western blotting of concentrated conditioned medium samples and cell lysates, respectively. Results: Serotonylated fibronectin was detected in ONHA. Exposure to stretch increased the amount of fibronectin that was serotonylated by 2.49-fold (p=0.0080). After stretching, extracellular FN levels were not changed. Extracellular TG2 levels were increased by 3.76-fold (p=0.0004). In cell lysates, post-stretch levels of both FN and TG2 were decreased by 5.56-fold (p=0.0181) and 2.51-fold (p=0.0441), respectively. Additionally, serotonin 2A and 2C (5HT2A, 5HT2C) receptor levels were unchanged, and serotonin transporter (SERT) levels decreased by 2.94-fold (p=0.0297). Conclusions: Increased FN serotonylation by TG2 is observed in ONHA after exposure to 24h of 0-12% cyclic stretch. Serotonylation may promote increased FN-crosslinking and fibrotic ECM remodeling, an important feature of glaucomatous pathology. The secreted TG2 – which was elevated in response to stretch – is likely to be the primary mediator of this increased serotonylation. The observed decrease in SERT may lead to increased extracellular 5HT levels, which increases the substrate availability for TG2-mediated serotonylation. Though unchanged, activity at the 5HT2A/C G-coupled protein receptors could increase the availability of intracellular calcium required for TG2 activity. Future experiments will be focused on furthering our understanding of how these proteins may interact to promote serotoynlation.Item It’s not a GIST: A Histopathological Investigation of a Rare Gastric Schwannoma(2023) Thai, Stephen; Phan, Jenny; Taft, Carter; Roth, Hannah; Tovar-Vidales, Tara; Crowe, NicoleBackground: Gastric schwannomas are rare gastrointestinal mesenchymal tumors, accounting for <0.2% of all gastric neoplasms. Gastric schwannomas are benign, arise from the gastrointestinal nerve plexus, and are often misdiagnosed as gastrointestinal stromal tumors (GISTs). Gastric schwannomas are typically incidental findings on conventional imaging studies, surgery, or autopsy. Reported patients are usually asymptomatic. Gastric schwannomas can be differentiated from GIST and other mesenchymal gastrointestinal tumors through the use of immuno-histochemical markers such as S100, Vimentin, and glial fibrillary acidic protein (GFAP). Case Information: During routine dissection of a 78-year-old female, a 1.5 x 1.2 x 1.0 cm tan, firm nodule protruding from the anterior stomach wall was identified. The nodule was located 11 cm from the gastroesophageal junction, 7 cm from the incisura angularis, and 6 cm from the pyloric sphincter. Sectioning revealed white, whorled, and rubbery cut surfaces. No hemorrhage, necrosis, or cystic spaces were grossly appreciated. Subsequently, the tissue underwent routine fixation, processing, paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. Using light microscopy, histopathologic findings included a well-circumscribed area containing mostly eosinophilic spindle cells with nuclear palisading, foci of lymphoid infiltration,and a micro-trabecular pattern noted centrally. Additionally, a peripheral lymphoid cuff was present. No mitotic figures were identified. Based on our histopathology findings, we hypothesized the presented nodule to be a gastric schwannoma. This diagnosis was corroborated by immunohistochemistry to demonstrate positive S100b protein expression. Common differential diagnoses include gastrointestinal stromal tumors (GISTs), leiomyomas, and solitary fibrous tumors, all of which lack S100 protein reactivity. Conclusion: This case aims to contribute an additional report about gastric schwannomas to better enhance our collective understanding of this rare lesion.Item miR-200b-3p and miR-211-5p downregulate the expression of extracellular matrix and associated proteins in human optic nerve head astrocytes(2018-03-14) Tovar-Vidales, Tara; Clark, Abbot F.; Lopez, NavitaPURPOSE: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is a leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the profibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eyes. Currently there is a lack of knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine: (a) differences in the expression of profibrotic and anti-fibrotic miRNAs in normal ONH astrocytes treated with or without TGFβ2 and (b) whether candidate miRNAs (miR-200b-3p and