Eye / Vision

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21624

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Decoding the anti-cataractogenic mechanism of grapes via a systemic pharmacology approach
(2019-03-05) Liu, Xiao; Wang, Duen; Garcia, Luis; Ssentamu, Frank; Lou, Alexander; Wu, Hong; Yu, Yu
Introduction: Our previous study has indicated that grapes may be able to protect against in vivo ultraviolet B (UV-B) radiation-induced cataract. To better understand the mechanism of action of grapes in cataract prevention, this follow-up study was designed to identify the molecular targets of grapes in the lens by using a systemic pharmacology approach. Methods: As recommended by the California Table Grape Commission (CTGC), we selected four compounds including resveratrol, catechin, quercetin, and anthocyanins as the major phytoconstituents of grapes for target prediction. All genes that can be regulated by grapes were obtained from NCBI (www.pubmed.gov) and TCMSP (http://lsp.nwu.edu.cn/tcmsp.php). Genes that are associated with cataracts were collected from GeneCards (www.GeneCards.org). The comparison between grape-related targets and cataract-associated genes was conducted using Cytoscape 3.2.1 with ClueGo plugin. Gene Ontology (GO) enrichment analysis of grape-regulated genes was conducted using Database for Annotation, Visualization, and Integrated Discovery (www.david.ncifcrf.gov). Results: A total of 332 targets that are grape regulated were identified and visualized by protein network. Subsequently, 147 GO functional pathways were clustered, including anti-apoptotic, anti-inflammation, PI3K-Akt signaling, ATP binding, and FOXO pathways. Among these protein targets, X-linked inhibitor of apoptosis (XIAP), heat shock protein (HSP) 90, and prostaglandin-endoperoxide synthase (PTGS) were correlated with all of the active ingredients of grapes. Comparison between grape targets and cataract disease genes showed that thirteen grape targets overlapped with cataract associated genes, including PTGS2, HSP90AA1, HSP90AA2P, mitogen-activated protein kinase 1 (MAPK1), MAPK14, MAPK3, amyloid precursor protein (APP), glycogen synthase kinase 3B (GSK3B), protein kinase α (PRKCA), protein kinase C delta (PRKCD), B-cell lymphoma 2 (BCL2), BCL2L1, and K-ras (KRAS). Conclusions: The anticataractogenesis effects of grapes may involve not only directly scavenging free radicals but also activating the antiapoptotic pathway.
The Endothelin Receptor Antagonist Macitentan Ameliorates Endothelin-Mediated Vasoconstriction and Promotes Neuroprotection of Retinal Ganglion Cells in Rats.
(2019-03-05) Krishnamoorthy, Vignesh; wei, Zhang; Kodati, Bindu; Chavala, Sai; Krishnamoorthy, Raghu; Stankowska, Dorota; Harris, Payton
The Endothelin Receptor Antagonist Macitentan Ameliorates Endothelin-Mediated Vasoconstriction and Promotes Neuroprotection of Retinal Ganglion Cells in Rats. Payton Harris Vignesh Krishnamoorthy Wei Zhang Bindu Kodati Sai Chavala Raghu Krishnamoorthy Dorota L. Stankowska 1. Texas College of Osteopathic Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth TX 76107 2. Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth TX 76107 Purpose: To determine if dietary administration of the dual ETA/ETB receptor antagonist, macitentan, could protect retinal ganglion cells (RGCs) following endothelin-1 mediated vasoconstriction in Brown Norway rats. Methods: Adult male and female Brown Norway rats were either untreated or treated with macitentan (5 mg/kg body weight) once a day for 3 days followed by intravitreal injection of either 4 µl of 500 mM ET-1 or vehicle in one eye. Imaging of the retinal vasculature using fluorescein angiography was carried out a various time points including 2, 5, 10 and 20 minutes. Following the imaging of the vasculature, treatment of rats was continued for 1 week with either macitentan (5 mg/kg/body weight) in dietary gels or untreated control gels. After euthanizing the rats, retinal flat mounts from the rats were prepared, immunostained for RGC marker Brn3a, imaged and surviving RGCs were counted in a masked manner. Results: Vasoconstrictive effects following intravitreal ET-1 injection were greatly reduced in rats administered with macitentan in the diet prior to the ET-1 administration. ET-1 intravitreal injection produced a 45% loss of RGCs which was significantly reduced in macitentan-treated rats and RGC counts were similar to that observed in control retinas. Conclusions: The endothelin receptor antagonist, macitentan, has neuroprotective effects in retinas of Brown Norway rats that occurs through different mechanisms, including, enhancement of RGC survival and reduction ET-1 mediated vasoconstriction preventing ischemia.
