Browsing by Author "He, Shaoqing"
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Item A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells(PLOS, 2017-09-22) Wang, Junming; Ma, Hai-Ying; Krishnamoorthy, Raghu R.; Yorio, Thomas; He, Shaoqingc-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein beta (C/EBPbeta) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPbeta in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPbeta as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPbeta augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPbeta was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPbeta and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPbeta appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.Item Detecting and Quantifying Oxidative DNA Damage using MinION Nanopore Sequencing(2018-05) Blessing, Alexandra M.; Phillips, Nicole; Planz, John; Allen, Michael; He, ShaoqingA common biomarker of damaged DNA, particularly mitochondrial DNA, 8-oxoguanine (8-oxoG) has been identified as a possible contributor to neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, preeclampsia, as well as type 1 and type 2 diabetes. Numerous methods have been developed to detect oxidative damage within the genome, including but not limited to immunological techniques, quantitative-polymerase chain reaction (qPCR), and in situ imaging. This study explores nanopore sequencing using the MinION Nanopore (Oxford Nanopore Technologies, Oxford, UK) as a more sensitive method of 8-oxoguanine detection, providing proof-of-concept for model training as well as preliminary model development.Item ENDOTHELIN B (ETB) RECEPTORS CONTRIBUTE TO NEURODEGENERATION IN A RODENT MODEL OF GLAUCOMA VIA UPREGULATION OF C-JUN AND BAX(2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.Item Endothelin-1 Induced the Reactivation of Primary Rat Ocular Astrocytes(2016-03-23) Ma, Hai-Ying; Park, Yong; Wang, Junming; Yorio, Thomas; He, ShaoqingPurposes: Astrocytes play a crucial role in cell survival and axon function of retinal ganglion cells (RGCs) by providing the structural support to neurons, secreting neurotrophic factors to regulate apoptosis and maintenance of the extracellular milieu. Endothelin-1(ET-1) and its receptors are found to be involved in the etiology of glaucoma. However, ET-mediated reactivation of astrocytes affecting RGC survival is still not fully understood. This study aimed to investigating the mechanisms by which ET-1 promotes the reactivation of primary rat ocular astrocytes. Methods: The primary astrocytes were isolated from retinas and optic nerve of rats. Immunostaining of glial fibrillary acid protein (GFAP), RNA binding protein with multiple splicing (RBPMS) and alpha smooth muscle actin (α-SMA) was performed on the cultured primary astrocytes to identify the purity of cells. The cultured primary astrocytes were treated with 100nM endothelin-1 for 24 hours followed the protein detection using Western Blot. ET-1-mediated influx of calcium was monitored in astrocytes using Fura-2 AM calcium imaging. Results: GFAP was uniformly stained on the primary astrocytes, and no staining of RBPMS and α-SMA was identified, whereas the staining of α-SMA was identified in NIH3T3 fibroblast cells. The treatment of ET-1 and ET-3 induced the upregulation of GFAP, neural cell adhesion molecule (NCAM), c-Jun, c-Jun N-terminal kinase (JNK) and Ki67 (a protein marker of cell proliferation). Administration of SP600125, an inhibitor of JNK, attenuated the increased GFAP induced by ET-1 in astrocytes. However, BQ788, an antagonist of ETB receptor, didn’t inhibit ET-1-mediated upregulation of GFAP. In addition, ET-1 triggered augment of intracellular calcium in the primary astrocytes, whereas the application of verapamil, an L-type calcium channel blocker, inhibited the influx of calcium. Conclusions: The hallmark of reactive astrocytes, GFAP, is tightly regulated in astrocytes. An increase in protein levels of GFAP and Ki67 induced by ET-1 reflected the reactivation of astrocytes. Meanwhile, other proteins were also found to be upregulated, such as NCAM, c-Jun and JNK. In addition, intracellular of calcium was also promoted with ET-1 treatment. Taken together, the results suggest that calcium-mediated signaling and JNK/c-Jun pathway are involved in reactivation of astrocytes. This reactivation could lead to dysfunction in the optic nerve and affect RGC survival.Item Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells.(2019-03-05) Stankowska, Dorota; He, Shaoqing; Kodati, Bindu; Krishnamoorthy, Raghu; Chaphalkar, Renuka M.; Chaphalkar, Renuka M.TITLE: Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells. Purpose: Endothelin-1 (ET-1) treatment has been shown to promote apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms underlying these effects are still unknown. The purpose of the study was to assess the changes in gene expression at the level of the translatome, occurring during ET-1 mediated neurodegeneration of RGCs. Methods: Primary RGCs isolated from post-natal day 5 rat pups were treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Polysomal RNA was isolated and libraries for RNA-Seq were prepared. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p Results: STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. Immunostaining of primary RGCs showed an appreciable decline in expression of COX17, while ATP5H expression was modestly decreased. A decreasing trend (three out of four rats) in immunostaining for ATP5H as well as COX17 was found in retinas of rats intravitreally injected with ET-1 (n=4). Conclusions: ET-1 treatment produced a decrease in expression of key components of mitochondrial electron transport chain. A compromise in bioenergetics could be one mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.Item 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 Neuroprotective effects of SB203580 (p38 MAP kinase inhibitor) against Endothelin-1-induced retinal ganglion cell death(2020) He, Shaoqing; Chaphalkar, Renuka; Kodati, Bindu; Krishnamoorthy, Raghu; Krishnamoorthy, VigneshPurpose: Endothelin-1 (ET-1) is a vasoactive peptide contributing to neurodegeneration in glaucoma, however, the underlying mechanisms are not completely understood. The current study tested the involvement of the p38 MAP kinase in ET-1-induced cell death in primary rat retinal ganglion cells (RGCs). Methods: Primary RGCs were treated for 24 hours with 100nM ET-1 either in the presence or absence of the p38 MAP kinase inhibitor SB203580. A Live/Dead assay was used to determine cell viability and the number of surviving and dying/dead cells were quantitated. Results: ET-1 induced RGC death following a 24 hour treatment (as seen by an increase in the dead/live ratio from 0.446 to 0.621), compared to untreated controls. Interestingly, compared to the untreated controls, an appreciable decrease in cell death was found in cells treated with SB203580 alone (a decrease in the dead/live ratio from 0.446 to 0.243). Following treatment with a combination of ET-1 and SB203580, cell counts showed a decrease in cell death when compared with cells treated with ET-1 alone (a change in the dead/live ratio from 0.621 to 0.333). Conclusions: The p38 MAP kinase pathway is known to be involved in signaling mechanisms underlying numerous pathways related to cellular stress. The current study suggests that p38 MAP kinase contributes to ET-1-mediated RGC death. Elucidation of endothelin-mediated signaling pathways will help understand mechanisms by which endothelins promote neurodegeneration in glaucoma.Item Overexpression of Endothelin A and B Receptors Enhances Calcium Mobilization in Ocular Astrocytes and Ciliary Epithelial Cells(2016-03-23) He, Shaoqing; Ma, Hai-Ying; Park, Yong; Yorio, Thomas; Broyles, Heather V.Purpose: Endothelin-1 (ET-1), a vasoactive peptide, binds ETA receptor and ETB receptor to exert its role in multiple cellular processes. A growing body of evidence suggests that elevated levels of ET-1 and activation of its receptors contributes to neurodegeneration in glaucoma, where reactive astrocytes are found to induce damage of retinal ganglion cells. Overexpression of c-Jun, a transcription factor, has been shown to increase levels of ETB receptor, suggesting that the expression of ETB receptor is regulated by c-Jun. This study attempts to determine if overexpression of ET-1 receptors affect calcium influx in response to ET-1 treatment. Methods: Primary astrocytes were isolated from retina and optic nerve of rat pups postnatal 4-7 days. Calcium imaging using Fura-2-AM fluorescent dye was used to determine calcium influx following treatment of ET-1, in the presence and absence of BQ610 (ETA selective antagonist), or BQ788 (ETB selective antagonist) or no treatment (control). ETA, ETB and c-Jun were also overexpressed in Human Non-Pigmented Epithelial (HNPE) cells using DNA transfection and calcium mobilization was measured. Results: Overexpression of ETA or ETB in HNPE cells significantly increased [Ca2+]i levels compared to control following ET-1 treatment at p2+]i in primary astrocytes. Treatment with either BQ610 or BQ788 in primary astrocytes significantly (p2+]i levels compared to control following ET-1 treatment. Conclusion: This study demonstrated that ETA and ETB can mediate calcium influx in HPNE cells and primary astrocytes. ETA receptor stimulation produced a similar calcium influx in HPNE cells as ETB receptor activation, suggesting that both receptors may be involved in [Ca2+]i signaling. The increase in calcium can result in activation of cell death pathways that may explain the ET-1 neurodegenerative actions.Item Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma(ARVO Journals, 2021-08-18) Li, Linya; He, Shaoqing; Liu, Yang; Yorio, Thomas; Ellis, Dorette Z.Purpose: The purpose of this study was to determine the effects of the Sigma-1R (sigma-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the sigma-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing sigma-1r expression and/or activate sigma-1r activity in sigma-1r K/O mice and wild type (WT) mice. AAV2-sigma-1r vector was used to increase sigma-1r expression and sigma-1r agonist used to activate the sigma-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with sigma-1r K/O mice. Pentazocine-induced effects and the effects of sigma-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and sigma-1r K/O mice, with sigma-1r K/O mice experiencing significant decreases compared with WT mice. The sigma-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with sigma-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The sigma-1r offers neuroprotection, as activation and/or transgenic expression of sigma-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.Item The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats(Frontiers Media S.A., 2023-01-01) Kodati, Bindu; Zhang, Wei; He, Shaoqing; Pham, Jennifer H.; Beall, Kallen