Browsing by Subject "glaucoma"
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Item A Novel Prodrug Approach for Central Nervous System-Selective Estrogen Therapy(MDPI, 2019-11-19) Prokai-Tatrai, Katalin; Prokai, LaszloBeneficial effects of estrogens in the central nervous system (CNS) results from the synergistic combination of their well-orchestrated genomic and non-genomic actions, making them potential broad-spectrum neurotherapeutic agents. However, owing to unwanted peripheral hormonal burdens by any currently known non-invasive drug administrations, the development of estrogens as safe pharmacotherapeutic modalities cannot be realized until they are confined specifically and selectively to the site of action. We have developed small-molecule bioprecursor prodrugs carrying the para-quinol scaffold on the steroidal A-ring that are preferentially metabolized in the CNS to the corresponding estrogens. Here, we give an overview of our discovery of these prodrugs. Selected examples are shown to illustrate that, independently of the route of administrations and duration of treatments, these agents produce high concentration of estrogens only in the CNS without peripheral hormonal liability. 10beta,17beta-Dihydroxyestra-1,4-dien-3-one (DHED) has been the best-studied representative of this novel type of prodrugs for brain and retina health. Specific applications in preclinical animal models of centrally-regulated and estrogen-responsive human diseases, including neurodegeneration, menopausal symptoms, cognitive decline and depression, are discussed to demonstrate the translational potential of our prodrug approach for CNS-selective and gender-independent estrogen therapy with inherent therapeutic safety.Item Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Crosstalk Between Dysfunctional Mitochondria and Inflammation in Glaucomatous Neurodegeneration(Frontiers Media S.A., 2021-07-21) Jassim, Assraa Hassan; Inman, Denise M.; Mitchell, Claire H.Mitochondrial dysfunction and excessive inflammatory responses are both sufficient to induce pathology in age-dependent neurodegenerations. However, emerging evidence indicates crosstalk between damaged mitochondrial and inflammatory signaling can exacerbate issues in chronic neurodegenerations. This review discusses evidence for the interaction between mitochondrial damage and inflammation, with a focus on glaucomatous neurodegeneration, and proposes that positive feedback resulting from this crosstalk drives pathology. Mitochondrial dysfunction exacerbates inflammatory signaling in multiple ways. Damaged mitochondrial DNA is a damage-associated molecular pattern, which activates the NLRP3 inflammasome; priming and activation of the NLRP3 inflammasome, and the resulting liberation of IL-1beta and IL-18 via the gasdermin D pore, is a major pathway to enhance inflammatory responses. The rise in reactive oxygen species induced by mitochondrial damage also activates inflammatory pathways, while blockage of Complex enzymes is sufficient to increase inflammatory signaling. Impaired mitophagy contributes to inflammation as the inability to turnover mitochondria in a timely manner increases levels of ROS and damaged mtDNA, with the latter likely to stimulate the cGAS-STING pathway to increase interferon signaling. Mitochondrial associated ER membrane contacts and the mitochondria-associated adaptor molecule MAVS can activate NLRP3 inflammasome signaling. In addition to dysfunctional mitochondria increasing inflammation, the corollary also occurs, with inflammation reducing mitochondrial function and ATP production; the resulting downward spiral accelerates degeneration. Evidence from several preclinical models including the DBA/2J mouse, microbead injection and transient elevation of IOP, in addition to patient data, implicates both mitochondrial damage and inflammation in glaucomatous neurodegeneration. The pressure-dependent hypoxia and the resulting metabolic vulnerability is associated with mitochondrial damage and IL-1beta release. Links between mitochondrial dysfunction and inflammation can occur in retinal ganglion cells, microglia cells and astrocytes. In summary, crosstalk between damaged mitochondria and increased inflammatory signaling enhances pathology in glaucomatous neurodegeneration, with implications for other complex age-dependent neurodegenerations like Alzheimer's and Parkinson's disease.Item Destabilizing COXIV in Muller Glia Increases Retinal Glycolysis and Alters Scotopic Electroretinogram(MDPI, 2022-12-12) Nsiah, Nana Yaa; Inman, Denise M.Muller glia (MG), the principal glial cell of the retina, have a metabolism that defies categorization into glycolytic versus oxidative. We showed that MG mount a strong hypoxia response to ocular hypertension, raising the question of their relative reliance on