Browsing by Subject "ET-1"
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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 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.