miR-211-5p) regulate the expression of ECM and ECM associated proteins in the ONH. METHODS: Primary normal human ONH astrocytes (ONA) were grown to 100% confluency. ONA were treated with 5ng/ml TGFβ2 or with control for 24hrs. miRNA qPCR arrays were performed to compare the expression levels of profibrotic and anti-fibrotic miRNAs in normal ONA treated with or without TGFβ2. ONA were transfected with miR-200b-3p and miR-211-5p mimics at 10nM, 5nM, and 1nM concentrations to confirm target predictions based on the TargetScan database. An all stars negative control siRNA was included which will not recognise any mammalian gene. RESULTS: The miRNA qPCR arrays analysed from normal ONA exposed to TGFβ2 showed that TGFβ2 downregulated the expression of hsa-miR-200b-3p and hsa-miR-211-5p. Transfection of miR-200b-3p mim downregulated fibronectin (FN), gremlin, and tissue transglutaminase II in ONA. Transfection of miR-211-5p downregulated FN and gremlin in ONA . CONCLUSIONS: Our results suggest that TGFβ2, which is elevated in the glaucomatous ONH, modulates the expression of miRNAs in ONA. These miRNAs target FN, TGM2, and gremlin to modify the ECM in the ONH. Downregulation of anti-fibrotic miRNAs may contribute to fibrosis of the ONH.Item Mirna Expression in Glaucomatous and TGFbeta2 Treated Lamina Cribrosa Cells(MDPI, 2021-06-08) Lopez, Navita N.; Rangan, Rajiv; Clark, Abbot F.; Tovar-Vidales, TaraGlaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins; however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFbeta2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFbeta2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFbeta2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.Item miRNA Profiling of Human Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2021-05) Rangan, Rajiv S.; Tovar-Vidales, Tara; Clark, Abbot F.; Liu, YangGlaucoma is a leading cause of irreversible blindness. Vision loss results from the degeneration and death of retinal ganglion cells (RGCs) and their axons. The primary risk factor for glaucoma is increased intraocular pressure (IOP) (2). Elevated IOP results in aberrations in the biomechanical properties of ocular tissues - including the transmission of biomechanical stretch through the reticulated, fibroelastic region of the optic nerve head (ONH) known as the lamina cribrosa (LC) (6). Cells of the LC are sensitive to biomechanical stretch and respond to increased stretch and pressure to promote the excessive synthesis of extracellular matrix (ECM) proteins and ECM remodeling (15,17). These responses promote a fibrotic environment within the LC that can cause mechanical damage to the axons of RGCs. ONH astrocytes represent one of the major cell types of the LC and are believed to contribute significantly to pathological ECM remodeling at the LC during glaucoma (11). ONH astrocytes also demonstrate a dysregulated pattern of protein expression when exposed to stretch (17). The mechanism that underlies this stretch-induced, aberrant dysregulation is unknown. MicroRNA (miRNA) dysregulation may represent one of the mechanisms contributing to the differential protein expression patterns seen in ONH astrocytes exposed to stretch. In this study we examine the miRNA profiles of ONH astrocytes exposed to cyclic stretch.Item miRNA Profiling of Human Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2021) Rangan, Rajiv; Tovar-Vidales, TaraPurpose: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness involving the progressive loss of retinal ganglion cells (RGCs) and their axons. Elevated IOP induces biomechanical aberrations within ocular tissues – including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small RNA molecules that can indirectly inhibit gene expression by binding messenger RNA. miRNA dysregulation may contribute to ECM remodeling during glaucoma progression. