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1.
Exp Eye Res ; 240: 109813, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38331016

RESUMEN

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.


Asunto(s)
Exosomas , Glaucoma , Disco Óptico , Ratas , Animales , Disco Óptico/metabolismo , Proteína-Lisina 6-Oxidasa/genética , Astrocitos/metabolismo , Exosomas/metabolismo , Gliosis/metabolismo , Glaucoma/metabolismo , Elastina/genética , Inflamación/metabolismo
2.
Artículo en Inglés | MEDLINE | ID: mdl-38935528

RESUMEN

Purpose: Antibody-drug conjugates (ADCs) are a relatively recent advance in the delivery of chemotherapeutics that improve targeting of cytotoxic agents. However, despite their antitumor activity, severe ocular adverse effects, including vision loss, have been reported for several ADCs. The nonspecific uptake of ADCs into human corneal epithelial cells (HCECs) and their precursors via macropinocytosis has been proposed to be the primary mechanism of ocular toxicity. In this study, we evaluated the ability of a novel polymer, poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), to decrease the ADC rituximab-mc monomethylauristatin F (MMAF) (RIX) uptake into human corneal epithelial (HCE-T) cells. Methods: HCE-T cells were exposed to increasing concentrations of RIX to determine inhibition of cell proliferation. HCE-T cells were treated with PLL-g-PEG, the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA), or vehicle. After 30 min of incubation, RIX was added. ADC was detected by fluorescent anti-human immunoglobulin G and fluorescently conjugated dextran as viewed by microscopy. Results: RIX caused dose-dependent inhibition of HCE-T cell proliferation. EIPA significantly reduced RIX uptake and decreased macropinocytosis as assessed by direct quantification of RIX using a fluorescently conjugated anti-human antibody as well as quantification of macropinocytosis using fluorescently conjugated dextran. PLL-g-PEG resulted in a dose-dependent inhibition of RIX uptake with half-maximal inhibitory concentrations of 0.022%-0.023% PLL-g-PEG. Conclusion: The data show PLL-g-PEG to be a potent inhibitor of RIX uptake by corneal epithelial cells and support its use as a novel therapeutic approach for the prevention of ocular adverse events associated with ADC therapy.

3.
J Ocul Pharmacol Ther ; 39(5): 303-316, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37253141

RESUMEN

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.


Asunto(s)
Antioxidantes , Síndromes de Ojo Seco , Humanos , Ratones , Animales , Antioxidantes/farmacología , Neuropatía Óptica Tóxica/patología , Ratones Endogámicos C57BL , Córnea , Estrés Oxidativo , Síndromes de Ojo Seco/metabolismo , Lágrimas/metabolismo , Fluoresceína/metabolismo , Consumo de Bebidas Alcohólicas/efectos adversos , Etanol/toxicidad , Dieta
4.
Antioxidants (Basel) ; 9(4)2020 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-32316287

RESUMEN

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

5.
Ocul Surf ; 17(2): 257-264, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30807830

RESUMEN

PURPOSE: To determine the efficacy of the superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro in human corneal epithelial (HCE-T) cells and in vivo in a preclinical mouse model for dry-eye disease (DED). METHODS: In vitro, HCE-T cultures were exposed either to tert-butylhydroperoxide (tBHP) to generate oxidative stress or to hyperosmolar conditions modeling cellular stress during DED. Cells were pre-treated with Mn-TM-2-PyP or vehicle. Mn-TM-2-PyP permeability across stratified HCE-T cells was assayed. In vivo, Mn-TM-2-PyP (0.1% w/v in saline) was delivered topically as eye drops in a desiccating stress/scopolamine model for DED. Preclinical efficacy was compared to untreated, vehicle- and ophthalmic cyclosporine emulsion-treated mice. RESULTS: Mn-TM-2-PyP protected HCE-T cells in a dose-dependent manner against tBHP-induced oxidative stress as determined by calculating the IC50 for tBHP in the resazurin, MTT and lactate dehydrogenase release cell viability assays. Mn-TM-2-PyP did not protect HCE-T cells from hyperosmolar insult. Its permeability coefficient across a barrier of HCE-T cells was 1.1 ±â€¯0.05 × 10-6 cm/s and the mass balance was 62 ±â€¯0.6%. In vivo, topical dosing with Mn-TM-2-PyP resulted in a statistically significant reduction of corneal fluorescein staining, similar to ophthalmic cyclosporine emulsion. Furthermore, Mn-TM-2-PyP significantly reduced leukocyte infiltration into lacrimal glands and prevented degeneration of parenchymal tissue. No protective effect against loss of conjunctival goblet cells was observed. Notably, Mn-TM-2-PyP did not produce ocular toxicity when administered topically. DISCUSSION: Our data suggest that Mn-TM-2-PyP, a prototypic synthetic metalloporphyrin compound with potent catalytic antioxidant activity, can improve signs of DED in vivo by reducing oxidative stress in corneal epithelial cells.


Asunto(s)
Síndromes de Ojo Seco/tratamiento farmacológico , Células Caliciformes/patología , Metaloporfirinas/administración & dosificación , Estrés Oxidativo , Animales , Antioxidantes , Recuento de Células , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Células Caliciformes/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Soluciones Oftálmicas/administración & dosificación , Índice de Severidad de la Enfermedad
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