Browsing by Subject "Glaucoma"
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Item BMP4 INDUCED ID PROTEIN PROTECTS TM FROM GLAUCOMATOUS EFFECTS OF TGFβ-2(2014-03) Mody, Avani A.; Wordinger, Robert J.; Clark, Abbot F.Insight into the BMP4 pathways in various disease models and different cell types has shown BMP4 to be a potent inducer of inhibitor of DNA binding proteins (ID1 and ID3). ID1 and ID3 are negative regulators of basic Helix loop Helix (bHLH) transcription factors and are known to control specific gene expression, including extracellular cellular matrix (ECM) genes. We previously have shown that BMP4 attenuates the pathogenic effects of TGFβ2 in the TM, an ocular tissue involved in regulation of intraocular pressure in glaucoma. We hypothesize that BMP-4 attenuates the effects TGFβ2 in the TM by inducing ID1 and ID3 expression. In our current study, we show that BMP4 induces ID1 and ID3 expression in TM cells. Over-expression and knockdown of ID1 and ID3 in TM cells show that ID1 and ID3 play a crucial role in attenuating the profibrotic effects of TGFβ2 in TM cells. Purpose (a): Increased aqueous humor (AH) outflow resistance causes high intraocular pressure (IOP), which is a critical risk factor in primary open-angle glaucoma. Elevated transforming growth factor b2 (TGFβ2) in the AH of glaucoma patients increases extracellular matrix (ECM) protein deposition in the trabecular meshwork (TM), thereby elevating IOP. Bone morphogenetic protein 4 (BMP4) inhibits the pathogenic effects of TGFβ2 in the TM. However, the underlying molecular mechanism for this BMP4 inhibition remains unknown. BMP4 regulates various cellular processes by induction of inhibitors of DNA binding proteins (ID1, ID3), which are transcriptional regulators that bind specific transcription factors and suppress their functions. This study will determine whether ID1/ID3 are downstream targets of BMP4, attenuating the TGFb-2 effects on TM cells. Methods (b): Cultured primary human TM cells and the GTM3 cell line were treated with BMP4 (5-10ng/ml) for 1-48 hrs. Q-PCR and western immunoblotting were performed to determine ID1 and ID3 expression. GTM3 and primary TM cells were transfected with ID1 and ID3 expression plasmids vectors or ID1 and ID3 siRNA to determine the effects of ID1 and ID3 on TGFβ2 induced extracellular matrix (ECM) proteins. The expression of fibronectin and plasminogen activator inhibitor-1 (PAI-1) was studied by western immunoblotting. Results (c): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma. Conclusions (d): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma.Item C1Q EXPRESSION AND GLIAL ACTIVITY IN THE MOUSE RETINA FOLLOWING ISCHEMIA/REPERFUSION INJURY(2014-03) Silverman, Sean; Kim, Byung-Jin; Wordinger, Robert J.; Clark, Abbot F.We are using a mouse model whereby blood flow to the eye is blocked by raising the pressure in the eye in order to mimic damage caused by glaucoma. Our interest is to see how levels of C1q, a protein typically associated with the immune system as well as injury responsive cells of the eye are changed. Purpose (a): The complement cascade has become of increasing interest in several neurodegenerative diseases, including glaucoma, a leading cause of blindness. C1q has been observed as one of the earliest upregulated genes in the optic nerve head, the initial site of glaucoma injury preceding pathological changes. Here we use a glaucoma-like model of retinal ischemia/reperfusion (I/R) to mimic clinical changes in visual function and cellular loss. Methods (b): Deeply anesthetized C57BL/6J received a cannula to the anterior chamber of their left eye, through which their intraocular pressure (IOP) was raised to 120mmHg for 60 minutes leading to complete retinal ischemia. The cannula was then removed and blood flow was naturally reperfused. The right eye was uninjured as a contralateral control. Mice were sacrificed and enucleated at 3, 7, 14, 21, and 28 days. Eyes were fixed in 4% PFA and frozen for immunofluorescence or in situ hybridization studies. Microglia and astrocytes were identified using Iba1 and GFAP, respectively. Quantifications were performed using ImageJ Analysis software(NIH). Results (c): Initial changes in C1q expression were observed as early as 72 hours following injury, with a nearly two-fold increase compared to uninjured controls. Upregulated C1q was observed only in the ganglion cell (GCL) and inner plexiform (IPL) layers. Maximum intensity of C1q expression was observed 14 days post injury. Fluorescent in situ hybridization (FISH) studies reveal primarily microglia, not