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Protection of cultured brain endothelial cells from cytokine-induced damage by ?-melanocyte stimulating hormone.

ABSTRACT: The blood-brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of ?-melanocyte stimulating hormone (?-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which ?-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-? and IL-1?, we hypothesized a protective effect of ?-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide ?-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-? and IL-1? induced cell damage, measured by impedance and MTT assay, which was attenuated by ?-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and ?-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-?B were also reduced by ?-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of ?-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB.

SUBMITTER: Harazin A

PROVIDER: S-EPMC5958884 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone.

Harazin András A Bocsik Alexandra A Barna Lilla L Kincses András A Váradi Judit J Fenyvesi Ferenc F Tubak Vilmos V Deli Maria A MA Vecsernyés Miklós M

PeerJ 20180515

The blood-brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Ba ...[more]

PMID: 29780671

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Project description:ImportanceCorneal endothelial cell (CEnC) damage and loss are major issues in eye banking and transplantation. The underlying mechanisms for CEnC loss are incompletely understood, and cytoprotective strategies that enhance CEnC viability could have a major effect on donor tissue quality and graft survival.ObjectiveTo investigate the cytoprotective role of neuropeptide α-melanocyte-stimulating hormone (α-MSH) in preventing CEnC loss in eye bank cold-stored corneas under oxidative and inflammatory cytokine-induced stress.Design, setting, and participantsThis single-center comparative research study conducted ex vivo experiments using 16 pairs of research-grade human donor corneas (courtesy of Eversight Eye Bank). Data were collected from June 2018 to November 2019, and data were analyzed from December 2019 to January 2020.ExposuresTwo corneas from the same donor were randomized to either control or 0.1 mmol/L of α-MSH treatment and then subjected to oxidative stress (1.4 mmol/L of hydrogen peroxide-phosphate-buffered saline for 15 minutes at 37 °C; n = 8 pairs) or cytokine-induced stress (100 ng/mL of tumor necrosis factor-α and 100 ng/mL of interferon γ for 18 hours at 37 °C; n = 8 pairs). Corneas were then stored at 4 °C. Specular images were taken at baseline and repeated twice per week using a calibrated wide-field specular microscope. CEnC viability was assessed using a fluorescent live/dead viability assay.Main outcome and measuresEndothelial morphometry analysis, central corneal thickness measurements, and percentage of dead cells at day 11.ResultsOf 16 donors who provided corneas, 9 (56%) were male, and the mean (SD) age was 57.9 (12.4) years. Corneas were paired, and baseline parameters were comparable between all groups. At all time points, CEnC loss was lower in the α-MSH groups compared with the control groups. This difference was statistically significant after cytokine-induced stress (20.2% vs 35.2%; sample estimate of median, -14.9; 95% CI, -23.6 to -6.3; P = .008). Compared with the control group, α-MSH treatment resulted in a smaller increase in central corneal thickness (cytokine-induced stress: 89.3 μm vs 169.8 μm; sample estimate of median, -84.9; 95% CI, -131.5 to -41.6; P = .008; oxidative stress: 43.6 μm vs 111.9 μm; sample estimate of median, -68.8; 95% CI, -100.0 to -34.5; P = .008) and a smaller proportion of cell death (cytokine-induced stress: 2.7% vs 10.4%; difference, -7.7; 95% CI, -13.1 to -2.4; P = .01; oxidative stress: 2.9% vs 12.4%; difference, 9.5; 95% CI, 5.1 to 13.9; P = .006).Conclusions and relevanceIn this study, α-MSH treatment attenuated CEnC loss during cold storage after acute oxidative and cytokine-induced stress in human eye bank cold-stored corneas. These data suggest that supplementation of corneal storage solution with α-MSH may positively affect CEnC survival after transplant and protect the endothelium from proinflammatory cytokines and oxidative stress after full-thickness or endothelial keratoplasty, which is particularly valuable in patients at high risk of graft failure.

Project description:AimsOxidative stress and apoptosis are among the earliest lesions of diabetic retinopathy. This study sought to examine the anti-oxidative and anti-apoptotic effects of α-melanocyte-stimulating hormone (α-MSH) in early diabetic retinas and to explore the underlying mechanisms in retinal vascular endothelial cells.MethodsSprague-Dawley rats were injected intravenously with streptozocin to induce diabetes. The diabetic rats were injected intravitreally with α-MSH or saline. At week 5 after diabetes, the retinas were analyzed for reactive oxygen species (ROS) and gene expression. One week later, the retinas were processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and transmission electron microscopy. Retinal vascular endothelial cells were stimulated by high glucose (HG) with or without α-MSH. The expression of Forkhead box O genes (Foxos) was examined through real-time PCR. The Foxo4 gene was overexpressed in endothelial cells by transient transfection prior to α-MSH or HG treatment, and oxidative stress and apoptosis were analyzed through CM-H2DCFDA and annexin-V assays, respectively.ResultsIn diabetic retinas, the levels of H2O2 and ROS and the total anti-oxidant capacity were normalized, the apoptotic cell number was reduced, and the ultrastructural injuries were ameliorated by α-MSH. Treatment with α-MSH also corrected the aberrant changes in eNOS, iNOS, ICAM-1, and TNF-α expression levels in diabetic retinas. Furthermore, α-MSH inhibited Foxo4 up-regulation in diabetic retinas and in endothelial cells exposed to HG, whereas Foxo4 overexpression abrogated the anti-oxidative and anti-apoptotic effects of α-MSH in HG-stimulated retinal vascular endothelial cells.Conclusionsα-MSH normalized oxidative stress, reduced apoptosis and ultrastructural injuries, and corrected gene expression levels in early diabetic retinas. The protective effects of α-MSH in retinal vascular endothelial cells may be mediated through the inhibition of Foxo4 up-regulation induced by HG. This study suggests an α-MSH-mediated potential intervention approach to early diabetic retinopathy and a novel regulatory mechanism involving Foxo4.

Dataset Information

Protection of cultured brain endothelial cells from cytokine-induced damage by ?-melanocyte stimulating hormone.

Publications

Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone.

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