Activation of TRPV4 channels reduces IOP and improves outflow facility by regulating eNOS dependent nitric oxide release from the trabecular meshwork
(2019-03-05) Kasetti, Ramesh; Maddineni, Prabhavathi; Millar, J. Cameron; Zode, Gulab; Patel, Pinkal
Purpose: Nitric oxide (NO) is known to reduce intraocular pressure (IOP) by relaxation of the trabecular meshwork (TM) and distal vessels of the conventional outflow pathway. However, the intrinsic mechanisms by which outflow pathway tissues regulate NO production is yet to be elucidated. In vascular endothelium, activation of mechanosensory transient receptor potential vanilloid 4 (TRPV4) channels results in endothelial nitic oxide synthase (eNOS) mediated NO release, which in turn promotes vasodilation. Here, we determined whether activation of TRPV4 regulates IOP and conventional outflow via NO release in the TM. Methods: In wildtype (WT) and glucocorticoid-induced ocular hypertensive (OHT) C57BL/6J mice, the effect of TRPV4 agonist GSK1016790A on IOP and outflow facility was determined using rebound tonometry and constant-flow infusion method respectively. Effect of TRPV4 agonist on eNOS activation and NO production was determined using Western blot and fluorometric DAF-FM assay in primary human TM cells and ex vivo cultured human TM donor tissues. We report for the first time a method for electrochemical measurement of NO in human anterior segment donor tissues using NO microsensors. Results: Topical administration of TRPV4 agonist GSK1016790A significantly reduced IOP (Pin WT and OHT mice compared to contralateral control eyes. In OHT mice, treatment with GSK1016790A resulted in increased outflow facility (P=0.02)compared to contralateral vehicle treated eyes. We further demonstrate that TRPV4 activation by GSK1016790A resulted in increased eNOS phosphorylation in GTM3 cells, primary human TM cells, and cultured human TM donor tissues. Activation of TRPV4 in primary TM cells and ex vivocultured human TM donor tissues resulted in increased DAF-FM fluorescence, which signifies increase in TRPV4-mediated NO production. Treatment of human anterior segments with TRPV4 agonist resulted in increased production of NO as detected electrochemically using NO microsensors. Nonselective inhibition of NOS by L-NAME abrogated the IOP lowering effect of TRPV4 agonist in mice and reduced TRPV4-mediated NO production in outflow pathway cells and donor tissues. Conclusion: TRPV4 activation improves IOP and outflow facility, perhaps by regulation of eNOS dependent NO release.