mitochondria for function. To explore the role of oxidative phosphorylation (OXPHOS) in MG energy production in vivo, we generated and characterized adult mice in which MG have impaired cytochrome c oxidase (COXIV) activity through knockout of the COXIV constituent COX10. Histochemistry and protein analysis showed that COXIV protein levels were significantly lower in knockout mouse retina compared to control. Loss of COXIV activity in MG did not induce structural abnormalities, though oxidative stress was increased. Electroretinography assessment showed that knocking out COX10 significantly impaired scotopic a- and b-wave responses. Inhibiting mitochondrial respiration in MG also altered the retinal glycolytic profile. However, blocking OXPHOS in MG did not significantly exacerbate retinal ganglion cell (RGC) loss or photopic negative response after ocular hypertension (OHT). These results suggest that MG were able to compensate for reduced COXIV stability by maintaining fundamental processes, but changes in retinal physiology and metabolism-associated proteins indicate subtle changes in MG function.Item Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation(MDPI, 2022-02-22) Kodati, Bindu; Merchant, Shawn A.; Millar, J. Cameron; Liu, YangMutations in PITX2 cause Axenfeld-Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.Item EFFECT OF TRANSFORMING GROWTH FACTOR BETA-2 SIGNALING AND GREMLIN INDUCTION ON FIBRONECTIN, OCULAR HYPERTENSION, AND OPTIC NERVE DAMAGE(2013-04-12) McDowell, ColleenPurpose: Transforming growth factor β2 (TGFβ2) induces extracellular matrix (ECM) remodeling and alters the cytoskeleton, which likely contribute to the inefficient function of the trabecular meshwork (TM) tissue leading to glaucomatous phenotypes. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFβ2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases intraocular pressure (IOP) in an ex vivo perfusion culture model. The purpose of this study was to determine whether TGFβ2 and gremlin regulate ECM proteins in the TM, induce ocular hypertension, and cause optic nerve damage in mice. Methods: Ad5.hTGFβ226/228 or Ad5.Gremlin (2 uL, 2 X 10^7 pfu) was injected intravitreally into one eye of A/J mice (n=7-13 mice per group), with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was assessed using the optic nerve damage score of PPD stained optic nerve cross sections. TGFβ2, fibronectin, and gremlin protein expression in the TM was determined by immunofluorescence and immunohistochemistry. Transduction of the TM with viral vector Ad5.hTGFβ2226/228 caused a prolonged, reproducible, and statistically significant IOP elevation. IOPs increased to approximately 25 mm Hg for 8 weeks (p<0.001). IOPs were stable (12-15 mm Hg) in the uninjected control eyes. The TGFP2 induced ocular hypertension also caused significant optic nerve damage with optic nerve damage scores (ONDS) > 3 (p<0.001) in the injected eye. Intraocular administration of viral vector Ad5.Gremlin also caused significant IOP elevation in A/J mice for 3 weeks (n=9, injected eye 23.2 +/- 5.6 mmmHg, uninjected eye 15.5 +/- 2.4; p<0.01). In addition, immunofluorescence and immunohistochemistry demonstrated that intraocular injection of Ad5.hTGFβ226/228 and Ad5.Gremlin increased TGFβ2 and fibronectin expression in the TM. Conclusions: These results demonstrate that intravitreal injections of Ad5.hTGFβ226/228 and Ad5.Gremlin in A/J mice elevate IOP and upregulate the ECM protein fibronectin. In addition, Ad5.hTGFβ226/228 expression induced significant optic nerve damage. These data demonstrate for the first time gremlin's role in inducing ocular hypertension in an in vivo model system and emphasize the importance of the TGFβ2 signaling pathway in ocular hypertension.Item Effects of Intravitreal Endothelin-1 on Anterograde Axonal Transport in Rat Optic Nerve: Evaluating a Possible Mechanism for Glaucomatous Optic Neuropathy(2002-05-01) Stokely, Martha Elise Lambert; Thomas Yorio; Scott T. Brady; Glenn DillonStokely, Martha Elise Lambert, Effects of Intravitreal Endothelin-1 on Anterograde Axonal Transport in Rat Optic Nerve: Evaluating a possible mechanism for glaucomatous optic neuropathy. Doctor of Philosophy (Biomedical Sciences and Neuroscience), May 2002; 114 pages; 1 table; 12 figures; bibliography, 274 titles. Glaucoma presents a distinctive dysfunction in anterograde axonal transport that disproportionately affects the delivery of specific types of cargo(s) into the optic nerve. Previous models for pathogenesis of glaucoma have failed to provide an adequate mechanism to explain the characteristic cargo-selectivity. A new theoretical model, the “endothelin receptor-mediated model of neuropathogenesis,” was developed to explain the cargo-selective axonal transport dysfunction seen in glaucomtous optic neuropathy. In