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Molecular and Cellular Analysis of Endothelial Permeability in Sepsis / YUEXI GUO

Swansea University Author: YUEXI GUO

  • E-Thesis – open access under embargo until: 7th May 2029

DOI (Published version): 10.23889/SUthesis.66391

Abstract

Sepsis is a common manifestation of multidrug-resistant (MDR) bacterial infections. Lipopolysaccharide (LPS), a bacterial membrane component, in sepsis is responsible for vascular endothelial barrier dysfunction. Thrombin plays a key role in sepsis, which causes increased endothelial cell (EC) perme...

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Published: Swansea, Wales, UK 2024
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
Supervisor: Kanamarlapudi, Venkateswarlu ; Wilkinson, Thomas
URI: https://cronfa.swan.ac.uk/Record/cronfa66391
first_indexed 2024-05-10T19:11:35Z
last_indexed 2024-11-25T14:18:02Z
id cronfa66391
recordtype RisThesis
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Coronavirus disease (COVID)-19, which is caused by severe acute respiratory syndrome coronavirus (SARS CoV)-2, is a &#x2018;vascular disease&#x2019; and it can also result in sepsis, which causes epithelial and endothelial barrier dysfunction and increases the secretion of von Willebrand factor (vWF) and zonulin. ADP-ribosylation factor (ARF)1 and ARF6 are potentially involved in LPS-, thrombin- or SARS-CoV-2 spike protein S1 receptor binding domain (RBD)-triggered-cellular response. In this study, we first analysed in vitro how LPS, thrombin and the RBD induce EC permeability and ARF1/6 activation by standardising a novel trans-monolayer permeability assay to assess the permeability of EA.hy 926 endothelial cells and the GST-Golgi-localised, gamma-ear-containing, ARF-binding protein (GGA)3 protein-binding domain (PBD) pull-down assay to evaluate the ARF activation, respectively. Secondly, we evaluated the secretion levels of cytokines [tumour necrosis factor (TNF)-&#x3B1;, interleukin (IL)-6, and IL-1&#x3B2;] or vWF in the LPS-, thrombin- or SARS-CoV-2 spike protein-treated EA.hy 926 cells. Thirdly, we analysed the downstream signalling cascade responsible for LPS- or thrombin-induced EC permeability and/or vWF secretion and the underlying mechanism response to the RBD-induced EC permeability and vWF secretion or epithelial cell permeability and zonulin secretion by using the inhibitory peptides and chemical inhibitors. In conclusion, we showed that LPS, thrombin and the RBD are sufficient to induce EC permeability and ARF1/6 activation but not cytokine secretion. We also showed that thrombin and the RBD markedly induce vWF secretion. However, we found that thrombin induces EC permeability rapidly and reversibly, which is different from the secretion of vWF levels induced by thrombin. In addition, we dissected the MyD88/cytohesin 2/ARF6 signalling cascade downstream of toll-like receptor (TLR)4 for LPS-induced EC permeability. We also found that Rac1 plays a role in the cAMP inhibition of EC permeability induced by thrombin. Moreover, we showed that the RBD induces EC permeability and vWF secretion through the angiotensin-converting enzyme (ACE)2 receptor in an ARF6 activation-dependent manner. However, the mutants, including those in South African and South Californian variants of SARS-CoV-2, of the spike protein didn&#x2019;t affect its induction of EC permeability or vWF secretion. We also identified a signalling cascade downstream of angiotensin-converting enzyme (ACE)2 for SARS CoV-2 spike protein-induced EC permeability and its vWF secretion, using pharmacological inhibitors. The RBD induced epithelial permeability and zonulin secretion but at negligible levels. However, the RBD altered neither ACE2 expression nor ARF6 activation in epithelial cells. Additionally, the inhibitors, which were used for identifying the signalling cascade downstream for the EC permeability and its vWF secretion induced by the RBD, did not affect the RBD-induced epithelial permeability or its zonulin secretion. However, a better epithelial model cell line is required to study epithelial cell permeability and zonulin secretion. 