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Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions
BACKGROUND: Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9843922/ https://www.ncbi.nlm.nih.gov/pubmed/36647012 http://dx.doi.org/10.1186/s11658-022-00413-w |
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author | Adzraku, Seyram Yao Wang, Guozhang Cao, Can Bao, Yurong Wang, Yizhou Smith, Alhaji Osman Du, Yuwei Wang, Haiyang Li, Yue Xu, Kailin Qiao, Jianlin Ju, Wen Zeng, Lingyu |
author_facet | Adzraku, Seyram Yao Wang, Guozhang Cao, Can Bao, Yurong Wang, Yizhou Smith, Alhaji Osman Du, Yuwei Wang, Haiyang Li, Yue Xu, Kailin Qiao, Jianlin Ju, Wen Zeng, Lingyu |
author_sort | Adzraku, Seyram Yao |
collection | PubMed |
description | BACKGROUND: Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable to overcome the preconditioning damages. This study aims to ascertain the role of Roundabout 4 (Robo4) in regulating irradiation-induced damage to the endothelium. METHODS: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. Robo4 expression in the endothelial cells was manipulated employing lentiviral-mediated RNAi and gene overexpression technology before irradiation treatment. The permeability of endothelial cells was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules, adherens junctions, tight junctions, and gap junctions. Using Transwell endothelial monolayer staining, FITC-Dextran permeability, and gap junction-mediated intercellular communication (GJIC) assays, we determined the changes in endothelial functions after Robo4 gene manipulation and irradiation. Moreover, we measured the proportion of CD31 expression in endothelial cells by flow cytometry. We analyzed variations between two or multiple groups using Student’s t-tests and ANOVA. RESULTS: Ionizing radiation upregulates Robo4 expression but disrupts endothelial junctional molecules. Robo4 deletion causes further degradation of endothelial junctions hence increasing the permeability of the endothelial cell monolayer. Robo4 knockdown in microvascular endothelial cells increases the degradation and delocalization of ZO-1, PECAM-1, occludin, and claudin-5 molecules after irradiation. Conversely, connexin 43 expression increases after silencing Robo4 in endothelial cells to induce permeability but are readily destroyed when exposed to 10 Gy of gamma radiation. Also, Robo4 knockdown enhances Y731-VE-cadherin phosphorylation leading to the depletion and destabilization of VE-cadherin at the endothelial junctions following irradiation. However, Robo4 overexpression mitigates irradiation-induced degradation of tight junctional proteins and stabilizes claudin-5 and ZO-1 distribution. Finally, the enhanced expression of Robo4 ameliorates the irradiation-induced depletion of VE-cadherin and connexin 43, improves the integrity of microvascular endothelial cell junctions, and decreases permeability. CONCLUSION: This study reveals that Robo4 maintains microvascular integrity after radiation preconditioning treatment by regulating endothelial permeability and protecting endothelial functions. Our results also provided a potential mechanism to repair the bone marrow vascular niche after irradiation by modulating Robo4 expression. |
format | Online Article Text |
id | pubmed-9843922 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-98439222023-01-18 Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions Adzraku, Seyram Yao Wang, Guozhang Cao, Can Bao, Yurong Wang, Yizhou Smith, Alhaji Osman Du, Yuwei Wang, Haiyang Li, Yue Xu, Kailin Qiao, Jianlin Ju, Wen Zeng, Lingyu Cell Mol Biol Lett Research Letter BACKGROUND: Hematopoietic stem cell transplantation involves irradiation preconditioning which causes bone marrow endothelial cell dysfunction. While much emphasis is on the reconstitution of hematopoietic stem cells in the bone marrow microenvironment, endothelial cell preservation is indispensable to overcome the preconditioning damages. This study aims to ascertain the role of Roundabout 4 (Robo4) in regulating irradiation-induced damage to the endothelium. METHODS: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. Robo4 expression in the endothelial cells was manipulated employing lentiviral-mediated RNAi and gene overexpression technology before irradiation treatment. The permeability of endothelial cells was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules, adherens junctions, tight junctions, and gap junctions. Using Transwell endothelial monolayer staining, FITC-Dextran permeability, and gap junction-mediated intercellular communication (GJIC) assays, we determined the changes in endothelial functions after Robo4 gene manipulation and irradiation. Moreover, we measured the proportion of CD31 expression in endothelial cells by flow cytometry. We analyzed variations between two or multiple groups using Student’s t-tests and ANOVA. RESULTS: Ionizing radiation upregulates Robo4 expression but disrupts endothelial junctional molecules. Robo4 deletion causes further degradation of endothelial junctions hence increasing the permeability of the endothelial cell monolayer. Robo4 knockdown in microvascular endothelial cells increases the degradation and delocalization of ZO-1, PECAM-1, occludin, and claudin-5 molecules after irradiation. Conversely, connexin 43 expression increases after silencing Robo4 in endothelial cells to induce permeability but are readily destroyed when exposed to 10 Gy of gamma radiation. Also, Robo4 knockdown enhances Y731-VE-cadherin phosphorylation leading to the depletion and destabilization of VE-cadherin at the endothelial junctions following irradiation. However, Robo4 overexpression mitigates irradiation-induced degradation of tight junctional proteins and stabilizes claudin-5 and ZO-1 distribution. Finally, the enhanced expression of Robo4 ameliorates the irradiation-induced depletion of VE-cadherin and connexin 43, improves the integrity of microvascular endothelial cell junctions, and decreases permeability. CONCLUSION: This study reveals that Robo4 maintains microvascular integrity after radiation preconditioning treatment by regulating endothelial permeability and protecting endothelial functions. Our results also provided a potential mechanism to repair the bone marrow vascular niche after irradiation by modulating Robo4 expression. BioMed Central 2023-01-16 /pmc/articles/PMC9843922/ /pubmed/36647012 http://dx.doi.org/10.1186/s11658-022-00413-w Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Letter Adzraku, Seyram Yao Wang, Guozhang Cao, Can Bao, Yurong Wang, Yizhou Smith, Alhaji Osman Du, Yuwei Wang, Haiyang Li, Yue Xu, Kailin Qiao, Jianlin Ju, Wen Zeng, Lingyu Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title | Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title_full | Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title_fullStr | Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title_full_unstemmed | Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title_short | Robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
title_sort | robo4 inhibits gamma radiation-induced permeability of a murine microvascular endothelial cell by regulating the junctions |
topic | Research Letter |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9843922/ https://www.ncbi.nlm.nih.gov/pubmed/36647012 http://dx.doi.org/10.1186/s11658-022-00413-w |
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