addition, a new experimental animal model, the “intravitreal endothlin/axonal transport” model was developed to test hypotheses generated by the new theoretical model. Intravitreal endothelin-1 significantly affected all of the known rate components and subcomponents of anterograde axonal transport in the rat optic nerve. Changes were seen in anterograde axonal transport in the rat optic nerve. Changes were seen in anterograde fast axonal transport for both the fastest moving small tubulovesicles, and slightly slower membrane bound organelles (MBOs), as well as in the slow transport of cytoplasmic matrix and cytoskeletal materials. Endothelin-1’s predominant effect was a severe depression in the mitochondrial subcomponent of fast anterograde axonal transport, which was most pronounced at 28 hours post-treatment. At that time, the effects of endothelin-1 were mimicked by endothelin-3, characteristic of the non-ischemic endothelin-B type of receptor. In addition, analysis of a cohort of 11 distinctive protein bands moving with the mitochondrial subcomponent demonstrated a cargo-selective effect of endothelin-1 and the delayed movement into the optic nerve for a chemically distinct subset of proteins, but not the majority of protein, in transport during this timeframe. These results appear to be consistent with what is known about the pathology of glaucomatous optic neuropathy and the neurochemistry of anterograde axonal transport and suggest that intravitreal may be an excellent model to study the mechanisms of neurodegeneration that occurs in glaucoma.Item EFFECTS OF TGF-BETA2 ON THE ELASTIC MODULUS OF TRABECULAR MESHWORK TISSUE(2013-04-12) Baradia, HusseinPurpose: The primary risk factor for developing glaucoma is elevated intraocular pressure (IOP.) The key outflow passage for aqueous humor (AH) in the human eye involves the trabecular meshwork (TM) and the Schlemm's canal (SC) with the main regulator of IOP being at the junction of the two. Elevated IOP has been attributed to increased resistance to flow at the TM/SC junction. Aqueous humor levels of transforming growth factor beta-2 (TGF-b2) are elevated in glaucoma patients, suggesting its contribution to the progression of the disease. Studies using cultured TM cells as well as ex vivo tissue have shown that TGF-b2 induces extracellular matrix (ECM) remodeling. A separate study used atomic force microscopy (AFM) to measure the elastic modulus (e.g. stiffness) of TM tissue obtained from glaucoma patients compared to age-matched controls. A marked increase in stiffness in the glaucoma tissue was observed compared to non-glaucomatous controls. Taken together, these findings imply that an elevated level of TGF-b2 may lead to ECM remodeling and increased stiffness thus reducing AH outflow and elevating IOP. The hypothesis of this study was that increased expression of TGF-b2 will cause increased stiffness (e.g. increased elastic modulus) of the TM. The goal of our study was to compare the stiffness of the TM cultured overnight with or without TGF-b2. Methods: Bovine eyes were obtained from an abattoir and the TM dissected from the anterior segment. The dissected TM was then cut into halves. One half was cultured in control media and the other half cultured in media containing TGF-b2 (5 ng/ml). Both TM halves were incubated at 37oC for a period of 72 hours. The TM was then loaded onto a force transducer and contraction induced with carbachol to measure stiffness. Results: The raw data obtained indicated that the TM exposed to TGF-b2 contracted with greater force than control indicating that TGF-b2 reduced the stiffness of the TM. However, using a Students T test at a p of 0.05 the results showed no statistical significance. Conclusions: The results obtained are from one experiment with an N =8 and since no statistical significance was observed, the data has to be considered inconclusive. The force transducer used was actually that designed for skeletal muscles which may not be the best system to test trabecular meshwork which has some smooth muscle components. The future goal of the study is to actually use force microscopy, the current standard for such experiments.Item Endothelin receptor-mediated neurodegeneration in glaucoma(2017-08-01) McGrady, Nolan; Krishnamoorthy, Raghu R.; Yorio, Thomas; Clark, Abbot F.Primary open-angle glaucoma (POAG) is a complex set of optic neuropathies which are characterized by the degeneration of the optic nerve, cupping of the optic disk and loss of retinal ganglion cells (RGCs). There are approximately 3 million Americans who currently suffer from this disease although this is most likely an underestimation since many individuals with glaucoma are unaware that they have the disease. POAG is an age-related disease progressing slowly over the course of several decades