Role of the ET B receptor in retinal ganglion cell death in glaucoma (original) (raw)
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Canadian Journal of Physiology and Pharmacology, 2008
Recent observations suggest that the vasoactive peptide endothelin-1 (ET-1) may be an important contributor to the etiology of glaucoma. ET-1 administration has been shown to produce optic nerve axonal loss and apoptosis of retinal ganglion cells. Ocular ET-1 levels are elevated in aqueous humor in response to elevated intraocular pressure both in glaucoma patients and in animal models of glaucoma; however, the precise mechanisms by which ET-1 mediates glaucomatous optic neuropathy are not clear. Presently we report that ET-1-mediated apoptosis was markedly attenuated in ETB receptor-deficient rats, suggesting a key role for ET B receptors in apoptosis of retinal ganglion cells by ET-1 treatment. Using virally transformed rat retinal ganglion cells (RGC-5 cells), we found that ET-1 (100 nmol/L) treatment produced apoptotic changes in these cells that was determined by flow cytometric analyses, release of mitochondrial cytochrome c to the cytosol, and increased phosphorylation of c-Jun N-terminal kinase. Pretreatment with the ET B -receptor antagonist BQ788 (1 mmol/L) was able to significantly attenuate ET-1-mediated apoptosis in RGC-5 cells. ET-1-mediated apoptotic changes in RGC-5 cells were associated with ETB-receptor activation and were accompanied by a significant upregulation of ETBreceptor expression. These studies suggest that ocular ET-1 acts through ET B receptors to mediate apoptosis of retinal ganglion cells, a key event in glaucoma and related optic neuropathies.
Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat
Experimental Eye Research, 2006
The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5 ml intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500 mM concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500 mM concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85 sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.
Endothelin B Receptors Contribute to Retinal Ganglion Cell Loss in a Rat Model of Glaucoma
PLoS ONE, 2012
Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET B ) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET B receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET B receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET B receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET B receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.
Endothelin B Receptor in Human Glaucoma and Experimentally Induced Optic Nerve Damage
Archives of Ophthalmology, 2006
To assess endothelin B receptor (ETbR) expression in human glaucomatous optic nerves and the spatial relationship between ETbR and astrocytes. Methods: Twenty-six eyes from 16 glaucoma patients and 10 normal control subjects were immunohistochemically labeled with antibodies to ETbR. The immunoreactivity was quantified and compared between normal and glaucomatous eyes with an image analysis system. Tissues were also double-labeled for ETbR and astrocytes. In addition, the optic nerve of a monkey with regional degeneration induced by laser coagulation was examined with the same techniques. Results: The frequency of positive ETbR immunoreactivity was higher in human glaucomatous optic nerves as compared with age-matched controls (9/16 vs 1/10, P = .02). The ETbR immunoreactivity colocalized with astrocytic processes and was quantitatively higher in the glaucomatous eyes (P =.02). In the monkey, the regions of degeneration showed increased ETbR associated with reactive astrocytes and was highest at the borders between normal areas and degeneration. Conclusion: Increased ETbR immunoreactivity in diseased optic nerves and its association with astrocytes suggest that the glia-endothelin system may be involved in the pathologic mechanisms of neuronal degeneration. Clinical Relevance: The study supports the clinical observation of endothelin involvement in glaucoma and provides direct evidence that the endothelin system is associated with glaucomatous pathologic abnormalities.
Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma
Pharmacological Research, 2005
The role of endothelin-1 (ET-1) a potent vasoactive peptide, in glaucoma pathogenesis is receiving increasing attention, particularly in astroglial activation in optic nerve damage. Our laboratory has also shown that ET-1 treatment causes proliferation of cultured human optic nerve head astrocytes to possibly initiate astrogliosis. ET-1 is distributed in retina, optic nerve, and ciliary epithelium, however the effects of elevated intraocular pressure (IOP) (as seen in glaucoma) on ET-1 and ET B receptors are not clearly understood. In the present study, the levels of immunoreactive ET-1 (ir-ET-1) in aqueous humour (AH) and optic nerve head (ONH) were determined in the Morrison elevated IOP model of glaucoma. Additionally in the ONH of these rats, immunohistochemical analyses of ET B receptors and glial fibrillary acidic protein (GFAP; a marker for astroglial cells and for astrogliosis) were performed. There was 2-to 2.5-fold increase in AH ir-ET-1 levels for rats subjected to elevated IOP, compared to their respective controls. In the Morrison rat model of glaucoma, elevated IOP increased optic nerve ir-ET-1 with concomitant increases in ir-ET B and ir-GFAP labelling (possibly indicative of astrogliosis and hypertrophy). As seen in brain astrocytes subjected to neurotrauma, the present findings are suggestive of ET-1's role in astroglial activation, particularly in response to elevated IOP in glaucoma.