astrocytes, colocalized with expression of C1q in the retina. Conclusions (d): Following retinal I/R injury, C1q expression is actively upregulated, which appears to spatio-temporally correlate with changes in microglial, astrocyte, and Mueller cell homeostasis. Our FISH studies identify microglial cells as the primary producers of C1q following I/R injury. This suggests the elevated levels of C1q may stimulate astrocyte activation. There appears to be an interplay between microglia and astrocytes, both of which have been directly implicated in neurodegenerative diseases, including loss of RGCs in glaucoma. We propose C1q is an integral part of this mechanism, and by removing C1q we hope to preserve visual function and prevent degeneration in the visual system following injury.Item CHANGES IN ENDOTHELIN RECEPTOR A EXPRESSION IN A RAT MODEL OF OCULAR HYPERTENSION(2014-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.Glaucoma is the leading cause of blindness in developed countries and is the second leading cause of blindness worldwide. The most common and currently only treatable symptom associated with glaucoma is an increase in intraocular pressure (pressure inside the eye). Rodent models have been routinely used to understand the effects elevated pressure has on the eye, and this study focuses on the changes in expression of a protein molecule (endothelin A receptor) within the retina due to elevated intraocular pressure in a rat model of elevated intraocular pressure. Purpose (a): The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression within the retina in the Morrison’s elevated IOP model of glaucoma in rats. Methods (b): IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison’s model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. The rats were maintained for two to four weeks following IOP elevation and sacrificed. Retinal sections were obtained from both control and IOP-elevated eyes, and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results (c): After two weeks, rat eyes with IOP elevation showed an increase in immunostaining for ETA receptors in several retinal layers including the inner and outer plexiform layers with a modest increase in the retinal ganglion cell layer. Following four weeks of IOP elevation, ETA receptor expression was modestly increased in the inner and outer plexiform layers of the retina, compared to that in the corresponding contralateral eyes. Conclusions (d): Elevated intraocular pressure results in a time-dependent change in ETA receptor expression. Increased ETA receptor expression is associated with neurodegenerative changes in glaucoma.Item Characterization and Function of Follistatin in Human Trabecular Meshwork Cell and Tissues(2013-05-01) Fitzgerald, Ashley M.; Robert WordingerPrimary Open Angle Glaucoma (POAG) is a leading cause of blindness affecting over 70 million people worldwide. The most important risk factor for developing POAG is elevated intraocular pressure (IOP), which results from increased resistance of aqueous humor (AH) through the trabecular meshwork (TM) outflow pathway. Transforming growth factor- beta II (TGF-β2) is elevated in the AH and TM of glaucoma patients. Recent evidence indicate an extracellular BMP antagonist, gremlin, regulates BMP signaling and TGF-β2 activity. Follistatin (FST), another secreted BMP antagonist is recognized for its ability to bind BMPs and their type I receptor, sequestering BMP signaling. The purpose is to evaluate the presence and relevant activity of follistatin in TM tissues and cells. We hypothesize expression of follistatin in human trabecular meshwork cells alters the expression of extracellular matrix (ECM) deposition seen in the pathogenesis of glaucoma. First, we examined differential FST expression in human trabecular meshwork cells and tissues. We observed a significant increase in expression of FST in glaucomatous as compared to normal protein and mRNA expression. Next, we determined if FST could be induced upon treatment of exogenous TGF-ß2 protein in human TM cells. Studies showed TGF-ß2 up-regulated FST mRNA transcript in a time dependent manner. FST protein secretion was increased in a time and does dependent manner. Third, we assessed FST effects on induction or inhibition of ECM proteins in human TM cells. ECM protein and mRNA expression was time dependent; nevertheless the response of ECM protein to FST treatment is different depending on isoform presence. Additional studies will be done to further elucidate these findings. Lastly, we evaluated FST-288 and FST-315 inhibition of BMP4 