Browsing by Subject "endothelin-1"
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Item Characterization and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells(1999-01-01) Finkley, Alvin; Thomas Yorio; S. Dan Dimitrijevich; Victoria J. RudickFinkley, Alvin, Characterization and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells. Master of Science (Biomedical Sciences). Endothelins (ETs) are potent vasoactive peptides, that are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-converting enzyme (ECE). Although the role of ocular ET’s are uncertain, ETs have been shown to lower the intraocular pressure. In the current study, ET-1 and Big-ET-1 were detected in SV-40 transformed human ciliary epithelial (HNPE) cells by immunofluorescence suggesting the presence of ECE activity. The presence of ECE was confirmed by Western blotting using polyclonal antibodies against ECE-1 which detected a 124 KDa protein in the membrane fraction and not in the cytosol. Further characterization of the enzymatic activity of ECE (conversion of Big ET-1 to ET-1) was performed using a novel assay involving 121I-Big ET-1 (substrate; 2fmloe) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as the ratio of 121^1-ET-1 produced to the total 125^I-Big ET-1 incubated X 100) was measured and corresponded to: 26% (0.5 3±0.02 fmole, 1 hr), 63% (1.26±0.07 fmole, 3hr) and 66% (1.33±0.11 fmole, 24 hr) compared to blank controls at 13% (0.25±0.03 fmole). Thiorphan (2mM), an inhibitor of ECE, abolished ECE-1 activity. These results suggest that ECE-1 is localized in HNPE cells and is essential for the production of ET-1. The physiological importance of the proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure. Key Words: endothelin converting enzyme-1; endothelin-1; Big endothelin-1; ciliary epithelium; aqueous humor dynamics; intraocular pressure, Western blotting, ECE-1Item 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 Endothelin-1-Induced Proliferation of Human Optic Nerve Head Astrocytes Under Hypoxia(2003-11-01) Desai, Devashish; Thomas Yorio; Ganesh Prasanna; Clark, Abbot F.Desai, Devashish, Endothelin-1-Induced Proliferation of Cultured Human Optic Nerve Head Astrocytes under Hypoxia. Master of Science (Biomedical Sciences). Purpose: Optic nerve head astrocytes (ONAs) normally support and protect the axons of retinal ganglion cells exiting the eye. Along with effects related to elevated intraocular pressure (IOP), proliferation and activation of ONAs, known as ‘astrogliosis’, is also thought to contribute to the pathophysiology of glaucoma by distributing axonal transport and preventing axon regeneration. Concentrations of endothelin-1 (ET-1) are elevated in glaucomatous eyes and in animal models for glaucoma. ET-1 injection into the eye causes reduction of ocular blood flow. ET-1 causes a time-dependent proliferation of human ONAs. Tumor necrosis factor-α (TNF-α), a cytokine, which is also elevated in glaucomatous optic nerve head, promotes ET-1 release from ocular cells and could potentially stimulate ET-1 secretion from the ONAs. Hypoxia resulting from ischemia, which is produced by the elevation of IOP or vasospasm in the retinal vasculature, is considered a significant factor contributing to the stress as the glaucomatous optic nerve head. Methods: Concentrations of ET-1 secreted by hONAs into cell culture media after hypoxia and TNF-α treatment was measured using an enzyme-linked immunosorbent assay (ELISA). Proliferation of hONAs was measured using a proliferation assay (formazan assay), performed at the end of various time periods of incubation with TNPα and ET-1 under normoxia or hypoxia. The involvement of mitogen activated protein kinase (MAPK) in hONA proliferation was examined using MAPK inhibitors and Western blot analyses. Results: Cell culture media collected from hONAs after 24-hour hypoxia with concurrent TNF-α treatment showed a 500% increase in the irET-1. Under normoxia, both TNF-α and ET-1 caused moderate proliferation of hONAs. Under hypoxia, TNF-α-induced proliferation was greatly increased. Conclusion: Hypoxia augments TNF-a and ET-1 growth of optic nerve head astrocytes, by way of increasing ET-1 synthesis and release as well as mitogenesis. Therefore reactive ONAs could be the common denominator underlying optic nerve damage in glaucoma since their localization makes them susceptible to mechanistic and ischemic influences in addition to influences of ET-1 and TNF-α. Keywords: astrocyte; endothelin-1; tumor-necrosis factor-α; hypoxia; proliferation; astrogliosis; glaucoma; optic nerveItem Regulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood Retinal Barrier(2003-08-28) Narayan, Santosh; Thomas Yorio; Glenn Dillon; Michael W. MartinRegulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood-Retinal Barrier. Santosh Narayan, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center Fort Worth, TX 76102. Summary The retinal pigment epithelium (RPE) constitutes the outer blood retinal barrier at the posterior segment of the eye. The RPE provides metabolic support to the photoreceptors in the neural retina. A breakdown in the barrier supported by RPE is a hallmark in several retinopathies including proliferative vitreoretinopathy, choroidal neovascularization and macular edema. Characteristic to all epithelial cells, mature RPE cells display a polarized phenotype both in culture (ARPE-19 cells) and in vivo, with specific apical and basolateral domains. This provides a testable model to study the RPE in vitro. The purpose of this study was to characterize the RPE as a source for endothelin-1, using both in vitro and in situ models. Endothelins (ET-1,-2, and -3) are known regulators of vascular tone, that are produced at sites close to their target, ET-1, being a potent vasoconstrictor may be involved in regulating blood supply to the choroid and the neural retina. We identified the RPE to be a major source for endothelin-1 (ET-1) in situ in the human retina as well as in pigmented and albino rat retinas. Additionally, using a cell-culture model of mature polarized ARPE-19 cells, we studied the synthesis and expression of ET-1 in response to muscarinic receptor stimulation, TNF-α and more recently to thrombin. We have identified other components involved in the synthesis and turnover of ET-1 in ARPE-19 cells including the proprotein convertase-furin, endothelin-converting enzyme-1 and its isoforms and the endothelin receptor B subtype. ARPE-19 cells grown on collagen filters helped determine if secretion of ET-1 was polarized or discriminative towards either the apical or basolateral surface. We consistently observed changes in cell shape and tight junction disassembly in ARPE-19 cells following TNF-α and thrombin addition. Additionally, thrombin caused an increase in preproET-1 mRNA at earlier time points that was dependent on the rhokinase (ROCK1/2) pathway. We report a novel signaling mechanism for regulating preproET-1 mRNA and mature ET-1 secretion in ARPE-19 cells that involves the thrombin receptor (protease activated receptor-1/PAR-1) dependent activation of the rho/ROCK1/2 signaling pathway that may also be involved in thrombin induced changes in the cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. ET-1 secreted by the RPE, under physiological conditions may provide an autoregulatory mechanism for controlling blood flow at the outer blood retinal barrier. Excessive ET-1 secretion following breakdown of the barrier may either promote wound repair or may mediate further damage to the retina, the substrates of which are presently unknown. Future experimental approaches are planned to address these possibilities.