Abstract BACKGROUND: This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. DESIGN AND SAMPLES: In vitro and ex vivo paradigms involving P6 and P60 Sprague-Dawley rat retinal explants and retinal ganglion cells were employed. METHODS: Postnatal day 6 (P6) and 60 (P60) Sprague-Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187. MAIN OUTCOME MEASURES: Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting. RESULTS: In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague-Dawley rat retina at P60 compared with P6. CONCLUSIONS: We demonstrate that neonatal Sprague-Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.
Effect of Erythropoietin Axotomy-Induced Apoptosis in Rat Retinal Ganglion Cells
Investigative Ophthalmology & Visual Science, 2004
PURPOSE. Erythropoietin (EPO) modulates erythropoiesis by inhibiting apoptosis in erythrocyte progenitors. Recently, EPO has been shown to be protective in experimental models of mechanical trauma, neuroinflammation, cerebral and retinal ischemia, and even in a human stroke trial. However, little is known about EPO signal transduction in vivo and the usefulness of EPO in the prevention of the chronic, purely apoptotic neuronal cell death that contributes to vision loss in glaucoma and the progression of neurodegenerative diseases. METHODS. EPO's effects and signaling in the retinal ganglion cell axotomy paradigm were studied by Western blot analysis and immunohistochemistry, receptor expression was characterized in the retina before and after lesion. EPO was injected into the vitreous body to investigate neuroprotection of axotomized rat RGCs. Moreover, EPO's effects were studied in cultures of immunopurified retinal ganglion cells. Signal-transduction pathways transmitting neuroprotective EPO effects in vivo were characterized by the use of specific kinase inhibitors, immunohistochemistry, and Western blot analysis. RESULTS. EPO receptors (EPORs) were expressed on RGC somata and dendrites in vivo. EPOR expression did not significantly change after axotomy. Application of EPO prevented death of neurotrophic-factor-deprived immunopurified rat RGCs in vitro, rescued axotomized RGCs in vivo, and prevented caspase-3 activation. EPO-induced Akt phosphorylation and survival-promoting EPO effects were completely abolished by inhibition of PI-3-kinase. EPO neuroprotection followed a bell-shaped dose-response curve in vitro and in vivo, whereas toxic EPO effects were never observed, even at high concentrations. CONCLUSIONS. These data support a potential role for EPO as a therapeutic molecule against predominantly apoptotic neuronal cell death in the context of glaucoma or neurodegenerative diseases and delineate the PI-3-K/Akt pathway as the predominant mediator of EPO neuroprotection in this in vivo paradigm of neuronal cell death. (Invest Ophthalmol Vis Sci. 2004;45: 1514 -1522
Molecular vision, 2010
The goal of this study is to determine whether increased optic atrophy type 1 (OPA1) expression protects against retinal ganglion cell (RGC) death in glaucomatous DBA/2J mice. Intraocular pressure in DBA/2J mice was measured, and pre-glaucomatous DBA/2J mice eyes were transfected with recombinant adeno-associated virus serotype 2 (AAV2) constructs including AAV2-wild type (WT) mOPA1 for two months. Increased OPA1 expression was confirmed by western blotting and RGC survival was assessed by retrograde labeling with FluoroGold. In addition, apoptotic cell death and mitochondrial structure were determined in AAV2-WT mOPA1-transfected differentiated RGC-5 cells exposed to elevated hydrostatic pressure (30 mmHg) for three days. WT AAV2-mOPA1 transfection significantly increased 90 kDa and 80 kDa OPA1 isoforms in the retina of glaucomatous DBA/2J mice. OPA1 immunoreactivity was increased in the inner nuclear layer, inner plexiform layer, and ganglion cell layer in nine month-old glaucomat...
Programmed cell death of retinal ganglion cells during experimental glaucoma
Experimental Eye Research, 1995
The death of retinal ganglion cells during glaucoma is thought to result from damage to their axons as they exit the eye through the lamina cribrosa. In this study, intraocular pressure in the rat was increased to twice the normal averge by cauterizing two limbal-derived veins. To investigate whether retinal ganglion cells in the glaucomatous eye follow an apoptotic type of death, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase to the 3′-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in numbers of biotin-labeled nuclei after administration of the protein synthesis inhibitor, cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when the intraocular pressure is markedly increased. This raises new possibilities in the treatment of glaucomatous damage to the retina, by the potential interruptibility of a program for neuronal death.