Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy

Miyazaki, Kyohei; Hashimoto, Koichi; Sato, Masatoki; Watanabe, Masahiro; Tomikawa, Naoki; Kanno, Shuto; Kawasaki, Yukihiko; Momoi, Nobuo; Hosoya, Mitsuaki

doi:10.1038/pr.2017.28

Cited by 28 publications

(20 citation statements)

References 39 publications

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“…TNF- α is produced by many cell types, including macrophages, lymphocytes, and fibroblasts in response to inflammation, infection, and other stresses [3]. In addition, TNF- α has been reported to be involved in the generation of reactive oxygen species (ROS), hyperpermeability, and apoptosis in vascular endothelial cells [4–6].…”

Section: Introductionmentioning

confidence: 99%

RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells

Sato

Takino

Nagamine

et al. 2019

The Scientific World Journal

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We have identified ras guanyl releasing protein 2 (rasgrp2) as a blood vessel related gene from Xenopus embryo. In addition, we reported that RASGRP2 is also expressed in human umbilical vein endothelial cells (HUVEC). It is known that RASGRP2 activates Ras-related protein 1 (Rap1). However, the function of RASGRP2 in human vascular endothelium remains unknown. Therefore, we performed functional analysis of RASGRP2 using immortalized HUVEC (TERT HUVEC). We established a stable RASGRP2 overexpressing cell line (TERT HUVEC R) and mock cell line (mock). Furthermore, we compared the activity of Rap1 and the generation of intracellular reactive oxygen species (ROS), which is related to cell death, in both cell lines. Significant increase in Rap1 activity was observed in the TERT HUVEC R compared to the mock. Furthermore, apoptosis by tumor necrosis factor-α (TNF-α) stimulation was significantly more reduced in the TERT HUVEC R than in the mock. In the mock, apoptosis induced by TNF-α stimulation was decreased by pretreatment with diphenyleneiodonium (DPI), which is an inhibitor of NADPH oxidase (NOX). However, in the TERT HUVEC R, apoptosis induced by TNF-α stimulation was not reduced after pretreatment of DPI. Furthermore, there was no reduction in ROS production in the TERT HUVEC R after DPI pretreatment. In addition, the difference in the degree of apoptosis induced by TNF-α stimulation in both cell lines was consistent with the difference in ROS production in the cell lines. From these results, it was suggested that RASGRP2 activates Rap1 and the activated Rap1 suppresses apoptosis via NOX inhibition.

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Section: Introductionmentioning

confidence: 99%

RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells

Sato

Takino

Nagamine

et al. 2019

The Scientific World Journal

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“…In addition, S1PC inhibited endothelial hyperpermeability induced by 1 U/mL thrombin (Additional file 2: Figure S2), that is a serine protease well known for its endothelial hyperpermeability effect [43,44]. Since the disruption of AJ and TJ proteins causes vascular hyperpermeability [1,9,10,45], we investigated the effect of S1PC on the amount of these proteins in the membrane fraction of HUVECs. As shown in Fig.…”

Section: S1pc Suppressed Endothelial Hyperpermeability By Improving Tmentioning

confidence: 99%

S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway

et al. 2021

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Background Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway. Methods Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

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“…After that, we added FITC-conjugated secondary antibody (1: 1000; eBioscience; USA) for 1 hour at RT. After PBS wash, the nuclei were stained with 1 µg/ml for 30 seconds [17].…”

Section: Measuring the Levels Of In Ammatory Cytokinesmentioning

confidence: 99%

Effect of docosahexaenoic acid plus insulin on atherosclerotic human endothelial cells

Abriz

Rahbarghazi‬

Nourazarian

et al. 2020

Preprint

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Background: Atherosclerosis is touted as one of the most critical consequences of diabetes mellitus indicated by local inflammation of endothelial cells. The Effect of Omega 3 fatty acids, mainly docosahexaenoic acid (DHA), has been investigated in cells after exposure to high doses of lipids. The current experiment aimed to address the modulatory effects of docosahexaenoic acid and insulin in palmitic-treated human endothelial cells. Methods: Human umbilical vein endothelial cells were treated with 1mM palmitic acid, 50μM insulin, 50μM docosahexaenoic acid, and their combination for 48 hours. Cell survival rate and apoptosis were measured using MTT and flow cytometry assays. The Griess assay detected NO levels. Protein levels of TNF-α, IL-6, and NF-κB were studied using ELISA and immunofluorescence imaging. The expression of genes participating in atherosclerosis was monitored using PCR array analysis. Results: Oil Red O staining showed the inhibitory effect of DHA and insulin to reduce the intracellular accumulation of palmitic acid. Both DHA and Insulin blunted palmitic acid detrimental effects on HUVECs indicated by an increased survival rate (p<0.05). The percent of apoptotic cells was decreased in palmitic-treated cells received insulin and DHA compared to palmitic-treated group (p<0.05). Based on our data, DHA and Insulin diminished the production of all inflammatory cytokines, TNF-α, IL-6, and NF-κB, in palmitic-treated cells (p<0.05). Similar to these data, NO production was also decreased in all groups treated with insulin and DHA compared to the palmitic-treated cells (p<0.05). PCR array analysis revealed the modulatory effect of DHA and insulin on the expression of atherosclerosis-related genes pre-treated with palmitic acid compared to the control group (p<0.05). Conclusion: DHA and Insulin could alter the dynamic growth and dysfunctional activity of human endothelial cells after treatment with palmitic acid. Taken together, Omega 3 fatty acids, along with insulin, could dictate specific cell behavior in endothelial cells in vitro

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Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy

Cited by 28 publications

References 39 publications

RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells

RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells

S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway

Effect of docosahexaenoic acid plus insulin on atherosclerotic human endothelial cells

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