and is most commonly associated with an elevation in intraocular pressure (IOP). Currently available treatments for glaucoma, both surgical and pharmacological, are solely focused on the regulation of IOP; nevertheless, some individuals continue to show progressive damage despite being on available therapies. In recent years, there has been increased momentum towards the development of neuroprotective strategies for POAG, particularly in preclinical models of glaucoma. Despite these efforts, there is still no neuroprotective treatment currently available for glaucoma patients. A potential target for the development of a neuroprotective approach is the endothelin system of peptides and their receptors. The endothelin (ET) system is composed of three vasoactive peptides (ET-1, ET-2 and ET-3) which are comprised of 21-amino acids. The peptides bind to two G-protein coupled receptors (ETA and ETB receptors) leading to activation of numerous signal transduction pathways. Although originally described for its role in the vasculature, all components of the ET system has been shown to be expressed in multiple tissues and cell types and are responsible for diverse cellular effects. Clinical studies have demonstrated an increase in ET-1 concentrations both in the aqueous humor and plasma of glaucoma patients. A previous study by our lab, using a rodent model of ocular hypertension, showed that endothelin B (ETB) receptor expression is increased when compared to control eyes and contributes to neurodegeneration (Minton et al., 2012). Preliminary data in the current study, using Brown Norway rats, demonstrated that ETA expression is also increased in the IOP elevated eyes, suggesting the possibility that the ETA receptor might also have a degenerative role during ocular hypertension. We hypothesize that the ETA expression increases following IOP elevation and contributes to the neurodegeneration of retinal ganglion cells and their axons. To test this hypothesis we employed a well-characterized in vivo model of glaucoma as well as multiple cellular and molecular approaches to understand the role of the ETA receptor in glaucomatous degeneration. Our data suggest that overexpression of the ETA receptor promotes cell death in cultured RGCs. Since both ETA and ETB receptors appear to contribute to neurodegeneration, we tested the ability of an FDA approved medication, macitentan, for neuroprotection in the Morrison model of glaucoma in rats and found it to promote RGC survival. Our studies raise the possibility of testing macitentan as a neuroprotective treatment for glaucoma patients.Item Endothelin-1 mediated decline in mitochondrial function contributes to neurodegeneration in glaucoma(2020-08) Chaphalkar, Renuka M.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.; Clark, Abbot F.; Zode, Gulab S.Glaucoma is an optic neuropathy with multifactorial etiologies, commonly associated with elevated intraocular pressure (IOP) and characterized by degeneration of the optic nerve, loss of retinal ganglion cells (RGC), cupping of optic disc and visual field deficits, which could ultimately lead to vision loss. In most cases, glaucoma is a chronic, asymptomatic and gradually progressing neurodegenerative disease, sometimes referred to as the "silent thief of sight," hence, routine eye examinations by an ophthalmologist are critical to determine if there is a likelihood of developing the disease. Elevated IOP is a primary and the only modifiable risk factor in glaucoma. Currently, reducing IOP remains the only proven treatment to delay the progression of RGC death; however, some patients continue to have neurodegenerative effects despite lowering IOP. Therefore, development of novel neuroprotection strategies as an adjunct therapy to IOP-lowering agents will provide a valuable therapeutic strategy in glaucoma. One of the promising targets for neuroprotection is the endothelin system of peptides and their receptors. The endothelin (ET) system comprises of three vasoactive peptides (ET-1, ET-2 and ET-3), which act through two types of G-protein coupled receptors, namely, ETA and ETB receptors. Originally discovered in the cardiovascular system, the diverse expression pattern of endothelin peptides and their receptors implicate their involvement in a variety of physiological processes in the body. A growing body of evidence suggests that endothelins and their receptors are associated with neurodegeneration in glaucoma. Previous studies have demonstrated that ET-1 levels are elevated in aqueous humor (AH) and plasma of glaucoma patients. Our lab previously demonstrated that in an ocular hypertension model in rats, there was an increase in ETB as well as ETA receptor expression primarily in RGCs compared to contralateral eyes. Following IOP elevation, RGC loss was significantly attenuated