attenuation of TGF-ß2 induced ECM expression. Data suggest FST-315 to suppress BMP-4 effects on TGF-ß2 induced ECM and FST-288 enhanced BMP-4 effects on TGF-ß2 induced ECM. The goal is to evaluate additional factors that contribute to the pathogenesis of POAG and assess how these factors can provide possible therapeutic mechanisms for the treatment of glaucoma.Item COMPLEMENT AND GLIAL ACTIVITY IN THE RETINOCOLLICULAR PATHWAY OF MICE USING A NOVEL MODEL OF GLAUCOMA(2013-04-12) Silverman, SeanPurpose: Glaucoma is a leading cause of irreversible visual impairment and blindness throughout the world. C1q is responsible for axonal pruning in early ocular development and is upregulated in glaucomatous eyes of mice, non-human primates, and humans. We used an inducible mouse model of human primary open angle glaucoma with elevated intraocular pressure (IOP) to examine expression levels of C1q in the retina and superior colliculus (SC), as well as identify changes in cellular homeostasis. Methods: Anesthetized A/J mice were given a single intravitreal injection of Ad5.MYOC.Y437H (5x107 pfu), a mutant glaucoma gene, or Ad5.null control virus. Following injections, conscious IOPs were measured weekly, using a TonoLab tonometer (iCare). Mice were sacrificed at time points between 3 days and 8 weeks. Brains and retinas were harvested for immunofluorescence or immunoblotting studies. Microglia and astrocytes cells were identified using Iba1 and GFAP, respectively. All quantifications were performed using ImageJ Analysis software(NIH). Results: IOPs were significantly increased in the Ad5.MYOC.Y437H eyes (p<0.01) compared to the contralateral un-injected eye and eyes receiving Ad5.null. Clq expression was significantly upregulated in retinas receiving Ad5.MYOC.Y437H (2.69-fold±0.38, p<0.0001) compared to contralateral control retinas (0.7-fold±0.29). Clq upregulation was additionally observed in SC hemispheres receiving neural connections from injected eyes. Mice given Ad5.null vector displayed no elevation of Clq in the visual axis. Additionally, colocalization studies demonstrated significant increases of inner retinal microglia density beginning 2 weeks post injection (0.61%±0.07, p<0.001) and continuing at 4 weeks (0.87%±0.09, p<0.0001) compared to untreated retinas (0.4l%±0.03 and 0.44%±0.03, respectively). No signs of astrogliosis were detected. Conclusions: C1q is actively upregulated in the retina and SC, following mutant myocilin induced ocular hypertention, whereas adenovirus alone had no effect. An increased microglial population in the retina accompanied these changes. This suggests that microglia may sense the increased IOP and play a role in upregulating endogenous C1q. Early glaucoma pathogenesis may result from the reactivation of the ocular developmental roles of C1q and microglia, suggesting new therapeutic targets for future neuroprotective studies.Item CORRELATIVE INCREASES IN ASTROCYTE, MICROGLIA, AND C1Q IN A MURINE MODEL OF ACUTE GLAUCOMATOUS INJURY(2014-03) Olarte, Neal; Silverman, Sean; Wordinger, Robert J.; Clark, Abbot F.Glaucoma is an injury to cells of the eye indicated by pressure within the eye which can lead to eventual blindness. Microglial cells are a special type of cell within the central nervous system (CNS) that cleans up cellular damage. Astrocytes are another specialized cell which plays a supportive role important for CNS function. C1q is a part of the immune system usually reserved for clearance of bacteria from the body, but has been recently demonstrated to serve other roles. Microglia and C1q have been implicated in mediating retinal damage in mouse models mimicking glaucoma. This study was conducted to investigate if C1q and either microglia or astrocytes, or both, could be involved in brain damage caused by a simulated glaucoma injury. Purpose (a): Glaucoma is a leading cause of blindness worldwide. Recent studies of glaucomatous retinal injury have observed a correlation of upregulated C1q and increased microglial activity. Using the optic nerve crush (ONC) model of glaucoma, we are investigating whether there is an injurious response involving C1q, microglia, and astrocytes within the superior colliculus (SC), the visual center of the mouse brain. Methods (b): Glaucomatous injury was simulated in mice using ONC of the left eye, while leaving the right eye intact. Brain tissue was harvested at 0, 7, 14, and 28 days post-injury, fixed overnight in 4% paraformaldehyde, and paraffin embedded. Following paraffin removal and antigen recovery, immunohistochemistry was performed to label astrocytes (GFAP), microglia (IBA1), and C1q in the SC. Results (c): Beginning 7 days post-injury, there was an increase in astrocytes, microglia, and C1q, with microglia assuming an activated morphology. Astrocytes and C1q remained elevated through 28 days post-injury, with a gradual reduction in microglial density. These results were observed only within the SC contralateral to the injured nerve, the main target of the retinal ganglion cell (RGC) axons from the ONC eye. Increased C1q and astrocyte activity was not observed in the ipsilateral hemisphere; however, there was a slight increase in microglial density. Conclusions (d): Our data support a similar response in the retina and SC of upregulated C1q, resulting from glaucomatous injury. Microglia and astrocytes also appear to be involved in the acute injury phase. Previous studies of retinal glaucomatous injury have shown that early reduction of C1q is protective. Future studies using a C1q-deficient mouse model might also show protective function against SC glaucomatous injury.Item DETERMINATION OF PROTEINS INVOLVED IN THE FORMATION OF CROSS-LINKED ACTIN NETWORKS IN THE TRABECULAR MESHWORK(2013-04-12) Bermudez, Jaclyn Y.Purpose: Glaucoma is a leading cause of blindness worldwide and the primary risk factor of glaucoma is increased intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor (a fluid that fills the anterior segment of the eye) production and outflow. In glaucoma patients, the outflow resistance through the trabecular meshwork (TM), a special tissue located at the angle between the cornea and iris, is abnormally elevated. Inside TM cells, actin proteins form cross-linked actin networks (CLANs). Excessive formation of these unique structures can be found in the glaucomatous TM. They can also be induced by dexamethasone (DEX) and TGFβ2. It is suggested that CLANs increase cell rigidity and therefore elevate aqueous humor outflow resistance. However, the proteins that are involved in CLANs formation are not fully identified. We hypothesize that by comparing CLANs enriched and un-enriched TM cells, we will be able to identify the proteins that are involved in CLANs formation. Methods: We treated cultured mouse TM cells with DEX (100 nM), TGFβ2, (5 ng/mL), or ethanol (as vehicle control), evaluated CLANs formation, and separated the cell proteins by 2D gel electrophoresis. Differentially expressed proteins were analyzed by the Redfin software, and gel spots were picked and assessed by spectrometry analysis. Western blotting was used to confirm 2D gel results. Results: We found a subset of proteins that are differentially expressed in CLANs-enriched TM cells, compared to non-enriched samples. Among these proteins, ferritin heavy chain, glial fibrillary acidic protein (GFAP), and integrin alpha V, were confirmed by mass spectrometry and Western immunoblot. Conclusions: We found a subset of proteins that are differentially expressed in CLANs-enriched mouse TM cells. Their contributions and involvements in CLANs formation and regulation of TM cell morphology and functions are being investigated. They may provide new insight of the pathogenesis of glaucoma.Item EFFECT OF CELLULAR AND PLASMA FIBRONECTIN ISOFORMS ON NORMAL HUMAN TRABECULAR MESHWORK CELLS(2013-04-12) Medina-Ortiz, Wanda E.Purpose: The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression. Methods: Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays. Results: GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes. Conclusions: Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.Item EFFECT OF LYSYL OXIDASE (LOX) AND TISSUE TRANSGLUTAMINASE (TGM2) GENES ON HUMAN TRABECULAR MESHWORK CELLS(2014-03) Kirkland, Kyle A.; Bermudez, Jaclyn Y.; Mao, Weiming; Clark, Abbot F.Purpose: Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM). Methods: LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells. Results: LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells. Conclusions: TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-β2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.Item EFFECTS OF TGF-BETA2, FOLLISTATIN AND ACTIVIN A ON EXTRACELLULAR MATRIX IN NORMAL HUMAN TRABECULAR MESHWORK CELLS AND TISSUES.