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Endothelin-1 (ET-1), a potent vaso-active peptide, mediates extracellular matrix regulation resulting in an increase in collagen deposition in various cell types and tissues and has been proposed to play a key role in glaucoma pathology. The role of ET-1 in the regulation of extracellular matrix collagens at the level of optic nerve head is not known. In this study we have examined the role of ET-1 in extracellular matrix collagen regulation in primary cultures of human lamina cribrosa cells. Our hypothesis is that ET-1 increases remodeling of the ECM of cells of the lamina cribrosa. Such actions could contribute to the development of optic neuropathy. QPCR analysis revealed that ET-1 mediated an increase in mRNA levels of collagen type I alpha1 and collagen type VI alpha1 chains at all doses of ET-1 with a significant increase at 1nM and 10nM concentration in LC cells. A dose-dependent increase in collagen type I and type VI protein deposition and secretion was also observed by Western blot in response to ET-1 and was significant at 10nM and 100nM concentrations of ET-1. ET-1 increased the [3H] proline uptake in LC cells suggesting that ET-1 contributed to an increase in total collagen synthesis in LC cells. ET-1-mediated increase in collagen type I, type VI and total collagen synthesis was significantly blocked by the ET(A) receptor antagonist, BQ610, as well as with the ET(B) receptor antagonist, BQ788, suggesting the involvement of both receptor subtypes in ET-1 mediated collagen synthesis in LC cells. These results suggest that ET-1 regulates extracellular matrix collagen synthesis in LC cells and may contribute to ECM remodeling at the level of LC of POAG subjects who have elevated plasma and aqueous humor levels of endothelin-1.

Primary open angle glaucoma (POAG) is a progressive optic neuropathy, characterized, in part by extensive extra cellular matrix remodeling and collapse of the lamina cribrosa (LC). Endothelin-1 (ET-1), a potent vasoactive peptide and its receptors, endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)), have been implicated in glaucomatous optic neuropathy. In this study we examined the expression of ET-1 and its receptors in GFAP negative LC cells. RT-PCR analysis revealed that LC cells express both ET(A), ET(B) receptors and prepro- ET-1, the primary gene transcript of ET-1. A dose-dependent increase in intra-cellular calcium concentrations was observed in the presence of 1, 10 and 100nM ET-1. Increased intracellular calcium concentrations were blocked by the ET(A) selective antagonist BQ610 but not by the ET(B) specific antagonist BQ788. Desensitization to ET(A)-mediated increase in intracellular calcium was observed in LC cells following pre-treatment with ET-1 for 24h. Western blot analysis of LC cells treated with ET-1 for 24h revealed a decreased expression of ET(A) receptor protein at 1, 10 and 100nM concentrations, while a dose dependent increase in the ET(B) receptor was observed with a significant increase at 100nM. Quantitative PCR showed a dose-dependent decrease in ET(A) receptor mRNA levels and an increase in the mRNA levels of ET(B) receptors. A Griess colorimetric assay was used to measure the NO released from LC cells and ET-1 induced a dose-dependent increase in NO release which was significant at 100nM concentration. ET-1 induced NO release was significantly blocked by BQ788, an ET(B) selective antagonist, and as well as BQ610, an ET(A) selective antagonist. These results suggested that human lamina cribrosa cells expressed functional ET(A) and ET(B) receptors and their expression and function was altered in response to prolong exposure to ET-1. This may have an implication in the normal physiology of LC cells and in POAG subjects where elevated levels of ET-1 could impact LC function.

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 ETB 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 ETB-receptor antagonist BQ788 (1 μmol/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 ETB-receptor expression. These studies suggest that ocular ET-1 acts through ETB receptors to mediate apoptosis of retinal ganglion cells, a key event in glaucoma and related optic neuropathies. De récentes observations conduisent à penser que le peptide vasoactif, endothéline-1 (ET-1), pourrait jouer un rôle un important dans l’étiologie du glaucome. L’administration d’ET-1 a entraîné une perte axonale dans le nerf optique et l’apoptose des cellules ganglionnaires rétiniennes (CGR). Les taux d’ET-1 oculaires sont augmentés dans l’humeur aqueuse et en réponse à une augmentation de la PIO, tant chez les patients atteints de glaucome que chez les modèles animaux de glaucome. Toutefois, les mécanismes précis par lesquels l’ET-1 médie la neuropathie optique glaucomateuse ne sont pas clairs. Dans le présent travail, nous soulignons que l’apoptose véhiculée par l’ET-1 est nettement atténuée chez les rats déficients en récepteurs ETB, ce qui laisse supposer que les récepteurs ETB jouent un rôle clé dans l’apoptose des CGR induite par le traitement à l’ET-1. Nous avons démontré, en utilisant des cellules ganglionnaires rétiniennes de rats transformées par un virus (cellules CGR-5), qu’un traitement à l’ET-1 (100 nmol/L) provoque des modifications liées à l’apoptose dans ces cellules, déterminées par la cytométrie en flux, la libération du cytochrome c mitochondrial dans le cytosol et l’augmentation de la phosphorylation de la c-Jun N-terminale kinase. Un prétraitement avec l’antagoniste des récepteurs ETB, BQ788 (1 μmol/L), a permis d’atténuer de manière significative l’apoptose véhiculée par l’ET-1 dans les cellules CGR-5. Les modifications apoptotiques induites par l’ET-1 dans ces cellules ont été associées à l’activation des récepteurs ETB et ont été accompagnées par une augmentation significative de leur expression. Ces résultats donnent à penser que l’ET-1 oculaire agit par l’intermédiaire des récepteurs ETB pour médier l’apoptose des cellules ganglionnaires rétiniennes, un événement clé dans le glaucome et les neuropathies optiques connexes. [ABSTRACT FROM AUTHOR]