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Thromboinflammation: in a Model of Hepatocyte Transplantation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hepatocyte transplantation is an attractive method for the treatment of metabolic liver disease and acute liver failure. The clinical application of this method has been hampered by a large initial loss of transplanted cells.

This thesis has identified and characterized an instant blood-mediated inflammatory reaction (IBMIR), which is a thromboinflammatory response from the innate immunity that may partly explain the observed loss of cells. In vitro perifusion experiments were performed and established that hepatocytes in contact with blood activate the complement and coagulation systems and induce clot formation in conjunction with the recruitment of neutrophils.  Within an hour, the hepatocytes were surrounded by platelets and entrapped in a clot infiltrated by neutrophils. Furthermore, hepatocytes expressed tissue factor (TF), and the reactions were shown to be initiated through the TF pathway. Monitoring of hepatocyte transplantation in vivo revealed activation of the same parameters as were noted in vitro.

For the first time, von Willebrand factor (vWF) was identified on the hepatocyte surface, being demonstrated by flow cytometry and confocal microscopy. mRNA for vWF was also confirmed in hepatocytes. Complex formation between platelets and hepatocytes was also identified. Addition of antibodies targeting the binding site for vWF on the platelets reduced the complex formation.

Two different strategies, systemic and local intervention, were applied to diminish the thromboinflammation elicited from the hepatocytes in contact with ABO-matched blood. Systemic inhibition with LMW-DS, in a clinically applicable dose, was found to be superior in controlling the IBMIR in vitro when compared to heparin. Cryopreserved hepatocytes elicited the IBMIR to the same extent as did fresh hepatocytes, and the IBMIR was equally well controlled with LMW-DS in both cryopreserved and fresh cells.

Hepatocytes were coated with two layers of immobilized heparin in an attempt to protect the cells from the IBMIR. In vitro perifusion experiments showed heparinized hepatocytes triggered a significantly lower degree of IBMIR.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 75 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 123
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-286869ISBN: 978-91-554-9592-3 (print)OAI: oai:DiVA.org:uu-286869DiVA: diva2:922111
Public defence
2016-06-10, Rosénsalen, Akademiska Barnsjukhuset Ing 95/96, Uppsala, 10:00 (Swedish)
Opponent
Supervisors
Available from: 2016-05-20 Created: 2016-04-22 Last updated: 2016-06-15Bibliographically approved
List of papers
1. The Instant Blood-Mediated Inflammatory Reaction Characterized in Hepatocyte Transplantation
Open this publication in new window or tab >>The Instant Blood-Mediated Inflammatory Reaction Characterized in Hepatocyte Transplantation
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2011 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 91, no 6, 632-638 p.Article in journal (Refereed) Published
Abstract [en]

Background. Hepatocyte transplantation (HcTx) has proven to be a safe procedure, although the functional results have been unsatisfactory, probably due to insufficient engraftment or a loss of transplanted mass or function. In this study, we investigate whether hepatocytes in contact with blood induce an inflammatory reaction leading to, similar to what happens in clinical islet transplantation, an instant blood-mediated inflammatory reaction (IBMIR) resulting in an early loss of transplanted cells. Methods. By using an experimental model that mimics the portal vein blood flow, we could study different parameters reflecting the effects on the innate immunity elicited by hepatocytes in contact with ABO-matched human blood. Results. We report that all aspects of the IBMIR such as platelet and granulocyte consumption, coagulation, and complement activation were demonstrated. Addition of various specific inhibitors of coagulation allowed us to clearly delineate the various stages of the hepatocyte-triggered IBMIR and show that the reaction was triggered by tissue factor. Analysis of a case of clinical HcTx showed that hepatocyte-induced IBMIR also occurs in vivo. Both the inflammatory and the coagulation aspects were controlled by low-molecular-weight dextran sulfate. Conclusion. Isolated hepatocytes in contact with blood induce the IBMIR in vitro, and there are indications that these events are also relevant in vivo. According to these findings, HcTx would benefit from controlling a wider range of signals from the innate immune system.