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spelling 2024-05-10T20:27:32.1927086 v2 66391 2024-05-10 Molecular and Cellular Analysis of Endothelial Permeability in Sepsis a4c2725136052b5a8ea8d4634d6cafa0 YUEXI GUO YUEXI GUO true false 2024-05-10 Sepsis is a common manifestation of multidrug-resistant (MDR) bacterial infections. Lipopolysaccharide (LPS), a bacterial membrane component, in sepsis is responsible for vascular endothelial barrier dysfunction. Thrombin plays a key role in sepsis, which causes increased endothelial cell (EC) permeability and von Willebrand factor (vWF) secretion. Coronavirus disease (COVID)-19, which is caused by severe acute respiratory syndrome coronavirus (SARS CoV)-2, is a ‘vascular disease’ and it can also result in sepsis, which causes epithelial and endothelial barrier dysfunction and increases the secretion of von Willebrand factor (vWF) and zonulin. ADP-ribosylation factor (ARF)1 and ARF6 are potentially involved in LPS-, thrombin- or SARS-CoV-2 spike protein S1 receptor binding domain (RBD)-triggered-cellular response. In this study, we first analysed in vitro how LPS, thrombin and the RBD induce EC permeability and ARF1/6 activation by standardising a novel trans-monolayer permeability assay to assess the permeability of EA.hy 926 endothelial cells and the GST-Golgi-localised, gamma-ear-containing, ARF-binding protein (GGA)3 protein-binding domain (PBD) pull-down assay to evaluate the ARF activation, respectively. Secondly, we evaluated the secretion levels of cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β] or vWF in the LPS-, thrombin- or SARS-CoV-2 spike protein-treated EA.hy 926 cells. Thirdly, we analysed the downstream signalling cascade responsible for LPS- or thrombin-induced EC permeability and/or vWF secretion and the underlying mechanism response to the RBD-induced EC permeability and vWF secretion or epithelial cell permeability and zonulin secretion by using the inhibitory peptides and chemical inhibitors. In conclusion, we showed that LPS, thrombin and the RBD are sufficient to induce EC permeability and ARF1/6 activation but not cytokine secretion. We also showed that thrombin and the RBD markedly induce vWF secretion. However, we found that thrombin induces EC permeability rapidly and reversibly, which is different from the secretion of vWF levels induced by thrombin. In addition, we dissected the MyD88/cytohesin 2/ARF6 signalling cascade downstream of toll-like receptor (TLR)4 for LPS-induced EC permeability. We also found that Rac1 plays a role in the cAMP inhibition of EC permeability induced by thrombin. Moreover, we showed that the RBD induces EC permeability and vWF secretion through the angiotensin-converting enzyme (ACE)2 receptor in an ARF6 activation-dependent manner. However, the mutants, including those in South African and South Californian variants of SARS-CoV-2, of the spike protein didn’t affect its induction of EC permeability or vWF secretion. We also identified a signalling cascade downstream of angiotensin-converting enzyme (ACE)2 for SARS CoV-2 spike protein-induced EC permeability and its vWF secretion, using pharmacological inhibitors. The RBD induced epithelial permeability and zonulin secretion but at negligible levels. However, the RBD altered neither ACE2 expression nor ARF6 activation in epithelial cells. Additionally, the inhibitors, which were used for identifying the signalling cascade downstream for the EC permeability and its vWF secretion induced by the RBD, did not affect the RBD-induced epithelial permeability or its zonulin secretion. However, a better epithelial model cell line is required to study epithelial cell permeability and zonulin secretion. The knowledge gained from this study could be useful in developing novel drugs or repurposing existing drugs for treating Sepsis and COVID-19. E-Thesis Swansea, Wales, UK Sepsis, COVID-19 and endothelial/epithelial vascular permeability 7 5 2024 2024-05-07 10.23889/SUthesis.66391 COLLEGE NANME COLLEGE CODE Swansea University Kanamarlapudi, Venkateswarlu ; Wilkinson, Thomas Doctoral Ph.D 2024-05-10T20:27:32.1927086 2024-05-10T19:50:20.9572674 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science YUEXI GUO 1 Under embargo Under embargo 2024-05-10T20:24:40.0776900 Output 7512617 application/pdf E-Thesis – open access true 2029-05-07T00:00:00.0000000 Copyright: The author, Yuexi Guo, 2024. Thesis released under the terms of a Creative Commons Attribution 4.0 (CC-BY) license. Third party content is excluded for use under the license terms. true eng https://creativecommons.org/licenses/by/4.0/deed.en
title Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
spellingShingle Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