(2013-04-12) Fisher, AndrewPurpose: Primary open angle glaucoma (POAG) is characterized as a group of eye diseases resulting in optic nerve head damage and irreversible blindness. A major risk factor for developing POAG is increased intraocular pressure (IOP) leading to decreased outflow of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor-beta2 (TGF-β2) is increased in the AH of glaucoma patients, and causes increased extracellular matrix (ECM) protein synthesis in the TM. Bone morphogenetic protein-4 (BMP-4) has been shown to inhibit TGF-β2 actions. Follistatin (FST) is an antagonist of BMP-4 and is elevated in the glaucomatous TM. Elevated levels of FST in the TM may block BMP-4 ability to attenuate TGF-β2 induction of ECM proteins. FST may also have a direct role in regulating ECM protein expression in human TM (HTM) cells. HTM cells also express Activin A (Act A). The purpose of this study was to assess the role of FST and Act A in HTM cells as related to TGF-β2/BMP-4 signaling. Understanding these interactions may provide possible new therapeutic targets for the treatment of glaucoma. Methods: Normal HTM cell lines were cultured and treated with TGF-β2 (5ng/ml), FST-315, FST-288, or Act A (each at 50ng/ml) alone and/or simultaneously for 24 and 48 hrs. Western blot analysis was used to evaluate the effects of FST-315/288, Act A, and TGF-β2 on ECM protein synthesis including fibronectin (FN), PAI-1, and collagen1A. Results: TGF-β2 induced expression of PAI-1 and FN.. ACT-A mildly induced PAI-1 and FN proteins as compared to TGF-β2. TGF-β2 and Act A treatment appeared to have a synergistic effect on the expression of PAI-1 and FN protein as compared to individual treatment (Tx) of TGF-β2 or Act A. FST 288 does not seem to change the Act A + TGF-β2 synergism. FST 315 inhibits the synergism of Act A + TGF-β2 showing a decrease in PAI-1 and FN protein. Conclusions: FST-315 decreased the induction of ECM proteins by TGF-β2 and Act-A. FST-288 increased induction of ECM proteins in cells treated with TGF-β2 and Act A. FST-288 treatment for 24 hours induced increased ECM proteins PAI-1 and FN, but there was no induction at 48 hours. FST-315 treatment for 24 hours slightly induced ECM PAI-1 and FN, but there was no induction at 48 hours. Act A treatment for 24 and 48 hours increased induction of PAI-1 or FN. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential roles in the pathogenesis of glaucoma.Item ENDOTHELIN A RECEPTOR EXPRESSION IN RAT MODEL OF OCULAR HYPERTENSION(2013-04-12) McGrady, NolanPurpose: The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression in the retina in the Morrison's elevated IOP model of glaucoma in rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison's model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the corresponding control. The rats were maintained for two weeks following IOP elevation and sacrificed. Retinal sections were obtained from both the control and IOP-elevated eyes and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results: Rat eyes with IOP elevation showed an increase in immunostaining ETA receptor in several retinal layers including retinal ganglion cells, the inner plexiform layer, and the outer plexiform layer. An increased co-immunostaining of ETA receptors with β-III-Tubulin was observed both in retinal ganglion cells and inner plexiform layer. Conclusions: Elevated intraocular pressure results in an increase in ETA receptor expression. Increased endothelin receptor expression is associated with neurodegeneration in glaucoma.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 EPIGENETIC REGULATION OF GLAUCOMA-ASSOCIATED GROWTH FACTORS IN THE TRABECULAR MESHWORK(2014-03) Bermudez, Jaclyn Y.; Webber, Hannah; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, WeimingGlaucoma is a leading cause of blindness in the U.S. and worldwide. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function. Therefore, it is important to study how glaucoma-associated growth factors in the TM are regulated. We investigated how heritable changes in gene activity regulate the TM without altering the DNA sequence. Purpose (a): Glaucoma is a leading cause of blindness in the U.S. and worldwide. This disease leads to progressive, irreversible damage to the optic nerve and visual function. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function, which increase aqueous humor outflow resistance. In the glaucomatous TM (GTM), there is excessive extracellular matrix (ECM) protein deposition. Many studies have suggested that cell signaling pathways, such as the transforming growth factor beta (TGF-β) and Wnt signaling pathways, play key roles in TM homeostasis.The growth factors that are associated with these pathways, including TGFβ2, Gremlin and sFRP1, are found to be at higher levels in the GTM cells compared to normal TM cells. Little is known about the role of epigenetics in regulating glaucoma-associated growth factors in the TM. One of the major epigenetic