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Impaired Autophagy Causes Severe Corneal Neovascularization

Purpose: To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). Methods: In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We...

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Autores principales: Yi, Kun, Yang, Yuping, Yuan, Ye, Xiang, Yingqian, Zhou, Shanbi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9737787/
https://www.ncbi.nlm.nih.gov/pubmed/36497153
http://dx.doi.org/10.3390/cells11233895
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author Yi, Kun
Yang, Yuping
Yuan, Ye
Xiang, Yingqian
Zhou, Shanbi
author_facet Yi, Kun
Yang, Yuping
Yuan, Ye
Xiang, Yingqian
Zhou, Shanbi
author_sort Yi, Kun
collection PubMed
description Purpose: To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). Methods: In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay. Results: In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon. Conclusions: Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy.
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spelling pubmed-97377872022-12-11 Impaired Autophagy Causes Severe Corneal Neovascularization Yi, Kun Yang, Yuping Yuan, Ye Xiang, Yingqian Zhou, Shanbi Cells Article Purpose: To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). Methods: In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay. Results: In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon. Conclusions: Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy. MDPI 2022-12-02 /pmc/articles/PMC9737787/ /pubmed/36497153 http://dx.doi.org/10.3390/cells11233895 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yi, Kun
Yang, Yuping
Yuan, Ye
Xiang, Yingqian
Zhou, Shanbi
Impaired Autophagy Causes Severe Corneal Neovascularization
title Impaired Autophagy Causes Severe Corneal Neovascularization
title_full Impaired Autophagy Causes Severe Corneal Neovascularization
title_fullStr Impaired Autophagy Causes Severe Corneal Neovascularization
title_full_unstemmed Impaired Autophagy Causes Severe Corneal Neovascularization
title_short Impaired Autophagy Causes Severe Corneal Neovascularization
title_sort impaired autophagy causes severe corneal neovascularization
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9737787/
https://www.ncbi.nlm.nih.gov/pubmed/36497153
http://dx.doi.org/10.3390/cells11233895
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