in the ETB receptor-deficient rats, pointing to a causative role of the ETB receptor in glaucomatous neurodegeneration. However, the precise cellular and molecular mechanisms by which ET-1 promotes neurodegeneration through its actions on the endothelin receptors are not completely understood. Previous studies have shown that ETB receptor stimulation increases the oxidative stress and production of superoxide anions, in sympathetic neurons. Several studies point to the role of mitochondrial dysfunction and oxidative stress as contributors to glaucomatous damage in animal models of glaucoma. To investigate various molecular events contributing to the ET-1 mediated RGC loss in glaucoma, we carried out RNA-seq analysis of the translatome in rat primary RGCs following ET-1 treatment. We identified several key mitochondrial and neurodegenerative gene candidates including Atp5h, Cox17, Foxo1, Moap1 and Map3k11 that were differentially expressed in the translatome by ET-1 treatment in RGCs. Based on our RNA-seq findings, we hypothesized that ET-1 causes an increase in reactive oxygen species (ROS) by acting through the ETB receptor that produces a subsequent decline in mitochondrial function and bioenergetics ultimately predisposing RGCs to cell death. To test this hypothesis, we used an in vitro approach by utilizing rat primary culture of RGCs treated with ET-1 as well as an in vivo approach by intravitreal ET-1 injections in rodents and the Morrison's model of glaucoma in rats. Our data showed that there is a significant decrease in the expression of cytochrome c oxidase 17 copper chaperone (COX17) and ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D (ATP5H), both of which are critical components of the electron transport chain and oxidative phosphorylation pathway. Using a Seahorse mitostress assay, we also found a significant decline of several mitochondrial parameters following ET-1 treatment in primary RGCs, which indicated the possibility of a disruption in the mitochondrial quality control machinery. Hence, we also explored the effect of the ET-1 treatment on the mitophagy pathway, specifically in RGCs. Our findings suggest that there is a decrease in mitophagosome formation in RGCs in the Morrison ocular hypertensive model as well as in GFP-LC3 mice injected with ET-1, indicating an impairment in the mitochondrial quality control mechanism. Our studies reveal several novel candidates that could be targeted for the development of neuroprotective approaches to treat glaucoma.Item Endothelin-1-Induced Proliferation of Human Optic Nerve Head Astrocytes Under Hypoxia(2003-11-01) Desai, Devashish; Thomas Yorio; Ganesh Prasanna; Clark, Abbot F.Desai, Devashish, Endothelin-1-Induced Proliferation of Cultured Human Optic Nerve Head Astrocytes under Hypoxia. Master of Science (Biomedical Sciences). Purpose: Optic nerve head astrocytes (ONAs) normally support and protect the axons of retinal ganglion cells exiting the eye. Along with effects related to elevated intraocular pressure (IOP), proliferation and activation of ONAs, known as ‘astrogliosis’, is also thought to contribute to the pathophysiology of glaucoma by distributing axonal transport and preventing axon regeneration. Concentrations of endothelin-1 (ET-1) are elevated in glaucomatous eyes and in animal models for glaucoma. ET-1 injection into the eye causes reduction of ocular blood flow. ET-1 causes a time-dependent proliferation of human ONAs. Tumor necrosis factor-α (TNF-α), a cytokine, which is also elevated in glaucomatous optic nerve head, promotes ET-1 release from ocular cells and could potentially stimulate ET-1 secretion from the ONAs. Hypoxia resulting from ischemia, which is produced by the elevation of IOP or vasospasm in the retinal vasculature, is considered a significant factor contributing to the stress as the glaucomatous optic nerve head. Methods: Concentrations of ET-1 secreted by hONAs into cell culture media after hypoxia and TNF-α treatment was measured using an enzyme-linked immunosorbent assay (ELISA). Proliferation of hONAs was measured using a proliferation assay (formazan assay), performed at the end of various time periods of incubation with TNPα and ET-1 under normoxia or hypoxia. The involvement of mitogen activated protein kinase (MAPK) in hONA proliferation was examined using MAPK inhibitors and Western blot analyses. Results: Cell culture media collected from hONAs after 24-hour hypoxia with concurrent TNF-α treatment showed a 500% increase in the irET-1. Under normoxia, both TNF-α and ET-1 caused moderate proliferation of hONAs. Under hypoxia, TNF-α-induced proliferation was greatly increased. Conclusion: Hypoxia augments TNF-a and ET-1 growth of optic nerve head astrocytes, by way of increasing ET-1 synthesis and release as well as mitogenesis. Therefore