regulatory mechanisms is histone acetylation.We hypothesize that histone acetylation is responsible for the increased expression of glaucoma associated factors in the TM. Methods (b): Primary human TM cell cultures were treated with 10nM Thailandepsin (TDP-A), a histone deacetylase inhibitor (HDACi), or 1% DMSO as vehicle control for 4 days. Cells were harvested for qPCR to compare gene expression levels or for ChIP assays to compare promoter associated histone acetylation status. We also treated paired perfusion cultured bovine anterior segments with DMSO or TDP-A for 7 to 10 days. The IOP change of the treated bovine eyes was monitored and recorded. Data were analyzed by using Student’s t-test or one-way ANOVA. P values less than 0.05 were considered significant. Results (c): TDP-A significantly elevated the expression of sFRP-1 and TGFβ2 (n=3, p2 as well as elevated IOP. Conclusions (d): Histone acetylation may play an important role in the dysregulation of growth factors in the TM. This mechanism provides a unique opportunity to elucidate the etiology of POAG. Also, TDP-A is a potent HDACi that can be used as a powerful tool in glaucoma research.Item Expression of Procollagen C Proteinase Enhancer Proteins in Trabecular Cells and Tissues(2013-05-01) Naik, Monal; Robert WordingerPrimary open angle glaucoma (POAG) is the most common form of glaucoma. Ocular hypertension is a major risk factor for POAG and is caused by increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM). Increased extracellular matrix (ECM) deposition within the TM is correlated with ocular hypertension. Transforming Growth Factor beta 2 (TGFb2) levels are elevated in the AH and TM of POAG patients, and TGFb2 increases ECM protein expression, aqueous outflow resistance, and intraocular pressure (IOP). Recently, TGFβ2 was found to induce bone morphogenetic protein 1 (BMP1) expression in TM cells suggesting that BMP1 activity might be involved in glaucoma pathogenesis. Procollagen C proteinase enhancers (PCOLCE 1 and PCOLCE 2) regulate BMP1 activity. Therefore, PCOLCE1 and PCOLCE2 may play an important role(s) in regulating ECM structural changes in the TM, and contribute to AH outflow resistance and elevated IOP in glaucoma. The purpose of this study was to determine if human TM cells and tissues express PCOLCE1 and PCOLCE2 and whether TGFb2 induces their expression. This is the first documentation that PCOLCE1 and PCOLCE2 are expressed in TM cells and tissues and that TGFb2 does induce expression of PCOLCE1.Item Fibronectin extra domain A (FN-EDA) causes glaucomatous trabecular meshwork, retina, and optic nerve damage in mice(BioMed Central Ltd., 2022-05-26) Mavlyutov, Timur A.; Myrah, Justin J.; Chauhan, Anil K.; Liu, Yang; McDowell, Colleen M.BACKGROUND: Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA(+/+)) that constitutively expresses only FN containing the EDA isoform. METHODS: Eyes from C57BL6/J (wild-type), B6.EDA+/+ (constitutively active EDA), B6.EDA-/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm's canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts. RESULTS: Ultrastructure analyses show the TM of B6.EDA(+/+) mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6 J mice (p < 0.05). SC basement membrane is thicker and more continuous in B6.EDA(+/+) mice compared to C57BL/6 J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA(+/+) mice compared to C57BL/6 J eyes (p < 0.05). IOP is significantly higher in B6.EDA(+/+) mice compared to C57BL/6 J eyes (p < 0.001), and significant ON damage (p < 0.001) and RGC loss (p < 0.05) detected at 1 year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA(+/+) mice compared to C57BL/6 J control eyes (p < 0.01). CONCLUSIONS: B6.EDA(+/+) mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA(+/+) mice recapitulate many aspects of glaucomatous damage.Item KNOCKOUT OF CASPASE-7 PROTECTS AGAINST OPTIC NERVE CRUSH-INDUCED RETINAL GANGLION CELL DEATH(2014-03) Choudhury, Shreyasi; Liu, Yang; Clark, Abbot F.; Pang, Iok-HouGlaucoma, a leading cause of blindness worldwide is characterized by injury to the nerve of the eye leading to the death of certain eye cells called retinal ganglion cells (RGCs) and vision loss. Currently available glaucoma therapies only attempt to reduce the eye pressure without addressing the associated RGC death problem. As a result, they do not always sufficiently slow the disease progression in all glaucoma patients. Thus, there is an urgent need to develop strategies for preventing glaucoma associated RGC death. Our preliminary studies have identified a novel