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Updated: Jul 1, 2025
Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells
Published on: May 11, 2020
[Pseudomonas Aeruginosa Affects the Function of Pulmonary Vascular Endothelial Cells]
Lei Zhang1,2,3, Jun-Yi Wang1,2,3, Xiang He2
1State Key Laboratory of Quality Research in Chinese Medicine,Macau University of Science and Technology,Macao 999078,China.
Abstract:
Objective To investigate the impact of Pseudomonas aeruginosa(PA) infection on the function of pulmonary vascular endothelial cells,and explore the mechanism of this bacterium in exacerbating lung inflammation in mice. Methods Two hours after human lung microvascular endothelial cell(HULEC-5a) were infected with the PA strain PAO1,the mRNA levels of autophagy-related gene 5(ATG5),6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3),and calcium adhesion protein 5(CDH5) were determined by reverse transcription real-time fluorescent quantitative PCR(RT-qPCR).The protein levels of ATG5,PFKFB3,and vascular endothelial calcium adhesion protein(VE-cadherin) were detected by immunofluorescence.After the expression of ATG5 and PFKFB3 was respectively knocked down by small interfering RNA(siRNA),RT-qPCR was employed to measure the mRNA levels of ATG5,PFKFB3,and CDH5,and immunofluorescence to detect the protein levels of PFKFB3 and VE-cadherin.In addition,the lactate assay kit was used to determine the level of lactate in the cells.After mice were infected with PAO1,lung inflammation was assessed through histopathological section staining.Confocal microscopy was employed to capture and analyze fluorescence-labeled PFKFB3 and VE-cadherin in endothelial cells. Results Compared with the control group,the HULEC-5a cells infected with PAO1 showed up-regulated mRNA and protein levels of PFKFB3(all P<0.05),down-regulated mRNA level of CDH5(P=0.023),disrupted continuity and down-regulated protein level of VE-cadherin(P<0.001),and elevated lactate level(P=0.017).Compared with PAO1-infected HULEC-5a cells,knocking down PFKFB3 led to the up-regulated mRNA level of CDH5(P=0.043),lowered lactate level(P=0.047),and restored continuity of VE-cadherin;knocking down ATG5 led to up-regulated mRNA and protein levels of PFKFB3(P=0.013 and P=0.003),elevated lactate level(P=0.015),and down-regulated mRNA level of CDH5(P=0.020) and protein level of VE-cadherin(P=0.001).The HE staining results showed obvious red blood cell leakage,inflammatory cell infiltration,alveolar septal widening,and partial detachment of vascular endothelial cells in the alveoli of PA-infected mice.Immunofluorescence staining showed up-regulated expression of PFKFB3 and decreased fluorescence signal of VE-cadherin in endothelial cells of infected mice compared with normal mice. Conclusion PA may regulate the PFKFB3 pathway via AGT5 to disrupt the function of pulmonary vascular endothelial cells,thereby exacerbating the inflammation in the lungs of mice.
Insights
Pseudomonas aeruginosa infection disrupts pulmonary vascular endothelial cell function by upregulating PFKFB3 via ATG5, worsening lung inflammation. This study reveals a key mechanism in bacterial exacerbation of lung injury.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Context:
- Pseudomonas aeruginosa (PA) infection is a significant cause of hospital-acquired pneumonia and ventilator-associated pneumonia.
- Pulmonary vascular endothelial cells play a critical role in maintaining lung homeostasis and regulating inflammation.
- Dysfunction of these cells contributes to the pathogenesis of acute lung injury and inflammation.
Purpose:
- To investigate the impact of Pseudomonas aeruginosa (PA) infection on human lung microvascular endothelial cell (HULEC-5a) function.
- To explore the underlying mechanism by which PA exacerbates lung inflammation in a mouse model.
- To examine the roles of autophagy-related gene 5 (ATG5) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in PA-induced endothelial cell dysfunction.
Summary:
- PAO1 infection of HULEC-5a cells upregulated PFKFB3, downregulated CDH5 and VE-cadherin, and increased lactate levels, indicating endothelial dysfunction.
- Knockdown of PFKFB3 partially restored CDH5 and VE-cadherin levels and reduced lactate. Knockdown of ATG5 exacerbated PFKFB3 upregulation and CDH5/VE-cadherin downregulation.
- PA-infected mice exhibited lung inflammation, increased PFKFB3, and decreased VE-cadherin in lung endothelial cells.
Impact:
- PA infection disrupts pulmonary vascular endothelial cell function, potentially through the ATG5-PFKFB3 pathway, contributing to lung inflammation.
- This study identifies PFKFB3 as a key mediator in PA-induced endothelial cell dysfunction and lung inflammation.
- Findings provide insights into the molecular mechanisms of bacterial pneumonia and suggest potential therapeutic targets for mitigating lung injury.