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours. miRNA PCR arrays were used to determine expression changes in profibrotic and anti-fibrotic miRNAs. Results: We found that specific miRNAs were consistently dysregulated across three independent strains of ONHA. Statistical significance could not be detected, but these patterns may represent biologically meaningful changes. Conclusion: Stretch modulates miRNA expression in cultured human ONHA and may be responsible for ECM alterations at the LC. Dysregulated miRNAs may serve as novel targets or models for future therapeutics.Item miRNA Profiling of Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2022) Rangan, Rajiv; Tovar-Vidales, TaraIntroduction: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness consequent to retinal ganglion cell (RGC) degeneration. Elevated IOP induces biomechanical aberrations within ocular tissues - including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling (fibrosis) and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small molecules that can inhibit protein expression by binding to and silencing mRNA. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. We hypothesized that cyclic stretch would induce upregulation of miRNAs that silence anti-fibrotic protein translation and downregulation of miRNAs that silence pro-fibrotic protein translation, promoting a net-fibrotic molecular signaling environment. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours; controls were exposed to 0% stretch. RNA samples were collected from stretched and control cells, and miRNA PCR arrays were used to determine expression changes for miRNAs associated with fibrosis. Expression fold changes were normalized to SNORD68. The bioinformatics tool TargetScan was used to predict mRNA targets for any dysregulated miRNAs. Induction of fibrotic cellular changes by cyclic stretch was confirmed by western blotting of conditioned media for secreted proteins. Results: miR-146b-5p was found to be significantly upregulated by +5.97-fold (P = 0.029) in stretched ONHA. Predicted mRNA targets for miR-146b-5p are known to be involved in fibrosis and cell survival, among other functions. Preliminary data indicates upregulation of secreted proteins associated with fibrosis (TGFβ2, Fibronectin, Transglutaminase 2) by stretched ONHA. Conclusions: Stretch modulates miRNA expression in cultured human ONHA, miR-146b may mediate ECM alterations and other pathological changes at the LC. Future experimental directions will include assessing co-expression of other miR-146 family miRNAs, validating putative mRNA targets and elucidating the mechanisms by which specific miRNA and their targets modulate ECM remodeling.Item Role of miR-29c-3p in regulation of extracellular matrix synthesis(2019-03-05) Clark, Abbot F.; Tovar-Vidales, Tara; Lopez, NavitaPurpose Glaucoma is a group of optic neuropathies characterized by cupping of the optic nerve head (ONH) and degeneration of retinal ganglion cell (RGC) axons that lead to loss of visual function. Primary open angle glaucoma (POAG) is the most common form of glaucoma, with a global prevalence of 65.5 million, approximately 74% of glaucoma cases. The initial site of damage in POAG is within the lamina cribrosa (LC) region of the ONH. There is upregulation of the pro-fibrotic cytokine, TGFβ2, and marked disparity in the distribution and organisation of extracellular matrix (ECM) proteins. TGFβ2 induced downregulation of miR-29 has been shown, in part, to drive ECM protein synthesis in trabecular meshwork cells. Our purpose was to determine the effect of TGFβ2 on miRNA expression, in cells that populate the LC. We hypothesise that increased TGFβ2 signalling downregulates the expression of anti-fibrotic miRNAs, stimulating a fibrotic response and remodelling of the glaucomatous LC. Methods Primary human LC cells were grown to 100% confluency, treated with TGFβ2 (5ng/ml) or control for 24hours and differences in expression of miRNAs were analysed by PCR arrays. LC cells were transfected with miR-29c-3p (10nM) mimic, inhibitor or non-targeting controls and analysed by Q-PCR to confirm overexpression or knockdown of miR-29c-3p. mRNA targets of miR-29c-3p were determined through protein expression analysis by immunocytochemistry. The effects of miR-29c-3p and TGFβ2 on collagen type (COL) I and IV protein expression were evaluated in cells transfected with miR-29c-3p mimic, inhibitor or control and treated with TGFβ2 expression. Results TGFβ2 treatment downregulated the expression of miR-29c-3p in LC cells (n=4, pa-smooth muscle actin,COL (collagen) I and IV. Transfection of miR-29c-3p mimic or inhibitor showed upregulation and downregulation of miR-29c-3p respectively, confirming transfection efficiency. miR-29c-3p was found to be a key regulator of COL I and IV synthesis. Overexpression