Glucocorticoid-induced glaucomatous neurodegeneration is associated with demyelination of optic nerve axons and infiltration of immune cells
(2019-03-05) Kasetti, Ramesh; Patel, Pinkal; Zode, Gulab; Maddineni, Prabhavathi
Purpose: Ocular hypertension (OHT) is a serious side effect of glucocorticoid (GC) therapy and if untreated, it can leads to secondary open-angle glaucoma. However, the precise mechanism of GC-induced glaucomatous neurodegeneration is not understood largely due to lack of proper mouse model that exhibits glaucomatous neurodegeneration similar to human glaucoma. Using a novel mouse model of GC-induced OHT, we determined whether prolonged GC-induced OHT leads to glaucomatous neurodegeneration and further explored the pathological mechanisms of axonal degeneration and role of neuroinflammation in glaucoma. Methods: C57BL/6J mice were injected with either Dexamethasone Acetate (Dex) or Vehicle (Veh) via periocular-route, once a week for 10-weeks. IOP was measured every week and glaucomatous neurodegeneration was examined at 5 and 10-weeks of treatment using pattern ERG (pERG), whole mount retina staining with RBPMS antibody and PPD staining and transmission electron microscopy (TEM) for optic nerve (ON) degeneration. Reactive astrocytes, axonal cytoskeleton changes and immune cells at ON head (ONH) were assessed by immunostaining. Cholera toxin B (CTB) was used to trace axon anterograde transport deficits. Results: Periocular injections of Dex caused significant and prolonged IOP elevation (Δ ≥3.5-5 mmHg) and outflow facility reduction (by ~40%) compared to Veh-injected mice. Dex-induced OHT was associated with increased ECM deposition and cytoskeleton changes in the TM. Interestingly, Dex-induced sustained OHT led to glaucomatous neurodegeneration after 10 weeks of treatment including significant functional and structural loss of RGCs as evident from reduced pERG amplitudes (10µV v/s 25µV) and ~36% loss of RGCs in whole mount retina RBPMS staining. Neuronal labelling with CT-B demonstrated anterograde transport deficits in Dex-treated eyes, with increased reactive astrocytes at ONH. We also observed ~40% loss of optic nerve axons in PPD staining. TEM analysis of ON further demonstrated chronic demyelination of optic nerve axons with mitochondrial accumulation and immune cells infiltration, which was further confirmed by immunostaining. Conclusions: These data highlights that GC-induced OHT causes inflammatory demyelination of the optic nerve axons, which results in glaucomatous neurodegeneration.
Glutaredoxin 2 (Grx2) protects the retina from light-induced photoreceptor damage via regulating the endothelin receptor B (Ednrb) pathway
(2019-03-05) Liu, Xiaobin; Ssentamu, Frank; Aguilera Garcia, Luis; Yu, Yu; Li, Yousong; Wu, Hongli; Wang, DuenShian
1.Purpose : Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase which is known to reduce S-glutathionylated proteins. In a previous study, we have found that Grx2 could protect the retina from light-induced retinal degeneration. However, the molecular mechanisms that coordinate thiol-repair processes and cell survival systems in the damaged retina remain largely unknown. To better understand the protective effects of Grx2 in the retina, our study was thus extended to analyze the full transcriptome changes of the retinal tissue in light-exposed Grx2 knockout (KO) mice. 2.Methods : Wild type (WT) and Grx2 KO mice were exposed to white light at 23,000 lux for 1 hour after dark adaptation for 10 hours. The retinal damage was confirmed by the electroretinogram (ERG) recording and spectral domain optical coherence tomography (SD-OCT) measurement. Protein glutathionylation level was evaluated by Western Blot. We then compared the full transcriptome of the retinal tissue in WT and Grx2 KO mice using transcriptome shotgun sequencing (RNA-seq). The gene network was analyzed using DESeq2 pathway analysis software, and real-time PCR and Western Blot further confirmed the selected genes of interest. 3.Results : Light-exposed Grx2 KO mice showed compromised visual function as indicated by severe loss of both a- and b-wave amplitudes and the thinning of the outer nuclear layer (ONL). Protein glutathionylation level was elevated in light-exposed Grx2 KO mice. We identified thousands of genes with statistically significant expression changes in light-exposed Grx2 KO mice and classified them into cellular processes and molecular pathways. Among these pathways, many genes that are related to complement activation, inflammation, and cell survival system were significantly upregulated. These genes include Bcl-3 and Fgf2 in NF-KappaB family pathway, C4b in classical activation pathway, Jak3 and STAT3 in JAK-STAT signaling pathway, Cdkn1a in DNA damage response pathway, Edn2 and Ednrb in endothelin 2 signaling pathway. 4.Conclusions : Collectively, our results suggest that Grx2 could protect the retina from light-induced retinal degeneration. Grx2 plays an important role in regulating light-induced retinal inflammation which may be associated with its ability to repair S-glutathionylated substrates.
Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells.
(2019-03-05) Stankowska, Dorota; He, Shaoqing; Kodati, Bindu; Krishnamoorthy, Raghu; Chaphalkar, Renuka M.; Chaphalkar, Renuka M.
TITLE: Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells. Purpose: Endothelin-1 (ET-1) treatment has been shown to promote apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms underlying these effects are still unknown. The purpose of the study was to assess the changes in gene expression at the level of the translatome, occurring during ET-1 mediated neurodegeneration of RGCs. Methods: Primary RGCs isolated from post-natal day 5 rat pups were treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Polysomal RNA was isolated and libraries for RNA-Seq were prepared. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p Results: STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. Immunostaining of primary RGCs showed an appreciable decline in expression of COX17, while ATP5H expression was modestly decreased. A decreasing trend (three out of four rats) in immunostaining for ATP5H as well as COX17 was found in retinas of rats intravitreally injected with ET-1 (n=4). Conclusions: ET-1 treatment produced a decrease in expression of key components of mitochondrial electron transport chain. A compromise in bioenergetics could be one mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.
TGFβ2-TLR4 Crosstalk Signaling in the Glaucomatous Trabecular Meshwork
(2019-03-05) Curry, Stacy; Clark, Abbot F.; McDowell, Colleen; Roberts, Amanda
Purpose: 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.
PREVALENCE OF VISUAL IMPAIRMENTS AND THEIR POTENTIAL IMPACT ON THE DAILY LIVES OF PRE-K CHILDREN.
(2019-03-05) Williamson, Catherine; Patel, Visha; Dossou, Sarah; Mozdbar, Sima; Aryal, Subhash; Clark, Abbot; Bugnariu, Nicoleta; Saavedra, Alvaro
1.Purpose/Hypothesis : The purpose of this study was to report the prevalence of visual impairments (myopia, hyperopia, astigmatism, amblyopia) in pre-school children and discuss the implications of those impairments on motor function in the academic environment. Number of Subjects : We collected data from1424 children, 698 female and 726 male, enrolled in Pre-K programs in 20 elementary schools within the Ft. Worth ISD. 2.Materials/Methods : Children were screened for myopia, hyperopia, astigmatism, and gaze asymmetry using the plusoptiX ™ hand-held vision-screening refractometer1 . Descriptive statistics provided prevalence of refractive errors and amblyopia. A Chi-squared test was used to compare proportions between male and female, and Hispanic and Non-Hispanic subjects, and those who needed a referral or not. 3.Results : Our sample had a similar proportion of male (51%) and female (49%) students (p = 0.4581); and the following breakdown by race: Asian 2.25%, Black 22.89%, Caucasian 12.57%, Hispanic 60.74%, and Mixed 1.54%. A total of 932 children (65.5%) passed the vision screening while 493 children (34.5%) were referred for further evaluation. For 184 subjects OD and OS sphere measurements were not recorded. We identified 493 children with isometropic refractive amblyopia risk factors: 9 children with myopia, 30 children with hyperopia, and 454 children with astigmatism. Additionally, 27 children were identified as being at-risk for anisometropic refractive amblyopia. We further evaluated our data for the four risk factors between Hispanic and Non-Hispanic groups. There was no significant difference in myopia (OR = 1.25, 95% CI 0.3361-4.7077) and anisometropia (OR= 1.08, 95%CI 0.49-2.38) between Hispanic and Non-Hispanic groups. There was significant difference in hyperopia (OR = 3.25, 95% CI 1.23- 8.57) and astigmatism (OR = 1.58 95% CI 1.25-2.00) between Hispanic and Non-Hispanic groups. 