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Abstract: 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 α1 and collagen type VI α1 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 ETA receptor antagonist, BQ610, as well as with the ETB 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. [Copyright &y& Elsevier]

Abstract: 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 (ETA) and endothelin receptor B (ETB), 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 ETA, ETB 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 ETA selective antagonist BQ610 but not by the ETB specific antagonist BQ788. Desensitization to ETA-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 ETA receptor protein at 1, 10 and 100nM concentrations, while a dose dependent increase in the ETB receptor was observed with a significant increase at 100nM. Quantitative PCR showed a dose-dependent decrease in ETA receptor mRNA levels and an increase in the mRNA levels of ETB 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 ETB selective antagonist, and as well as BQ610, an ETA selective antagonist. These results suggested that human lamina cribrosa cells expressed functional ETA and ETB 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. [Copyright &y& Elsevier]

Abstract: Objective: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. Methods: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) — (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood–retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. Results: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. Conclusion: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy. [Copyright &y& Elsevier]

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro , positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis. • Reactive astrocytosis reduces expression of lysyl oxidase like 1 (Loxl1) in ONHA. • Loxl1 knock-down is sufficient to induce reactive astrocytosis in ONHA. • ONHA-derived exosomes are readily taken up by ONHA and cultured neurons. • ONHA-derived exosomes increase neurite outgrowth of cultured primary neurons. • Exosomes from Loxl1-deficient ONHA have diminished trophic effects on neurons. [ABSTRACT FROM AUTHOR]