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Vascular Interactions in Innate Immunity and Immunothrombosis:: Models of Endothelial Protection
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The phenomenon known as immunothrombosis has garnered increased attention over the last few years. Much work has been done to characterize the cross talk between hemostasis and the innate immune system. This thesis outlines the role of the vascular endothelial cells during immunothrombotic events as regulators of coagulation, platelet-, and leukocyte recruitment.

A newly developed method for investigating the interaction between endothelial cells and the blood compartment illustrated the procoagulant and proinflammatory effects elicited by tumor necrosis factor α activated endothelial cells upon exposure to whole blood. The method was utilized in evaluating treatment of endothelial dysfunction and disruption with a heparin conjugate. Damaged or hypoxic endothelial cells, in addition to basement membrane collagen, that were pretreated with the heparin conjugate prior to contact with blood were found to have reduced activation of coagulation, platelet-, and leukocyte recruitment; in contrast to unfractionated heparin, which had no effect on the aforementioned parameters. The treatment was then investigated in the setting of ischemia reperfusion injury during kidney transplantation and the heparin conjugate was found to bind cultured endothelial cells with high avidity under cold storage conditions. Furthermore, it was found to bind to the renal vasculature during static cold storage and was subsequently found to be beneficial with regard to early graft function in an experimental mouse model of syngeneic kidney transplantation. Recipients of kidneys treated with the heparin conjugate had reduced serum creatinine compared to controls 24 hours after transplantation. Lastly, the anticoagulant properties of the heparin conjugate were investigated in comparison to unfractionated heparin. While the conjugate exerted reduced capacity with regard to thrombin inhibition, it rapidly inhibited the binding of platelets to exposed collagen. The conjugate was furthermore found to preferentially locate to sites of endothelial cell activation at early stage during endotoxic shock in mice.

In conclusion, this thesis demonstrates that disrupted functioning of the vascular endothelial cells actively contributes to immunothrombosis, and that it is possible to model endothelial cell function using whole blood assays. Furthermore, this thesis presents a treatment that enhances the hemocompatibility of damaged endothelial cells and subsequently improves the early renal function after kidney transplantation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1220
Keyword [en]
Endothelial cells, Thrombosis, Innate immunity, Immunothrombosis, Thromboinflammation
National Category
Immunology in the medical area Cell and Molecular Biology
Research subject
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-283548ISBN: 978-91-554-9564-0 (print)OAI: oai:DiVA.org:uu-283548DiVA: diva2:919309
Public defence
2016-06-03, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-05-13 Created: 2016-04-13 Last updated: 2016-06-01
List of papers
1. A novel in vitro model for studying the interactions between human whole blood and endothelium
Open this publication in new window or tab >>A novel in vitro model for studying the interactions between human whole blood and endothelium
2014 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, Vol. 21, no 93, e52112Article in journal (Refereed) Published
Abstract [en]

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel. We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16(+) cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

National Category
Medical and Health Sciences
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-244316 (URN)10.3791/52112 (DOI)000349311400057 ()25489671 (PubMedID)
Funder
Swedish Research Council, 90293501, A0290401, A0290402EU, FP7, Seventh Framework Programme, 602699Åke Wiberg FoundationNovo Nordisk
Available from: 2015-02-13 Created: 2015-02-13 Last updated: 2017-12-04Bibliographically approved
2. Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation
Open this publication in new window or tab >>Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation
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2015 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 113, no 6, 1312-1322 p.Article in journal (Refereed) Published
Abstract [en]

Ischaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vas, culature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a Significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

Keyword
Blood coagulation, endothelial cells, heparin, surface modification, collagen
National Category
Cardiac and Cardiovascular Systems Hematology
Identifiers
urn:nbn:se:uu:diva-258043 (URN)10.1160/TH14-09-0724 (DOI)000355776400021 ()25740465 (PubMedID)
Funder
Swedish Research Council, 90293501, A0290401, A0290402
Available from: 2015-07-13 Created: 2015-07-10 Last updated: 2017-12-04Bibliographically approved
3. Enhanced protection of the vascular endothelium in porcine and murine kidneys against ischemia reperfusion injury prior to transplantation
Open this publication in new window or tab >>Enhanced protection of the vascular endothelium in porcine and murine kidneys against ischemia reperfusion injury prior to transplantation
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-283451 (URN)
Available from: 2016-04-13 Created: 2016-04-13 Last updated: 2016-06-01
4. A novel anticoagulant targeting vascular wall damage
Open this publication in new window or tab >>A novel anticoagulant targeting vascular wall damage
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-283455 (URN)
Available from: 2016-04-13 Created: 2016-04-13 Last updated: 2016-06-01

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