of miR-29c-3p decreased TGFβ2 induced COL I and IV expression in LC cells. Inhibition of miR-29c-3p exacerbated the effects of TGFβ2 on COL I and IV expression. Conclusion This suggests that elevated TGFβ2 signalling may stimulate a pro-fibrotic response through downregulation of miR-29c-3p. Although miR-29c-3p may be protective by decreasing the effects of TGFβ2 induced ECM protein synthesis, we will need to further elucidate the role of TGFβ2 and miR-29c-3p in maintaining the balance of ECM synthesis.Item Role of TGFβ2 and miRNAs in optic nerve head fibrosis(2020) Tovar-Vidales, Tara; Clark, Abbot; Lopez, NavitaPurpose: Glaucoma is characterised by degeneration of retinal ganglion cells (RGCs) and loss of visual function. This is associated with extracellular matrix (ECM) remodelling of the optic nerve head (ONH). The ONH provides mechanical and biochemical support to unmyelinated RGC axons as they exit the eye to form the optic nerve; thus, changes in the ONH affect the physiology of axons and may damage the visual circuitry. In glaucoma, mechanosensitive cells in the ONH respond to elevated pressure by secreting growth factors, ECM and inflammatory cytokines that damage RGCs and remodel the ONH. A key upregulated pathway in glaucoma is TGFβ2, which leads to increased expression of profibrotic genes. We hypothesised that impaired regulation of TGFβ2 and miRNAs is involved in glaucomatous remodelling of the ONH ECM. The purpose of this study was to identify cell-specific changes in glaucoma pathophysiology. Method: ONH astrocytes and lamina cribrosa (LC) were treated with TGFβ2 (5ng/ml) or vehicle and analyzed by miRNA qPCR arrays and NGS. Cells were transfected with candidate miRNA mimics, inhibitors, or non-targeting siRNA (10nM) to determine ECM expression. Results: TGFβ2 treatment decreased the expression of multiple antifibrotic miRNAs, which was associated with increased expression of several profibrotic genes including collagen I and IV and fibronectin. Overexpression of antifibrotic miRNAs including miR-29c-3p and miR-200b-3p decreased TGFβ2 induced collagen I and IV and fibronectin expression. Conclusion: TGFβ2 modulated the expression of several miRNAs to stimulate a profibrotic response; this may lead to pathogenic ONH remodelling.Item The Effects of Ad5.CMV.hTGFβ2C226/228S on AHD in Mice(2021-05) Stevenson, Cooper H.; Millar, J. Cameron; Tovar-Vidales, Tara; Stankowska, Dorota L.Elevated intraocular pressure (IOP) is a key risk factor for the development of primary open-angle glaucoma (POAG), a leading cause of blindness in people over the age of 40 years. Transforming growth factor beta-2 is a cytokine known to contribute to the pathogenesis of POAG due to its deleterious effects on aqueous humor outflow via the conventional, or trabecular, outflow pathway in the eye. However, its effects on the rate of aqueous outflow (Fu) via the unconventional or uveoscleral outflow pathway, rate of aqueous humor production (Fin), and episcleral venous pressure (Pe) are unknown. Further, effects of euthanasia and enucleation in our hands on TGFβ2-mediated effects on Fu are also unknown. The goal of the present study was to quantify the impact of over-expression of TGFβ2 on aqueous humor dynamics (AHD) in the mouse eye, with special emphasis on Fu, Fin, and Pe in the mouse eye. To simulate TGFβ2 over-expression, left (OS) eyes were injected intravitreally (IVT) with a mutant form of TGFβ2 (Ad5.CMV.hTGFβ2C226/228S, 2×10⁷pfu in 2μL), while right (OD) eyes were injected IVT with a null virus (Ad5.CMV.null, same titer and volume). Following 14 days, after which time mean IOP (determined tonometrically in conscious mice) had become elevated in TGFβ2-injected eyes (84.29% increase in IOP, P < 0.001), Fu was determined directly by cannulating the anterior aqueous chamber (AC) and perfusing it with fluorescein isothiocyanate-dextran (1×10⁻⁹ M), followed by dissection of the retina/choroid/iris-ciliary body/scleral shell, homogenization, and measurement of each sample's fluorescence, and then inference of flow rate using a standard curve. Those perfusion were performed in living eyes, also in eyes in situ in the animal immediately following euthanasia, and enucleated eyes perfused in vivo either (i) exposed to air, or (ii) submerged in PBS. In a further group of experiments in living animals