Human adenovirus type 7 (HAdV-7) infection induces damage to pulmonary vascular endothelial cells by activating autophagy in human lung microvascular endothelial cells
Authors: Chen zhihe , Yang Zhongying , Rao Lifen , Li Changgen , Zang Na , Liu Enmei 
Published: June 2024

Abstract

Background HAdV-7 is a prevalent pathogen that can cause severe pneumonia in children. Previous clinical studies have shown a significant increase in serum levels of vascular endothelial growth factor (VEGF) and viral load in pediatric patients with fatal HAdV-7 infection, suggesting potential damage to pulmonary vascular endothelial cells and increased vascular permeability. Further research is necessary to elucidate the underlying mechanism behind this finding.
Methods The human lung microvascular endothelial cell line-5a (HULEC-5a) and human CD46 mice (hCD46) were utilized for in vitro and in vivo experiments, respectively. Correlative omics analysis was performed using gene set enrichment analysis (GSEA) and gene ontology (GO) enrichment analysis. Transmission electron microscope (TEM) was employed to observe cellular ultrastructure and virus particles, while immunofluorescence was used to detect the viral protein Hexon to investigate viral infection status. Various methods, including ELISAs for VEGF and other injury markers, qPCR for HAdV-7 fiber gene levels, the CCK8 assay for cell viability, and flow cytometry analysis for endothelial cell numbers, were employed to evaluate endothelial cell damage. Acute lung injury (ALI) severity was evaluated by scoring pathological inflammation and measuring pulmonary vascular permeability. Autophagy activation was assessed by observing autophagosomes and validating marker proteins.
Results GSEA analysis revealed significant enrichment in gene sets associated with endothelial functions (barrier, defense, and regeneration) and ALI in the HAdV-7-infected group. GO analysis suggested an enrichment of autophagy-related pathways associated with cell death. Subsequently, successful signs of infection were observed in lung endothelial cells with HAdV-7, including intracellular virions in HULEC-5a cells and perivascular viral protein Hexon in mouse lungs. Lung endothelial cell injury was observed after HAdV-7 infection, showing cytopathic effects, mitochondrial damage, and elevated levels of endothelial injury markers such as sICAM-1, sVCAM-1, E-selectin, ESM1, MCP1, and IL1β. Furthermore, a decrease in the number of CD45-CD31+ endothelial cells was noted. Moreover, increased VEGF expression and HAdV-7 fiber gene copy number were detected in the cellular supernatant, mice blood, bronchoalveolar lavage fluid/lung tissue. Additionally, evidence of leaky lung blood vessels and ALI were observed, including progressive weight loss, elevated pulmonary vascular permeability, significant inflammatory cells infiltration, alveolar congestion, structural destruction, and lung consolidation. Furthermore, HAdV-7 infection induced autophagosome formation in endothelial cells and triggered complete cell autophagy. Importantly, inhibiting autophagic flux reduced VEGF levels and other markers of endothelial injury, decreased the copy number of the HAdV-7 fiber gene, improved cell survival rate, reduced pulmonary vessel leakage, and alleviated inflammation in lung tissue.
Conclusions HAdV-7 infection induces damage to pulmonary endothelial cells, resulting in elevated VEGF levels, a high viral load, and ALI/ARDS. Activation of endothelial autophagy may be an important mechanism underlying these observations.Targeting autophagy could be a novel approach to rescue pulmonary endothelial cell damage and improve the poor prognosis in severe HAdV-7 infection.