reactive ONAs could be the common denominator underlying optic nerve damage in glaucoma since their localization makes them susceptible to mechanistic and ischemic influences in addition to influences of ET-1 and TNF-α. Keywords: astrocyte; endothelin-1; tumor-necrosis factor-α; hypoxia; proliferation; astrogliosis; glaucoma; optic nerveItem Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Exploring Trabecular Meshwork Molecular Pathogenic Mechanisms In Primary Open Angle Glaucoma And Glucocorticoid Induced Glaucoma(2016-08-01) Bermudez, Jaclyn Y.; Clark, Abbot F.; Mao, Weiming; Singh, MeharvanIn a normal functioning eye, the aqueous humor, a fluid secreted by the ciliary body, drains through the trabecular meshwork (TM), a multilayered tissue in the anterior segment of the eye. The TM is the initial site of damage in glaucoma. Damaged TM results in higher aqueous humor outflow resistance and causes elevated IOP, the latter of which leads to optic nerve damage. Numerous clinical studies have shown that lowering IOP can prevent neuronal damage and slow/stop the progression of the disease. In the glaucomatous TM (GTM), there is excessive extracellular matrix protein deposition, cytoskeletal changes and altered cell function. The transforming growth factor β (TGFβ) pathway is activated by TGFβ2 which has been found to be more abundant in the GTM. Additionally, formation of cross-linked actin networks (CLANs) in the GTM is increased compared to non-glaucoma TM. Primary open angle glaucoma (POAG), glucocorticoid-induced glaucoma (GIG) and glucocorticoid-induced ocular hypertension (GCOHT), share similar pathophysiologies. GC-OHT differs from POAG in that about 40% of the population develops GC-OHT after topical treatment with glucocorticoids however, the mechanism that differentiates steroid responders from non-responders is unknown. In our studies we have explored trabecular meshwork molecular pathogenic mechanisms that are responsible for the disease pathology. We have studied epigenetics as a regulatory mechanism for increasing TGFβ2 expression. We have also used proteomics to determine proteins that are associated with CLANs. Lastly, we studied genes that are differentially expressed in glucocorticoid responders versus non-responders in our bovine model of GC-OHT. Overall, our research has enhanced our understanding of the TM and the molecular mechanisms that play a role in glaucoma. We hope to use this information to find new disease modifying therapies.Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Glucororticoid Receptor Alternative Splicing: Key Players and Role in TM and Glaucoma(2012-12-01) Jain, Ankur; Clark, Abbot F.Elevated intraocular pressure (IOP) is the primary risk factor in glaucoma, a leading cause of irreversible blindness. Various morphological and biochemical changes in the trabecular meshwork (TM) appear to be responsible for blocking aqueous humor outflow, thereby elevating IOP. Glucocorticoids (GCs) are known to induce ocular hypertension and other biochemical changes associated with glaucoma. Interestingly, there are differences in steroid responsiveness among the population, with 40% people known as responders who significantly elevate IOP upon GC treatment and others being classified as nonresponders. The steroid-responders are at higher risk of developing primary open angle glaucoma (POAG) as compared to the steroid nonresponders. At the same time, almost all POAG patients are moderate to high steroid responders. GC responsiveness is regulated by the relative ratios of the GC activated transcription factor GC receptor alpha (GRα) and the alternatively spliced dominant negative regulator isoform of this receptor (GRβ). Glaucomatous TM cell strains have a higher GRα/GRβ ratio compared to normal TM cells making them more sensitive to GCs. Regulation of the GRα/GRβ splicing is not very well documented. The role of splicing factors that regulate spliceosome assembly seems to be one of the key factors regulating the process of alternative splicing. We have shown that the relative levels of the different serine-arginine (SR) proteins (SRps) in the TM regulate the differential expression of the two alternatively spliced isoforms of GR, GRα and GRβ and that expression of these SR proteins regulates GC responsiveness in TM cells. In addition, we evaluated a special class of compounds (thailanstatins or TSTs) and found them to modulate this splicing process to enhance GRβ levels in TM cells. These splicing modulators increased GRβ/GRα in TM, decreased GC response and provide potential glaucoma therapeutic agents.Item Histological Investigation of Human Glaucomatous Eyes: Extracellular Fibrotic Changes and Galectin3 Expression in the Trabecular Meshwork and Optic Nerve Head(2018-05) Belmares, Ricardo; Clark, Abbot F.; Rosales, Armando; Lovely, Rehana