aqueous humor outflow conductance (C) (also known as aqueous humor outflow facility), and Pe were measured, and then Fin and Fu were calculated using a constant flow infusion method. Further, we sought to determine whether IOP elevation would lead to a reduction in RGC numbers in the retina, so retinal flat mounts from both treated and untreated eyes from 5 of our animals were prepared and RGC counts were made. For eyes perfused in-vivo, Fu was reduced in OS (0.0048 ± 0.0017 μL/min) compared to OD (0.0987 ± 0.0126 μL/min, P = 0.025). For eyes perfused in euthanatized mice, Fu was reduced in OS (0.0215 ± 0.0101 μL/min) compared to OD (0.1543 ± 0.0241 μL/min, P = 0.010). For eyes perfused ex-vivo while submerged in PBS, there was no difference in Fu between OS (0.0222 ± 0.0065 μL/min) and OD (0.0137 ± 0.0078 μL/min, P = 0.175). For eyes perfused ex-vivo while exposed to air, Fu was reduced in OS (0.0702 ± 0.0087 μL/min) compared to OD (0.1377 ± 0.0106 μL/min, P = 0.008). Fin showed a trend towards a reduction in the eyes in which TGFβ2 was over-expressed, but this effect did not reach statistical significance. There was a significant increase in Pe in eyes in which TGFβ2 was expressed (8.6 ± 0.7 mmHg in OS to 6.4 ± 0.2 mmHg in OD, P = 0.015). Given these results, the present study further quantifies the effect of TGFβ2 in POAG, providing more insight into its mechanism of action in this disease.Item The Neuroprotective Effects of SA-2-NP in a Mouse Model of RGC Injury(2021-05) Ferguson, Jonathan L.; Stankowska, Dorota L.; Millar, J. Cameron; Tovar-Vidales, TaraDetermine if a novel hybrid compound SA-2 can be delivered to the retina in a nanoparticle formulation and have protective effects on retinal ganglion cells (RGCs) following an optic nerve crush (ONC) model of RGC death. Pattern Electroretinography (PERG) was performed on six- to twelve-week-old female (C57BL/6) mice (n = 1-8 mice per group) prior to performing ONC on the left eye to promote RGC death similar to that seen in normotensive glaucoma. Mice were dosed topically for seven or fourteen days either with SA-2 in polylactic glycolic acid (PLGA) nanoparticles, or empty PLGA nanoparticles. Subsequent PERG was performed at seven day following ONC to reassess RGC function after the optic nerve injury and treatments. The mice were subsequently euthanized and both eyes we enucleated and fixed with paraformaldehyde. The retinas were removed, flat mounts were prepared and immunostained with RBPMS antibody to quantify surviving RGCs. Our study demonstrated that SA-2 can be delivered to the retinal tissue with PLGA nanoparticles. However, following optic nerve crush in mice, at the selected doses and delivery regimen of SA-2, neuroprotective effects determined by RGC counts and PERG analysis were not statistically significant. Following ONC in mice, topically delivered SA-2 loaded nanoparticles demonstrated some trend in neuroprotection without statistical significance. Further investigation is required to delineate the efficacious delivery mode and dose.Item The regulation of transforming growth factor beta-2-induced ocular hypertension and glaucomatous environment in the trabecular meshwork(2020-12) Roberts, Amanda L.; Clark, Abbot F.; Krishnamoorthy, Raghu R.; Pang, Iok-Hou; Tovar-Vidales, Tara; Gatch, Michael B.Glaucoma is a heterogeneous group of optic neuropathies that damage the optic nerve that leads to progressive visual loss and irreversible blindness in over 80 million people worldwide. Primary open angle glaucoma (POAG) is the most common form of glaucoma, and elevated intraocular pressure (IOP) is the major risk factor for the development and progression of this ocular disease. One of the potential mechanisms responsible for elevated IOP in POAG patients is the excessive accumulation of extracellular matrix (ECM) proteins within the trabecular meshwork (TM). Damage to the TM impairs the aqueous humor outflow and increases aqueous humor outflow resistance, and elevates IOP. Discovering potential new disease modifying targets to lower IOP is necessary to develop effective therapies to inhibit the progression of glaucoma. Here, we explored a novel molecular mechanism involved in the development of glaucomatous TM ECM damage. The effects of transforming growth factor beta 2 (TGF[beta]2) signaling pathways in the TM ECM have been extensively