4.Conclusions : Preliminary results from this sample indicate that myopia and anisometropia have similar prevalence across race groups, but hyperopia and astigmatism are more prevalent in the Hispanic vs. Non-Hispanic group. Clinical Relevance : Visual impairments have been associated with delayed development and decreased performance in areas including gross motor skill, fine motor, and academic performance.2,3,4 Other studies have shown that there is an association between visual impairments and delayed gross motor skills leading to decreased physical activity and participation.2 Further studies have shown that with proper visual correction, academic performance can improve to age-matched peers.5 Gross motor skill delay is often due to lack of opportunity to practice skills rather than just the impairment itself.6 Therefore screening and correcting visual impairments is pivotal to facilitate typical motor function and development, increase physical activity and participation, and improve academic performance in children
Neuroprotective properties of sigma-1 Receptor in Retinal Ganglion cells from Optic Crush Model
(2019-03-05) Li, Linya; Ellis, Dorette; He, Shaqing; Yorio, Thomas; Lee, Deidra
Glaucoma is a group of neurodegenerative disorders that lead to the death of the retinal ganglion cells (RGC), optic nerve damage, and may ultimately cause blindness. Elevated intraocular pressure (IOP) is one of the major contributors to glaucoma1. As a result, the majority of treatments available target lowering IOP, as a therapy for glaucoma2. Purpose: Studying the neuroprotection of RGCs is important to provide alternative therapies for pro-survival which mitigates RGC death and irreversible optic nerve damage. This study will investigate the sigma-1 receptor as an alternate additional therapeutic target to provide protection of Retinal ganglion cells (RGCs) and possible treatment for glaucoma. Hypothesis: It is hypothesized that the sigma-1 receptor (σ-1r) offers neuroprotection to retinal ganglion cells, therefore, it will restore RGCs and mitigate cell death. Methods: Treatment groups: wild type mice and σ-1r KO mice. Transfected mice with AAV2-σ-1r vector in order to induce overexpression of sigma-1 receptor (σ-1r), activated σ-1r through agonist- pentazocine, afterward induced injury via Optic Nerve Crush (ONC) protocol, then assessed RGC function and survival. Expression of σ-1r assessed using immunohistostaining & western blot analysis. Pattern Electrocardiogram (PERG) was used to assess visual function of mice retina pre and post- ONC. Adobe Photoshop software was used post retina staining and flat mount to assess the density of RGCs. Results & Conclusion: When σ-1r was activated and overexpressed, retinal ganglion cells death was mitigated in σ-1r knock out mice. This data suggests that σ-1r has neuroprotective functions to the retinal ganglion cells. Studying the mitigation of retinal ganglion cell death may be beneficial as an alternate target in the treatment of Glaucoma and other optic neuropathies.
Restoration of vision by chemically reprogrammed photoreceptors
(2019-03-05) Kaya, Koray; Fan, Yan; Sumien, Nathalie; Shetty, Ritu; wei, Zhang; Davis, Delaney; Thomas Mock, Thomas; Batabyal, Subrata; Ni, Aiguo; Mohanty, Samarendra; Han, Zongchao; Farjo, Rafal; Forster, Michael; Swaroop, Anand; Chavala, Sai; Mahato, Biraj
Purpose: Many retinopathies such as Retinitis Pigmentosa, Stargardt disease, Cone-rod dystrophy, Achromatopsia, Chroideremia and Labor congenital Amaurosis (LCA) comprise a wide range of genetically and phenotypically heterogeneous conditions that share common progressive loss of photoreceptor function accompanied by irreversible vision loss. Majority of the patients affected by these diseases present with uncorrectable decreased visual acuity during their childhood years, which most often progress to legal blindness. Strategy to restore vision with photoreceptor like replacement cell has the advantage of being applied to these patients, regardless of their genetic dysfunction or stage of disease. Currently no FDA approved treatments are available to treat these disorders. We have discovered a chemical engineering method that can convert fibroblasts to chemically induced photoreceptors (CiPCs) with their ability to restore vision in retinal degeneration mouse model. Methods: A combination of small molecule (5C) was used to convert fibroblasts to CiPCs. Gene expression of CiPCs was analyzed by RNA sequencing, RT-PCR and immunofluorescence. Light