S.; Reeves, Rustin E.; Jung, Marianna; Yao, HaiGlaucoma is a leading cause of vision loss and is associated with fibrotic changes in two ocular tissues: the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences of extracellular components of the two ocular tissues in human glaucomatous eyes to determine fibrotic changes. The extracellular components studied included: collagen, elastin, Galectin 3 (Gal3), and Transforming Growth Factor beta-2 Type II receptor (TGFβ-2 RII). We hypothesized that these components will be increased in glaucomatous eyes using chemical staining and immunohistochemistry. Chemical staining included: Masson's Trichrome and Sirius Red stains (collagen) and Vernhoeff-Van Giesen (elastin). Immunohistochemistry was used to determine expression of Gal3 and TGFβ-2 RII. Data was analyzed using Image J software to quantify expression of the extracellular components. The results from Image J analysis of extracellular components demonstrated an overall increase in glaucomatous tissue. TM studies showed an increase of collagen (P=0.0469), and Gal3 (P [less than] 0.0001), and TGFβ-2 RII (P=0.0005) in glaucomatous eyes. Collagen was apparently increased in ONH ((P=0.0517) and Galectin3 (P=0.041) was increased in myelin transition zone of the glaucomatous optic nerve. Vernhoeff-Van Giesen stain showed increased thickness and irregular arrangement of elastic fibers in ONH. Vernoeff-van Giesen and Sirius Red stains also showed increased staining in glaucomatous tissue, but were not quantifiable with Image J software. Analysis showed a correlation of TGFβ-2 RII with Gal3 in TM (P [less than ]0.0001) and myelin transition zone of optic nerve (P=0.0003). Analysis of extracellular components of TM and ONH showed that glaucomatous eyes demonstrate a fibrotic state. Increased collagen deposition and thickened elastic fibers are recognized features of fibrosis and both TM and ONH revealed these features through chemical staining. Galectin3, another known marker for fibrosis, was also elevated in TM and optic nerve. Moreover, Gal3 co-localization with TGFβ-2 RII suggests that it may be involved with the pro-fibrotic TGFβ-2 signaling pathway.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 Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork(2000-12-01) Shade, Debra L.; Pang, Iok-Hou; Yorio, Thomas; Dillon, GlennShade, Debra L., Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 2000, 140 pp., 13 tables, 30 figures, references, 156 titles. This research provides important insights into the means by which interleukin-1alpha (IL-1α) regulates TM cell functions and enhances aqueous outflow, thus lowering IOP. The studies reported herein represent the first known characterization of the central role of the AP-1 transcription factor pathway in IL-1α-mediated production of proMMP-3 by TM cells, as well as the first known evidence that IL-1α can also enhance TM phagocytosis. Using these results as a stepping stone, this research has furthermore led to the identification of “AP-1 activators” as a novel compound class which may be useful in the treatment of glaucoma; it also points to the potential for compounds which regulate MEK, p38, and PKCμ activity as additional means of treatment. Based on these results, it is postulated that such compounds would be expected to lower IOP via upregulations of MMP production, followed by ECM degradation, and potentially, enhanced clearance of degraded ECM via phagocytosis.Item Mechanisms of Glucocorticoid-induced ocular hypertension(2003-08-01) Zhang, Xinyu; Thomas YorioZhang, Xinyu, Mechanisms of glucocorticoid-induced ocular hypertension. Doctor of Philosophy (Pharmacology & Neuroscience). August 2003; 163p; 4 tables; 24 figures; 102 titles. Glucocorticoids, frequently used anti-inflammatory and immunosuppressive agents, are associated with ocular hypertension and glaucoma. Endothelin-1 (ET-1) is also implicated in glaucoma pathology and optic neuropathy as its concentration is elevated in glaucoma patients and in animal models of glaucoma and chronic administration of ET-1 produces damage to the optic nerve head in rats. Glucocorticoids have been reported to regulate the expression of ET-1 gene and ET receptors in the cardiovascular system. However in the eye, the interactions between glucocorticoids and ET-1 have been implicated in the regulation of intraocular pressure and contribute to glaucoma pathology. Therefore, the purpose of the investigations described herein was to determine the novel mechanisms that may be involved in the regulation of intraocular pressure by glucocorticoids with interactions with ET-1 and ET receptors in NPE cells, a source of ET-1, and in TM cells where both glucocorticoids and ET-1 effect aqueous humor outflow. The hypothesis was that ET-1 exacerbates the actions of