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Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability

BACKGROUND: Lipopolysaccharide (LPS) was shown to induce an increase in caveolin-1 (Cav-1) expression in endothelial cells; however, the mechanisms regarding this response and the consequences on caveolae-mediated transcellular transport have not been completely investigated. This study aims to inve...

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Autores principales: Wang, Nan, Zhang, Dan, Sun, Gengyun, Zhang, Hong, You, Qinghai, Shao, Min, Yue, Yang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Dove Medical Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560510/
https://www.ncbi.nlm.nih.gov/pubmed/26357463
http://dx.doi.org/10.2147/DDDT.S77646
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author Wang, Nan
Zhang, Dan
Sun, Gengyun
Zhang, Hong
You, Qinghai
Shao, Min
Yue, Yang
author_facet Wang, Nan
Zhang, Dan
Sun, Gengyun
Zhang, Hong
You, Qinghai
Shao, Min
Yue, Yang
author_sort Wang, Nan
collection PubMed
description BACKGROUND: Lipopolysaccharide (LPS) was shown to induce an increase in caveolin-1 (Cav-1) expression in endothelial cells; however, the mechanisms regarding this response and the consequences on caveolae-mediated transcellular transport have not been completely investigated. This study aims to investigate the role of LPS-induced Cav-1 phosphorylation in pulmonary microvascular permeability in pulmonary microvascular endothelial cells (PMVECs). METHODS: Rat PMVECs were isolated, cultured, and identified. Endocytosis experiments were employed to stain the nuclei by DAPI, and images were obtained with a fluorescence microscope. Permeability of endothelial cultures was measured to analyze the barrier function of endothelial monolayer. Western blot assay was used to examine the expression of Cav-1, pCav-1, triton-insoluble Cav-1, and triton-soluble Cav-1 protein. RESULTS: The LPS treatment induced phosphorylation of Cav-1, but did not alter the total Cav-1 level till 60 min in both rat and human PMVECs. LPS treatment also increased the triton-insoluble Cav-1 level, which peaked 15 min after LPS treatment in both rat and human PMVECs. LPS treatment increases the intercellular cell adhesion molecule-1 expression. Src inhibitors, including PP2, PP1, Saracatinib, and Quercetin, partially inhibited LPS-induced phosphorylation of Cav-1. In addition, both PP2 and caveolae disruptor MβCD inhibited LPS-induced increase of triton-insoluble Cav-1. LPS induces permeability by activating interleukin-8 and vascular endothelial growth factor and targeting other adhesion markers, such as ZO-1 and occludin. LPS treatment also significantly increased the endocytosis of albumin, which could be blocked by PP2 or MβCD. Furthermore, LPS treatment for 15 min significantly elevated Evans Blue-labeled BSA transport in advance of a decrease in transendothelial electrical resistance of PMVEC monolayer at this time point. After LPS treatment for 30 min, transendothelial electrical resistance decreased significantly. Moreover, PP2 and MβCD blocked LPS-induced increase in Evans Blue-labeled BSA level. CONCLUSION: Our study demonstrates that LPS-induced Cav-1 phosphorylation may lead to the increase of transcellular permeability prior to the increase of paracellular permeability in a Src-dependent manner. Thus, LPS-induced Cav-1 phosphorylation may be a therapeutic target for the treatment of inflammatory lung disease associated with elevated microvascular permeability.
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spelling pubmed-45605102015-09-09 Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability Wang, Nan Zhang, Dan Sun, Gengyun Zhang, Hong You, Qinghai Shao, Min Yue, Yang Drug Des Devel Ther Original Research BACKGROUND: Lipopolysaccharide (LPS) was shown to induce an increase in caveolin-1 (Cav-1) expression in endothelial cells; however, the mechanisms regarding this response and the consequences on caveolae-mediated transcellular transport have not been completely investigated. This study aims to investigate the role of LPS-induced Cav-1 phosphorylation in pulmonary microvascular permeability in pulmonary microvascular endothelial cells (PMVECs). METHODS: Rat PMVECs were isolated, cultured, and identified. Endocytosis experiments were employed to stain the nuclei by DAPI, and images were obtained with a fluorescence microscope. Permeability of endothelial cultures was measured to analyze the barrier function of endothelial monolayer. Western blot assay was used to examine the expression of Cav-1, pCav-1, triton-insoluble Cav-1, and triton-soluble Cav-1 protein. RESULTS: The LPS treatment induced phosphorylation of Cav-1, but did not alter the total Cav-1 level till 60 min in both rat and human PMVECs. LPS treatment also increased the triton-insoluble Cav-1 level, which peaked 15 min after LPS treatment in both rat and human PMVECs. LPS treatment increases the intercellular cell adhesion molecule-1 expression. Src inhibitors, including PP2, PP1, Saracatinib, and Quercetin, partially inhibited LPS-induced phosphorylation of Cav-1. In addition, both PP2 and caveolae disruptor MβCD inhibited LPS-induced increase of triton-insoluble Cav-1. LPS induces permeability by activating interleukin-8 and vascular endothelial growth factor and targeting other adhesion markers, such as ZO-1 and occludin. LPS treatment also significantly increased the endocytosis of albumin, which could be blocked by PP2 or MβCD. Furthermore, LPS treatment for 15 min significantly elevated Evans Blue-labeled BSA transport in advance of a decrease in transendothelial electrical resistance of PMVEC monolayer at this time point. After LPS treatment for 30 min, transendothelial electrical resistance decreased significantly. Moreover, PP2 and MβCD blocked LPS-induced increase in Evans Blue-labeled BSA level. CONCLUSION: Our study demonstrates that LPS-induced Cav-1 phosphorylation may lead to the increase of transcellular permeability prior to the increase of paracellular permeability in a Src-dependent manner. Thus, LPS-induced Cav-1 phosphorylation may be a therapeutic target for the treatment of inflammatory lung disease associated with elevated microvascular permeability. Dove Medical Press 2015-08-28 /pmc/articles/PMC4560510/ /pubmed/26357463 http://dx.doi.org/10.2147/DDDT.S77646 Text en © 2015 Wang et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.
spellingShingle Original Research
Wang, Nan
Zhang, Dan
Sun, Gengyun
Zhang, Hong
You, Qinghai
Shao, Min
Yue, Yang
Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title_full Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title_fullStr Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title_full_unstemmed Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title_short Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
title_sort lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560510/
https://www.ncbi.nlm.nih.gov/pubmed/26357463
http://dx.doi.org/10.2147/DDDT.S77646
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