YUEXI GUO
title_short Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
title_full Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
title_fullStr Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
title_full_unstemmed Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
title_sort Molecular and Cellular Analysis of Endothelial Permeability in Sepsis
author_id_str_mv a4c2725136052b5a8ea8d4634d6cafa0
author_id_fullname_str_mv a4c2725136052b5a8ea8d4634d6cafa0_***_YUEXI GUO
author YUEXI GUO
author2 YUEXI GUO
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institution Swansea University
doi_str_mv 10.23889/SUthesis.66391
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
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description Sepsis is a common manifestation of multidrug-resistant (MDR) bacterial infections. Lipopolysaccharide (LPS), a bacterial membrane component, in sepsis is responsible for vascular endothelial barrier dysfunction. Thrombin plays a key role in sepsis, which causes increased endothelial cell (EC) permeability and von Willebrand factor (vWF) secretion. Coronavirus disease (COVID)-19, which is caused by severe acute respiratory syndrome coronavirus (SARS CoV)-2, is a ‘vascular disease’ and it can also result in sepsis, which causes epithelial and endothelial barrier dysfunction and increases the secretion of von Willebrand factor (vWF) and zonulin. ADP-ribosylation factor (ARF)1 and ARF6 are potentially involved in LPS-, thrombin- or SARS-CoV-2 spike protein S1 receptor binding domain (RBD)-triggered-cellular response. In this study, we first analysed in vitro how LPS, thrombin and the RBD induce EC permeability and ARF1/6 activation by standardising a novel trans-monolayer permeability assay to assess the permeability of EA.hy 926 endothelial cells and the GST-Golgi-localised, gamma-ear-containing, ARF-binding protein (GGA)3 protein-binding domain (PBD) pull-down assay to evaluate the ARF activation, respectively. Secondly, we evaluated the secretion levels of cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β] or vWF in the LPS-, thrombin- or SARS-CoV-2 spike protein-treated EA.hy 926 cells. Thirdly, we analysed the downstream signalling cascade responsible for LPS- or thrombin-induced EC permeability and/or vWF secretion and the underlying mechanism response to the RBD-induced EC permeability and vWF secretion or epithelial cell permeability and zonulin secretion by using the inhibitory peptides and chemical inhibitors. In conclusion, we showed that LPS, thrombin and the RBD are sufficient to induce EC permeability and ARF1/6 activation but not cytokine secretion. We also showed that thrombin and the RBD markedly induce vWF secretion. However, we found that thrombin induces EC permeability rapidly and reversibly, which is different from the secretion of vWF levels induced by thrombin. In addition, we dissected the MyD88/cytohesin 2/ARF6 signalling cascade downstream of toll-like receptor (TLR)4 for LPS-induced EC permeability. We also found that Rac1 plays a role in the cAMP inhibition of EC permeability induced by thrombin. Moreover, we showed that the RBD induces EC permeability and vWF secretion through the angiotensin-converting enzyme (ACE)2 receptor in an ARF6 activation-dependent manner. However, the mutants, including those in South African and South Californian variants of SARS-CoV-2, of the spike protein didn’t affect its induction of EC permeability or vWF secretion. We also identified a signalling cascade downstream of angiotensin-converting enzyme (ACE)2 for SARS CoV-2 spike protein-induced EC permeability and its vWF secretion, using pharmacological inhibitors. The RBD induced epithelial permeability and zonulin secretion but at negligible levels. However, the RBD altered neither ACE2 expression nor ARF6 activation in epithelial cells. Additionally, the inhibitors, which were used for identifying the signalling cascade downstream for the EC permeability and its vWF secretion induced by the RBD, did not affect the RBD-induced epithelial permeability or its zonulin secretion. However, a better epithelial model cell line is required to study epithelial cell permeability and zonulin secretion. The knowledge gained from this study could be useful in developing novel drugs or repurposing existing drugs for treating Sepsis and COVID-19.
published_date 2024-05-07T20:38:27Z
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