responsiveness of CiPCs was tested by single cell patch clamp recording upon stimulation with light. In vivo CiPC function was examined by pupil analysis, light aversion test, visual acuity and contrast sensitivity measurement after injecting them into retinal degeneration mouse model. Results: We have identified a set of five small molecules (5C) that induces mouse embryonic fibroblasts (MEFs) and human adult dermal fibroblasts (HADF) into CiPCs both rods and cones, without the use of pluripotent cells or viral transcription factors in less than two weeks time. Detailed analyses have been performed in mouse cells, but in brief, these cells express transcript and proteins consistent with youthful photoreceptors (postnatal day 5). In vitro functional analysis indicated that CiPCs are light responsive. Moreover, when mouse ciPCs are injected into the subretinal space of retinal degeneration mutant mice (rd1), in some mice (approximately 50%) we observed cell survival for several months (more than 120 days), restored light-dark preference/discrimination, improved scotopic-Optomotry testing, fleeting but partial ERG recovery, and restoration of pupillary reflexes. Conclusions: Based upon these observations we demonstrate restoration of visual functions by CiPCs that carry extraordinary translational potential for millions of visually impaired patients worldwide.
Role of miR-29c-3p in regulation of extracellular matrix synthesis
(2019-03-05) Clark, Abbot F.; Tovar-Vidales, Tara; Lopez, Navita
Purpose 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.
Ocular Findings and Management in a Steven-Johnson Syndrome Patient
(2019-03-05) Jenkins, Mark MD; Kahlon, Hania
Ocular Findings and Management in a Steven-Johnson Syndrome Patient Hania Kahlon1, Dr. Mark Jenkins, MD2 1University of North Texas Health Science Center, Fort Worth, TX, 2Houston Eye Associates at the Woodlands, TX, Background: Steven Johnson Syndrome (SJS) is a rare, severe mucocutaneous reaction that commonly occurs in response to medications. The response involves necrosis of the epidermis with severe effects on the patient’s mucous membranes. SJS occurs in two to seven people per million each year, with overall mortality at 30%. In our case, we focus on the ocular findings of SJS. Reported ocular findings of SJS commonly include photophobia, severe conjunctivitis, and corneal ulceration. We present a case of SJS to discuss ocular findings in SJS and emphasize the importance of proper recognition of the clinical indications and management of SJS. Case Information: We present the case of a 52 year old patient who was diagnosed with Steven Johnson Syndrome in January of 1984, 24 hours after taking a sulfa drug. Though the patient was initially misdiagnosed, the patient was eventually admitted with SJS for a 6-week inpatient stay. The patient was placed on a course of outpatient steroids after his hospital stay. It was not until April of 1985 that the patient began seeing an ophthalmologist for his deteriorating vision. The ophthalmologist found that the patient’s ocular findings included trichiasis without entropion of both left and right eyelids, corneal epithelial defect in both eyes, pinguecula of the conjunctiva of both eyes, and hyperemia with lid eversion in both eyes. The patient presents to the office every week for epilation of his lashes, as he has for over 20 years. We followed the patient for a period of 6 months to monitor for any changes in his ocular findings. The management of the patient’s ocular findings includes weekly epilation of his lashes, along with medications. Conclusion: This case highlights the ocular findings that can be found in the rare SJS patient. Furthermore, our case indicates the need for further research on the ocular management of SJS. Since our patient’s SJS was initially misdiagnosed, our case also indicates the importance of clinical knowledge of the early symptoms of SJS. We hope that, by sharing this information, we can add to the limited research and documentation that we currently have of this rare condition.
TGFβ2 induces chronic endoplasmic reticulum stress in trabecular meshwork cells.