studied. TGF[beta]2 is a profibrotic signaling component in ocular hypertension development that increases ECM deposition in the TM. As a member of the toll-like receptor family, toll-like receptor 4 (TLR4) was originally identified as the receptor for lipopolysaccharide (LPS). TLR4 can also be activated by endogenous ligands known as damaged associated molecular patterns (DAMPS), which are generated as a result of injury, cell damage, and ECM remodeling. DAMP-induced TLR4 activation has been linked to fibrosis, ECM protein deposition, and augmented TGF[beta]2 signaling and downstream fibrotic responses. Recently, we identified TGF[beta]2-TLR4 signaling crosstalk that regulates the TM ECM, and mutation in TLR4 rescues TGF[beta]2-induced ocular hypertension in mice. The goal of this work was to investigate the role of the endogenous TLR4 ligand fibronectin containing extra domain A isoform (FN-EDA), and the TLR4 downstream signaling molecule NF[kappa]B in TGF[beta]2-induced ocular hypertension in mice. Overall, we discovered that TLR4, FNEDA and NF[kappa]B are necessary for TGF[beta]2-induced ocular hypertension and ECM deposition in mice. In addition, constitutively active EDA mice spontaneously develop ocular hypertension. These data provide new targets to lower IOP and inhibit glaucoma disease progression.Item The Role of LLT1 (CLEC2D, OCIL) in Ewing Sarcoma(2021-12) Buller, Casey W.; Mathew, Stephen O.; Mathew, Porunelloor A.; Jones, Harlan P.; Basha, Riyaz; Tovar-Vidales, TaraEwing Sarcoma (EWS) is a pediatric bone cancer that is characterized by a chromosomal translocation giving rise to a neomorphic gene fusion. Although treatment of EWS has a 5-year incident free survival rate of 66%, treatment has plateaued since the 1980's. Natural killer (NK) cells are an important innate immune cell due to their ability to recognize and lyse virally infected and cancerous cells. Unfortunately, cancerous cells often employ strategies such as the downregulation of major histocompatibility complex (MHC) I, or upregulation of inhibitory ligands enabling the escape of T-cells and NK cell mediated killing. LLT1 is an inhibitory ligand our lab had previously shown that it is expressed on TNBC and prostate cancer cell lines. The expression of LLT1 and its function in Ewing Sarcoma (EWS) cell lines has yet to be performed. Our hypothesis is that LLT1 is increased in EWS cell lines TC-32 and CHLA-258 when treated with chemotherapeutic drugs vincristine and etoposide. Our results show that LLT1 is expressed on EWS cell lines and inhibition of LLT1 increases NK cell cytotoxicity. These results indicate that LLT1 is a potential immunotherapy target that needs to be further evaluated in an in vivo model.Item The Role of Transforming Growth Factor Beta 2 Signaling and MicroRNAs in Optic Nerve Head Remodeling(2020-08) Lopez, Navita N.; Clark, Abbot F.; Tovar-Vidales, Tara; Liu, Yang; Pang, Iok-HouPrimary open-angle glaucoma (POAG) is a prevalent age-related neurodegenerative disease of the visual system. There are functional and morphological changes in the retina, optic nerve head (ONH) and brain that lead to an irreversible loss of vision. POAG is characterised by degeneration of retinal ganglion cells (RGC), thinning of the neuro-retinal rim and structural deformation of the ONH. The primary site of injury is the lamina cribrosa, which is a fibro-elastic connective tissue that supports the ONH and unmyelinated RGC axons as they exit the intraocular space. Pathological changes to the lamina cribrosa include posterior displacement of the lamina cribrosa, loss of trophic support, and remodeling of the extracellular matrix (ECM). An important growth factor associated with tissue remodeling is TGFb2. TGFb2 activates the SMAD-dependent TGFb2 pathway and increases transcription of several ECM genes including collagen, fibronectin and crosslinking enzymes. In POAG, the levels of TGFb2 are increased in the lamina cribrosa and is associated with excess deposition of ECM molecules. This study proposes to investigate the intermediary mechanisms that lead to tissue remodeling. microRNAs (miRNAs) regulate gene expression by inhibiting protein translation. We hypothesized that miRNAs are dysregulated in POAG and in response to TGFb2, which leads to excess ECM synthesis and tissue remodeling. We isolated primary human ONH