J.; Swanger, Zoe E.; Krishnamoorthy, Vignesh R.; Harris, Payton E.; Hall, Trent; Tran, Ashley V.; Chaphalkar, Renuka M.; Chavala, Sai H.; Stankowska, Dorota L.; Krishnamoorthy, Raghu R.Glaucoma is a chronic and progressive eye disease, commonly associated with elevated intraocular pressure (IOP) and characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The pathological changes in glaucoma are triggered by multiple mechanisms and both mechanical effects and vascular factors are thought to contribute to the etiology of glaucoma. Various studies have shown that endothelin-1 (ET-1), a vasoactive peptide, acting through its G protein coupled receptors, ET(A) and ET(B), plays a pathophysiologic role in glaucoma. However, the mechanisms by which ET-1 contribute to neurodegeneration remain to be completely understood. Our laboratory and others demonstrated that macitentan (MAC), a pan endothelin receptor antagonist, has neuroprotective effects in rodent models of IOP elevation. The current study aimed to determine if oral administration of a dual endothelin antagonist, macitentan, could promote neuroprotection in an acute model of intravitreal administration of ET-1. We demonstrate that vasoconstriction following the intravitreal administration of ET-1 was attenuated by dietary administration of the ET(A)/ET(B) dual receptor antagonist, macitentan (5 mg/kg body weight) in retired breeder Brown Norway rats. ET-1 intravitreal injection produced a 40% loss of RGCs, which was significantly lower in macitentan-treated rats. We also evaluated the expression levels of glial fibrillary acidic protein (GFAP) at 24 h and 7 days post intravitreal administration of ET-1 in Brown Norway rats as well as following ET-1 treatment in cultured human optic nerve head astrocytes. We observed that at the 24 h time point the expression levels of GFAP was upregulated (indicative of glial activation) following intravitreal ET-1 administration in both retina and optic nerve head regions. However, following macitentan administration for 7 days after intravitreal ET-1 administration, we observed an upregulation of GFAP expression, compared to untreated rats injected intravitreally with ET-1 alone. Macitentan treatment in ET-1 administered rats showed protection of RGC somas but was not able to preserve axonal integrity and functionality. The endothelin receptor antagonist, macitentan, has neuroprotective effects in the retinas of Brown Norway rats acting through different mechanisms, including enhancement of RGC survival and reduction of ET-1 mediated vasoconstriction.Item Upregulation of the endothelin A (ETA) receptor and its association with neurodegeneration in a rodent model of glaucoma(BioMed Central Ltd., 2017-03-01) McGrady, Nolan R.; Minton, Alena Z.; Stankowska, Dorota L.; He, Shaoqing; Jefferies, Hayden B.; Krishnamoorthy, Raghu R.BACKGROUND: Primary open angle glaucoma is a heterogeneous group of optic neuropathies that results in optic nerve degeneration and a loss of retinal ganglion cells (RGCs) ultimately causing blindness if allowed to progress. Elevation of intraocular pressure (IOP) is the most attributable risk factor for developing glaucoma and lowering of IOP is currently the only available therapy. However, despite lowering IOP, neurodegenerative effects persist in some patients. Hence, it would be beneficial to develop approaches to promote neuroprotection of RGCs in addition to IOP lowering therapies. The endothelin system is a key target for intervention against glaucomatous neurodegeneration. The endothelin family of peptides and receptors, particularly endothelin-1 (ET-1) and endothelin B (ETB) receptor, has been shown to have neurodegenerative roles in glaucoma. The purpose of this study was to examine changes in endothelin A (ETA) receptor protein expression in the retinas of adult male Brown Norway rats following IOP elevation by the Morrison's model of ocular hypertension and the impact of ETA receptor overexpression on RGC viability in vitro. RESULTS: IOP elevation was carried out in one eye of Brown Norway rats by injection of hypertonic saline through episcleral veins. After 2 weeks of IOP elevation, immunohistochemical analysis of retinal sections from rat eyes showed an increasing trend in immunostaining for ETA receptors in multiple retinal layers including the inner plexiform layer, ganglion cell layer and outer plexiform layer. Following 4 weeks of IOP elevation, a significant increase in immunostaining for ETA receptor expression was found in the retina, primarily in the inner plexiform layer and ganglion cells. A modest increase in staining for ETA receptors was also found in the outer plexiform layer in the retina of rats with IOP elevation. Cell culture studies showed that overexpression of ETA receptors in 661W cells as well as primary RGCs decreases cell viability, compared to empty vector transfected cells. Adeno-associated virus mediated overexpression of the ETA receptor produced an increase in the ETB receptor in primary RGCs. CONCLUSIONS: Elevated IOP results in an appreciable change in ETA receptor expression in the retina. Overexpression of the ETA receptor results in an overall decrease in cell viability, accompanied by an increase in ETB receptor levels, suggesting the involvement of both ETA and ETB receptors in mediating cell death. These findings raise possibilities for the development of ETA/ETB dual receptor antagonists as neuroprotective treatments for glaucomatous neuropathy.