Eye/Vision
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Browsing Eye/Vision 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 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 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 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 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 TRANSFORMING GROWTH FACTOR-β2 EFFECTS EXPRESSION OF PERIOSTIN AND PROCOLLAGEN C-ENDOPEPTIDASE ENHANCER 1 IN HUMAN TRABECULAR MESHWORK CELLS(2014-03) Tovar, Tara; Naik, Monal; Clark, Abbot F.; Wordinger, Robert J.Transforming growth factor-beta 2 (TGF-β2) has been implicated in the development of elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Glaucoma patients have increased levels of TGF-β2 in their aqueous humor and trabecular meshwork (TM), and TGF-β2 increases extracellular matrix (ECM) proteins. Thus, we are interested in growth factors that are associated with glaucoma and the ECM. Purpose (a): Transforming growth beta-2 (TGF-β2) has been associated with increased extracellular matrix (ECM) deposition, which is attributed to increased aqueous humor outflow resistance through the trabecular meshwork (TM). We have previously demonstrated that bone morphogenetic protein 1 (BMP1) (an enzyme responsible for the cleavage and maturation of ECM proteins) is expressed and regulated by TGF-β2 in the human TM and that BMP1 regulates lysyl oxidase activity. Also, other factors associated with the ECM remodeling include periostin (POSTN) and procollagen c-endopeptidase enhancer 1 (PCOLCE1). The purpose of this study was to determine whether human TM cells (a) express POSTN and PCOLCE1 (b) whether expression of POSTN and PCOLCE1 are regulated by TGF-β2. Methods (b): Primary human normal (NTM) and glaucomatous (GTM) cells were isolated and subjected to qPCR and Western immunoblotting (WB) for POSTN and PCOLCE1 expression. qPCR was used to determine POSTN and PCOLCE1 expression between control and TGF-β2 treated (5ng/ml for 24 hours) TM cells. WBs of cell lysates and conditioned medium were used to compare POSTN and PCOLCE1 protein expression between control and TGF-β2 treated NTM and GTM cells. Results (c): Human TM cells expressed POSTN and PCOLCE1 mRNA and protein. Exogenous TGF-β2 increased POSTN mRNA expression (p<0.05) and decreased PCOLCE1 expression (p<0.0005) compared to control cells. WB analysis showed increased POSTN secretion in NTM compared to GTM cells (p<0.05). TGF-β2 induced POSTN in NTM cells (p<0.05). However, no POSTN was detected in cell lysates of TM cells. WB analysis showed decreased PCOLCE1 secretion in NTM cells compared to GTM cells (p<0.05). Conclusions (d): POSTN and PCOLCE1 are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of POSTN and PCOLCE1 may lead to structural and functional changes in the ECM within the TM.