Keyword
IBMIR, Islet transplantation, Cell transplantation, Innate immunity, Engraftment
National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-149561 (URN)10.1097/TP.0b013e31820ae459 (DOI)000288115800016 ()21289595 (PubMedID)
Available from: 2011-03-22 Created: 2011-03-21 Last updated: 2017-12-11Bibliographically approved
2. von Willebrand factor: a factor to consider in hepatocyte transplantation
Open this publication in new window or tab >>von Willebrand factor: a factor to consider in hepatocyte transplantation
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-286867 (URN)
Available from: 2016-04-22 Created: 2016-04-22 Last updated: 2016-06-15
3. Control of IBMIR induced by fresh and cryopreserved hepatocytes by low molecular weight dextran sulfate
Open this publication in new window or tab >>Control of IBMIR induced by fresh and cryopreserved hepatocytes by low molecular weight dextran sulfate
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2017 (English)In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 26, no 1, 71-81 p.Article in journal (Refereed) Published
Abstract [en]

Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 mu g/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 mu g/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.

Keyword
Innate immunity, IBMIR, Thromboinflammation, Hepatocyte transplantation, Low molecular weight dextran sulfate (LMW-DS)
National Category
Other Medical Sciences
Identifiers
urn:nbn:se:uu:diva-286868 (URN)10.3727/096368916X692609 (DOI)000392785000007 ()
Note

The manusripct of this article is part of the thesis Thromboinflammation: in a Model of Hepatocyte Transplantation http://uu.diva-portal.org/smash/record.jsf?pid=diva2:922111

Available from: 2016-04-22 Created: 2016-04-22 Last updated: 2017-11-30Bibliographically approved
4. Heparinization of cell surfaces with short pepetide-conjugated PEG-lipid regulates thromboinflammation in thransplantation of human MSCs and hepatocytes
Open this publication in new window or tab >>Heparinization of cell surfaces with short pepetide-conjugated PEG-lipid regulates thromboinflammation in thransplantation of human MSCs and hepatocytes
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2016 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 35, 194-205 p.Article in journal (Refereed) Published
Abstract [en]

Infusion of therapeutic cells into humans is associated with immune responses, including thromboinflammation, which result in a large loss of transplanted cells\ To address these problems, heparinization of the cell surfaces was achieved by a cell-surface modification technique using polyethylene glycol conjugated phospholipid (PEG-lipid) derivatives. A short heparin-binding peptide was conjugated to the PEG-lipid for immobilization of heparin conjugates on the surface of human mesenchymal stem cells (hMSCs) and human hepatocytes. Here three kinds of heparin-binding peptides were used for immobilizing heparin conjugates and examined for the antithrombogenic effects on the cell surface. The heparinized cells were incubated in human whole blood to evaluate their hemocompatibility by measuring blood parameters such as platelet count, coagulation markers, complement markers, and Factor Xa activity. We found that one of the heparin-binding peptides did not show cytotoxicity after the immobilization with heparin conjugates. The degree of binding of the heparin conjugates on the cell surface (analyzed by flow cytometer) depended on the ratio of the active peptide to control peptide. For both human MSCs and hepatocytes in whole-blood experiments, no platelet aggregation was seen in the heparin conjugate-immobilized cell group vs. the controls (non-coated cells or control peptide). Also, the levels of thrombin-antithrombin complex (TAT), C3a, and sC5b-9 were significantly lower than those of the controls, indicating a lower activation of coagulation and complement. Factor Xa analysis indicated that the heparin conjugate was still active on the cell surface at 24 h post-coating. It is possible to immobilize heparin conjugates onto hMSC and human hepatocyte surfaces and thereby protect the cell surfaces from damaging thromboinflammation. Statement of Signigficance We present a promising approach to enhance the biocompatibility of therapeutic cells. Here we used short peptide-conjugated PEG-lipid for cell surface modification and heparin conjugates for the coating of human hepatocytes and MSCs. We screened the short peptides to find higher affinity for heparinization of cell surface and performed hemocompatibility assay of heparinized human hepatocytes and human MSCs in human whole blood. Using heparin-binding peptide with higher affinity, not only coagulation activation but also complement activation was significantly suppressed. Thus, it was possible to protect human hepatocytes and human MSCs from the attack of thromboinflammatory activation, which can contribute to the improvement graft survival.

Keyword
Cell surface modification; Heparinization; Thromboinflammation; MSCs; Hepatocyte; Polyethylene glycol-conjugated phospholipid (PEG-lipid)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-279420 (URN)10.1016/j.actbio.2016.02.018 (DOI)000375162200018 ()26876877 (PubMedID)
Funder
Swedish Research CouncilThe Swedish Foundation for International Cooperation in Research and Higher Education (STINT)
Available from: 2016-03-01 Created: 2016-03-01 Last updated: 2017-11-30Bibliographically approved

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