glucocorticoids on TM cells and contributes to increased outflow resistance. Furthermore, individual sensitivities to glucocorticoids differ considerably. About one in every three people in the general population is considered potential steroid responders while almost all primary open angle glaucoma (POAG) patients are steroid responders and develop ocular hypertension after ocular administration of glucocorticoids. The molecular mechanisms underlying the higher glucocorticoid responsiveness among POAG patients remain unknown. The glucocorticoid receptor beta isoform (hGRβ) has become a candidate for glucocorticoid resistance in some diseases, especially in asthma, based on the reports of its negative activity. The purpose of this segment of the investigations was to test the hypothesis that glucocorticoid responsiveness was regulated by the expression of hGRβ in TM cells. We demonstrated that dexamethaosone (Dex), a synthetic glucocorticoid, increased ET-1 synthesis and release from human non-pigmented ciliary epithelial (HNPE) cells. Dex also suppressed ETB receptor protein expression and attenuated ET-1 mediated increase in nitric oxide (NO) while Dex had no effect on ETA receptor expression and ETA receptor mediated intracellular Ca2+ mobilization in TM cells. The increase in the release of ET-1 from HNPE cells with a concomitant decrease of ETB receptor protein expression and ETB receptor mediated NO release by Dex in TM could result in an increase in the contraction and decrease in relaxation of trabecular meshwork thus reducing the intratrabecular space. Such actions by ET-1 may exacerbate Dex effects on the outflow pathway leading to increased outflow resistance and consequently elevated intraocular pressure that typically is associated with glucocorticoids. We have also found a significant difference in hGRβ levels among normal versus glaucomatous TM cell lines, with the POAG TM cell lines having lower hGRβ receptor expression. This is coincidence with the fact that in the normal population, there is a low rate of glucocorticoid responders as compared to almost all POAG patients considered as glucocorticoid responders. Overexpression of hGRβ in TM cells, produced by transfecting a hGRβ expression construct, inhibited Dex-induced expression of myocilin, a glaucomatous gene, supporting the contention that hGRβ acts as a negative regulator of glucocorticoid activity. In addition, we studied the machinery of cytoplasm to nuclear transport of hGRβ. We identified that a chaperon protein, hsp90, is a requirement for the nuclear translocation of hGRβ. In conclusion, we have described a novel-signaling pathway for glucocorticoids through the regulation of ET-1 and ET receptors in the anterior segment which have consequences on aqueous humor outflow. We have also demonstrated a possible molecular mechanism by which glucocorticoid responsiveness in POAG patients is achieved as a result of the low level of nuclear hGRβ receptor isoform expression. Furthermore, we have, for the first time, identified hap90 as a chaperon protein for the translocation of hGRβ from the cytoplasm to the nucleus.Item Mitochondria-associated endoplasmic reticulum membranes (MAMs) and their role in glaucomatous retinal ganglion cell degeneration-a mini review(Frontiers Media S.A., 2023-05-30) Pham, Jennifer H.; Stankowska, Dorota L.Glaucoma is a leading cause of blindness worldwide, commonly associated with elevated intraocular pressure (IOP), leading to degeneration of the optic nerve and death of retinal ganglion cells, the output neurons in the eye. In recent years, many studies have implicated mitochondrial dysfunction as a crucial player in glaucomatous neurodegeneration. Mitochondrial function has been an increasingly researched topic in glaucoma, given its vital role in bioenergetics and propagation of action potentials. One of the most metabolically active tissues in the body characterized by high oxygen consumption is the retina, particularly the retinal ganglion cells (RGCs). RGCs, which have long axons that extend from the eyes to the brain, rely heavily on the energy generated by oxidative phosphorylation for signal transduction, rendering them more vulnerable to oxidative damage. In various glaucoma models, mitochondrial dysfunction and stress from protein aggregates in the endoplasmic reticulum (ER) have been observed in the RGCs. However, it has been shown that the two organelles are connected through a network called mitochondria-associated ER membranes (MAMs); hence this crosstalk in a pathophysiological condition such as glaucoma should be evaluated. Here, we review the current literature suggestive of mitochondrial and ER stress related to glaucoma, indicating potential cross-signaling and the potential roles of MAMs.
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