protein, caspase-7 as a major player in RGC death pathways. We are studying the role of caspase-7 in RGC death in a mouse glaucoma model and whether it can be targeted for better therapeutic outcome. This project is significant because it will identify a new and potentially critical component of RGC death. This will aid in the design of better therapeutic treatment for glaucoma and other degenerative diseases. Purpose (a): Optic nerve (ON) injury is involved in various ocular diseases, such as glaucoma, which leads to apoptotic death of retinal ganglion cells (RGC) and loss of vision. Caspases have been implicated previously in glaucoma and RGC death. However, the role of caspase-7, a functionally unique caspase, in ON injury and glaucomatous damage has not been studied. Therefore, the purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis. Methods (b): C57BL/6 (Wt) and caspase-7knockout (casp7KO) mice were used for this study. Optic nerve crush (ONC) was performed on left eyes; right eyes served as control. Western blots of the isolated retinas of Wt mice were used to assess the activation of caspase-7 at 3h, 6h, 12h, 1d, 3d, and 7d after ONC. Immunohistochemistry was performed to detect the localization of caspase-7 in RGC. RGC survival was determined by counting the RBPMS (RGC marker) labeled cells in flat-mounted retinas of Wt and casp7KO mice at 7d, 14d and 28d post injury. Both Wt and casp7KO mice were subjected to spectral-domain optical coherence tomography (SD-OCT) and scotopic threshold response of electroretinography (STR-ERG) to evaluate the retinal structural and RGC functional changes at 7d, 14d, and 28d after ONC. Results (c): Western blot data demonstrated that caspase-7 was activated in Wt retina at 12h, 1d, 3d, and 7d after ONC compared to the uninjured control retinas. The number of surviving RGCs was significantly more (3173±59 cells/mm2, mean±SEM, n=6, p<0.001) in casp7KO retinas compared to Wt retinas (1693±84 cells/mm2) at 28d post ONC. SD-OCT analysis revealed that the thickness of the inner retinal layer (ganglion cell layer, nerve fiber layer, and inner plexiform layer) in casp7KO mice was greater (54±1.1 μm, p<0.05) compared to Wt mice (42.3±1.5 μm). Most importantly, analysis of the STR-ERG response demonstrated a decline in amplitude in Wt ONC eyes (10.5±1.9 μv), whereas the response was significantly higher (20.7±2.3 μv, p<0.05) in casp7KO mice even at 28d post injury. Conclusions (d): The current study indicates that injury to the ON activates caspase-7 and knockout of caspase-7 protects inner retinal layer morphology and RGC function after ONC. Thus, caspase-7 appears to play a critical role in ONC-induced RGC death and inhibition of caspase-7 activity may be a novel therapeutic target for glaucoma and other neurodegenerative diseases of the retina.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 NEUROPROTECTIVE PROPERTIES OF SIGMA-1 RECEPTOR IN GLAUCOMA(2014-05-01) Mueller, Brett H.; Thomas YorioGlaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) that affects over 70 million individuals worldwide. Glaucoma pathology is manifested as cupping of the optic disk, damage to the nerve fiber layer, and visual field deficits. The final pathological step of this disease contributing to visual field loss is the apoptosis of retinal ganglion cells (RGCs). Currently, the only therapeutic agents that are used to treat glaucoma are IOP lowering drugs. However, even when IOP is brought within normal range, a significant number of patients still have progression of visual deficits. Currently, there are no treatment options that have the ability to sustain the viability of RGCs during the disease process of glaucoma. Therefore, neuroprotective drugs that protect RGCs need to be developed as adjunct therapeutic agents to IOP lowering drugs. The sigma-1 receptor (σ-1r) is a non-opioid receptor that has been shown to have the ability to bind to benzomorphans, steroids, and psychotropic drugs. This receptor is ubiquitously expressed throughout the entire body; however, the endogenous ligand and function of σ-1r is not yet known. Several in vitro and in vivo studies have demonstrated the neuroprotective effects of σ-1r stimulation in several models of retinal neurodegenerative diseases including glaucoma and diabetic retinopathy. Numerous studies have linked the neuroprotective effects of σ-1r to its ability to block cytotoxic calcium ion influx through ligand gated and voltage gated ion channels, modulation of ER stress, maintenance of mitochondrial homeostasis, and stimulation of pro-survival intracellular