(2019-03-05) Kasetti, Ramesh; Zode, Gulab S.; Patil, Shruti
Purpose: TGFβ2-induced extracellular matrix (ECM) accumulation in trabecular meshwork (TM) is associated with aqueous humor outflow resistance and IOP elevation. Recently, we have demonstrated that abnormal ECM accumulation leads to endoplasmic reticulum (ER) stress in TM. Here, we examined whether TGFβ2 induces ER stress in human TM cells. Methods: GTM3 or primary human TM cells (n=2) were treated with vehicle or recombinant TGFβ2 (5 ng/ml) in 0.5% FBS containing DMEM medium for 3 days & 7 days respectively. ER stress markers (Grp78, Grp94, ATF4 and CHOP) and ECM proteins (Fibronectin & Collagen IV) were examined by Western blot and immunostaining. GTM3 cells were transfected with plasmids expressing CRISPR-Cas9 targeting ATF4 or CHOP and subsequently treated with TGFβ2 for 48 hours. Cellular lysates were examined for ER stress and ECM proteins. Results: Western blot analysis demonstrated that TGFβ2 treatment led to ER stress as evident from increased levels of Grp78, Grp94, ATF4 and CHOP proteins compared to the vehicle treatment. TGFβ2-induced ER stress markers were also associated with ECM protein (fibronectin and Collagen IV) levels. Moreover, TGFβ2 treatment increased fibronectin staining and its colocalization with ER stress markers, suggesting TGFβ2- induced ECM proteins are associated with ER stress. Knockdown of key chronic ER stress transcriptional factors, ATF4 or CHOP prevented TGFβ2-induced ECM deposition and also reduced ER stress in GTM3 cells. Conclusions: Our preliminary findings clearly indicate that TGFβ2 can directly induce chronic ER stress in cultured human TM cells.
Ex-vivo human anterior segment culture: a suitable model for glaucoma research
(2019-03-05) Patel, Pinkal; Zode, Gulab; Kasetti, Ramesh
Purpose: The available human models to study the trabecular meshwork (TM) pathology in glaucoma are very limited. The well-established ex-vivo human anterior segment perfusion system requires intact eyes and cost intensive. Here we tested the feasibility of ex-vivo human anterior segment culture to use as a model for glaucoma research. Methods: Human rejected corneas with intact TM ring were obtained from the Lions Eye Institute (Tampla, Florida) in accordance with Declaration of Helsinki guidelines for research involving human tissue. The anterior segments were dissected into 4 equal quadrants and each quadrant was cultured in a 24 well plate using DMEM media added with 10% FBS and 1% Pen-strep. The anterior segment quadrants either treated with 100nM dexamethasone or 5ng per ml recombinant Tgfβ2 (in 0.5% FBS containing media) with appropriate vehicles or transduced with Adenoviral mediated wild type or mutant myocilin for up to 7 days. The spent media was collected for western blot analysis and cultured tissues were fixed and paraffin sections were utilized for immnostaining. Results: The TM morphology is well preserved in human rejected corneas before and after 7 days of culture, observed in H&E staining. The dexamethasone, Tgfβ2 and mutant myocilin induced glaucomatous changes like increased extracellular matrix proteins, increased ER stress were observed in the TM of dexamethasone, Tgfβ2 and mutant myocilin treated tissue sections compared to their respective controls. The western blot analysis of spent media clearly showed an increase of myocilin and fibronectin levels in dexamethasone treated samples whereas an increase in fibronectin observed in Tgfβ2 treated samples. As expected mutant myocilin secretion is hampered in Ad5-mutant-myocilin transduced quadrant tissue samples compared to that of Ad5-wildtype-myocilin. The tissue disintegration rate in an ex-vivo culture was monitored by counting tunnel positive cells at the TM region at different time points. Conclusion: The ex-vivo human anterior segment culture is an effective model to study the TM pathology in glaucoma research. Moreover this model is cost-effective and the rejected corneas available with ease.

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