astrocytes and lamina cribrosa cells from POAG and normal donor eyes. We used miRNA PCR arrays to determine differentially expressed miRNAs in POAG and TGFb2 treated cells. Several anti-fibrotic miRNAs were downregulated, including downregulation of miR-29c-3p in POAG and TGFb2 treated lamina cribrosa cells, and downregulation of miR-200b-3p in TGFb2 treated ONH astrocytes. To validate mRNA targets and determine the functional role of differentially expressed miRNAs, we modulated miRNA biology using miRNA mimics and inhibitors. Overexpression of miR-29c-3p and miR-200b- decreased the expression of ECM proteins. Treatment with TGFb2 increased the expression of collagens and fibronectin and overexpression of miR-29c-3p and miR-200b-3p decreased this effect, suggesting that miR-29c-3p and miR-200b-3p regulate the TGFb2 signaling pathway. It is possible that increased TGFb2 is responsible for tissue remodeling through inhibition of anti-fibrotic miRNAs and a subsequent increase in ECM synthesis.Item Tissue Transglutaminase in Glaucoma(2008-08-01) Tovar-Vidales, Tara; Clark, Abbot F.; Reeves, Rustin E.; Sheedlo, HaroldTovar-Vidales, Tara, Tissue Transglutaminase in Glaucoma. Doctor of Philosophy (Cell Biology and Genetics), August 2008; 113pp; 0 tables, 23 illustrations, 165 bibliography, 25 titles. Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is the major risk factor for POAG and is due to resistance of aqueous humor (AH) outflow through the trabecular meshwork ™ and Schlemm’s canal. Transforming growth factor-beta2 (TGF-β2) is elevated in the aqueous humor of glaucomatous eyes compared to normal eyes. Thus, TGF-β2 may play a role in regulating IOP. Tissue transglutaminase (TGM2) is a member of the transglutaminase family involved in cross-linking ECM proteins. In POAG, there are increased cross-linked extracellular matrix proteins (ECM) in the TM, and therefore, may result in elevated AH outflow resistance and elevated IOP. In this study, we examined the differences in both protein expression and enzyme activity of TGM2 between normal and glaucomatous TM cells and tissues. The findings demonstrated the presence of TGM2 in normal and glaucomatous cultured TM cells. We also showed that glaucomatous cultured TM cells and tissues have elevated levels of TGM2. Thus, this data suggest that TGM2 may have a pathogenic role in elevated outflow resistance and elevated IOP. Second, we observed the induction of TGM2 by TGF-β1, β2, and β3 in cultured TM cells, suggesting TGF-β isoforms regulate TGM2 protein levels. Finally, we observed that R-Smads and O38 regulated TGM2 protein levels, suggesting TGF-β2 acts through both its canonical and non-canonical signaling pathway to regulate TGM2.Item Transforming Growth Factor β2 Regulates the Expression of microRNAs (miRNAs) in Human Optic Nerve Head Cells(2017-03-14) Tovar-Vidales, Tara; Clark, Abbot; Lopez, NavitaPurpose: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is the leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the pro-fibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eye. However, there currently is little knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine if there are differences in expression of pro-fibrotic and anti-fibrotic miRNAs in normal ONH cells treated with or without TGFβ2. Methods: Primary human ONH cell strains derived from normal donor eyes were grown to 100% confluency. ONH cells were treated with 5ng/ml TGFβ2 or with control medium for 24hrs. RNA was isolated and cDNA synthesis performed for miRNA qPCR arrays to compare expression levels of pro-fibrotic and anti-fibrotic miRNAs in normal human ONH cells treated with or without TGFβ2. Results: Normal ONH cells exposed to TGFβ2 showed that several anti-fibrotic miRNAs were downregulated (hsa-miR-107, hsa-miR-132-3p, hsa-miR-141-3p hsa-miR-18a-5p, hsa-miR-194-5p, hsa-miR-204-5p) compared to control cells. In contrast, only one pro-fibrotic miRNA was upregulated (hsa-miR-34a-5p) in ONH cells treated with TGFβ2 compared to control. The most prominent targets of these miRNAs include connective tissue growth factor (CTGF), gremlin 2 and lysyl oxidase-like 3 (LOX-L3). Conclusions: Our results suggest that miRNAs expressed by ONH cells may be regulated by TGFβ2. These miRNAs may target CTGF, crosslinking enzymes and BMP antagonists to modify the ECM in the ONH.