signaling pathways. However in primary RGCs, there have been no studies demonstrating σ-1 receptor mechanism of action. The only proposed neuroprotective mechanism of action of σ-1r that has been performed in retinal flat mounts is blockage of calcium ion influx through activated NMDA receptors. This present research project investigated the mechanism of neuroprotective effects of σ-1rs in primary RGCs, particularly involving L-type voltage gated calcium channels (VGCCs) and activation of extracellular-signal-regulated kinases (ERK 1/2). We demonstrated that VGCCs were activated using KCl (20mM). Pre-treatment with a known L-type VGCC blocker produced a 57% decrease in calcium ion influx through activated VGCCs (following depolarization by KCl). In addition, calcium imaging showed that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Treatment with a σ-1r antagonist, BD1047, produced a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs. This confirms that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms of action of both these agents. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in primary RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in primary RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in primary RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in primary RGCs implies that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs Another signaling pathway that was studied as a potential target of σ-1r mediated neuroprotection was the MAP kinase pathway, in particular, ERK phosphorylation as an index of cell survival. RGCs subjected to oxygen and glucose deprivation (OGD) for 6 hours induced 50% cell death in primary RGCs and inhibited pERK1/2 expression by 65%. Cell death was attenuated when RGCs were treated with pentazocine under OGD and pERK1/2 expression was increased by 1.6 fold compared to OGD treated RGCs without pentazocine treatment. The co-treatment of with an ERK1/2 inhibitor PD098059 with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. These results established a link between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. In conclusion, we have established two novel mechanisms underlying σ-1 receptor mediated neuroprotection in primary RGCs. These findings suggest that activation of the σ-1 receptor in RGCs has a role in calcium regulation and the activation of the ERK1/2 pathway. In addition, this study also demonstrates the robust neuroprotective effects of σ-1 receptor in RGCs when subjected to OGD. These data also provide evidence suggesting that σ-1 receptor may be a therapeutic target to protect RGCs during ocular neurodegenerative diseases like glaucoma.Item OVEREXPRESSION OF POU DOMAIN TRANSCRIPTION FACTOR, BRN3B CAUSES NEURITE OUTGROWTH IN CULTURED PC 12 CELLS(2013-04-12) Phatak, NitashaPurpose: Brn3b is a POU domain transcription factor shown to play key role in regulating retinal ganglion cell axon outgrowth during development of the retina. The purpose of this study was to determine if overexpression of Brn3b could promote neurite outgrowth in cultured PC 12 cells. Methods: Rat Pheochromocytoma cells ( PC 12) were seeded and grown on Poly-D-lysine coated 100 mm dish and transfected either with pCMV6-Brn3b (an expression vector encoding Brn3b cDNA) or pCMV6-MCS (empty vector). Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Protein extracts were isolated from these cells and analyzed for Brn3b and GAP43, TUBA-1 protein expression in 24 hours by immunoblot analysis. In another set of experiments, PC 12 cells were seeded on Poly-D-Lysine coated 25mm cover slip and transfected with either pCMV6-Brn3b or pCMV6 -MCS. Medium changed to complete medium with NGF (100ng/ml) after 6 hours of transfection. Brn3b, GAP43 and TUBA-1 expression in 24 hours were analyzed by using immunocytochemistry in the transfected cells. Morphological changes in PC 12 cells transfected with Brn3b were studied by using confocal microscopy. Results: Immunoblot analysis showed overexpression of Brn3b in PC12 cells transfected with Brn3b cDNA. Overexpression of transcription factor Brn3b in PC12 cells produced morphological changes including increased neurite outgrowth. An increased immunostaining for Brn3b and neurite-specific GAP43, TUBA-1 were also observed in PC12 cells overexpressing Brn3b. Conclusions: The POU domain transcription factor, Brn3b, could promote neurite outgrowth in PC12 cells.Item Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma(MDPI, 2023-08-26) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloElevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.