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Molecular mechanisms of platelet-mediated liver regeneration after partial hepatectomyKirschbaum, Marc

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Molecular mechanisms of platelet-mediated

liver regeneration after partial hepatectomy

Marc Kirschbaum

ISBN: 978-94-92679-10-9Cover design: Marc en Corline KirschbaumLay out by Sinds1961Printed by Print Service Ede

No part of this thesis may reproduced without prior permission of the author.

Molecular mechanisms of platelet-mediated liver regeneration after partial hepatectomy

Proefschrift

ter verkrijging van de graad van doctor aan de

Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 4 oktober 2017 om 12:45 uur

door

Marc Kirschbaum

geboren op 24 juni 1987

te Tönisvorst, Duitsland

SupervisorsProf. dr. J.A. LismanProf. dr. R.J. Porte

Assessment CommitteeProf. dr. K.N. FaberProf. dr. S.C.D. van IJzendoornProf. dr. J.C.M. Meijers

ParanimfenC.M. Kirschbaum - de VosD. Hoeksma

Table of contents

Chapter 1 Introduction and outline of this thesis

Chapter 2 The role of platelets in liver regeneration – what don’t we know?

Chapter 3 Horizontal RNA transfer mediates platelet-induced hepatocyte proliferation

Chapter 4 Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice

Chapter 5 Vitamin E attenuates the progression of non-alcoholic fatty liver disease caused by partial hepatectomy in mice

Chapter 6 Evidence against a role for platelet-derived molecules in liver regeneration after partial hepatectomy in humans

Chapter 7 Intermezzo: In vitro uptake of recombinant factor VIIa by megakaryocytes with subsequent production of platelets containing hemostatically active drug

Chapter 8 Summary and discussion

Nederlandse Samenvatting/ Dutch Summary

Author affilations

Dankwoord

About the author

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IntroductIon and outlIne of thIs thesIs1

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Chapter 1 IntroduCtIon and outlIne of thIs thesIs

1Liver regenerationThe liver has a unique regenerative capacity. The ultimate regenerative response of the liver occurs after a partial liver resection. Up to 70% of liver tissue can be safely removed in patients that require a liver resection for removal of a liver tumor (1,2). Also, healthy individuals can donate part of their liver for transplantation purposes, resulting in partial livers in both donor and recipient. Following partial liver resection, the liver eventually regenerates to its original size with substantial regeneration in humans already after one week, while regeneration is virtually complete after three months. In rodents, liver regeneration is even faster, with complete regeneration in mice after ~5 days.

Liver regenerative responses also occur when liver tissue is damaged by for example toxins or viruses, or by ischemia/reperfusion injury. Diseases associated with damage to liver tissue are either chronic or acute. Chronic liver disease, for example causes formation of fibrous tissue replacing healthy liver tissue leading to fibrosis and eventually cirrhosis. Acute liver failure, for example caused by intoxication with acetaminophen, results in rapid necrosis by a mechanism involving sterile inflammation. The most severe cases lead to necrosis of virtually the entire liver.In the context of liver transplantation, livers also suffer from acute hepatocellular injury as a consequence of combined warm and cold ischemia and the subsequent reperfusion injury. Livers that have suffered substantial damage in the process of transplantation can also fully recover. Animal experiments as well as biopsy studies in humans have shown that moderate fibrosis can resolve when the initiating trigger is removed or when successful treatment is given (3,4). The liver can thus regenerate not only following physical removal of liver tissue but also following damage from an acute or chronic insult. Although the liver can regenerate and sustain vital functions, it sometimes fails to regenerate and fails to maintain the metabolic demand of the body. This condition is associated with high mortality and morbidity rate in patients. In those patients little therapeutic options are available and patients die from liver insufficiency. A liver transplantation is the only lifesaving procedure. Therefore, strategies for preventing liver damage and accelerating liver regeneration after a partial liver resection or liver transplantation are of great interest.

PlateletsPlates are anucleated, discoid cellular fragments derived from the cytoplasm of megakaryocytes in the bone marrow. They are the smallest of the many types of cells in circulating blood, averaging only from 2 to 5 micrometer in diameter and 0.5 micrometer in thickness. In contrast, the number of platelets in the circulation is enormous. A normal human platelet count in healthy individuals ranges from 150.000 to 450.000 platelets per microliter of blood. Per day megakaryocytes release approximately 1011 platelets into the bloodstream and the average life span of circulating platelets is between 7 and 10 days.Although platelets lack a nucleus, and they are per definition no cells, they contain a

numerous amounts of intracellular organelles, which are also present in cells. Beside platelets specific cytoplasmic compartments, alpha and dense granules, platelets contain mitochondria to maintain their energy balance and also an endoplasmic reticulum and ribosomes. Because of their anatomy platelets are often considered simple, noncomplex “cells” with their primary responsibilities to stop bleeding. Platelet functions in primary hemostasis have been investigated extensively in the last decades. Circulating platelets are recruited to the site of injury, where they become a major component of the developing thrombus. However platelets are more than bleeding stoppers. Platelets are multifunctional “cells” that are involved in a variety of biologic and pathologic processes, including host defense, angiogenesis, wound healing, inflammation, and also liver regeneration (5-8).

Platelet-mediated liver regenerationLiver regeneration after partial liver resection is a complex and well-organized process, which involves the participation of all liver cells, immune cells and also platelets. It is well-known that in experimental animal models in which platelets were depleted or functionally impaired, liver regeneration is substantially delayed after a partial liver resection (9,10). In a clinical study, our research group showed that a low platelet count immediately after partial liver resection is an independent predictor of delayed postoperative liver function recovery following liver surgery, suggesting that platelets stimulate liver regeneration also in humans (11). Nevertheless the molecular mechanisms behind platelet-mediated stimulation of liver regeneration are largely unexplored. It seems that platelets use various mechanisms to perform these extra-hemostatic functions.Platelets store a variety of growth factors in their alpha granules, including platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and tissue growth factor (TGF)-β (12,13). Upon platelet activation granules become excreted and growth factors are released. It has been demonstrated in vitro that hepatocytes show a mitogenic response to various growth factors stored in platelets (14). It seems plausible that local release of platelet stored growth factors after liver resection is partly responsible for platelet-mediated liver regeneration. Nevertheless it has to been proven in vivo, that platelet growth factors are actually responsible. Another potential player in platelet-mediated liver regeneration seems to be serotonin (9). Platelets carry serotonin in blood, which is not only a neurotransmitter but also a hormone with various extraneuronal functions. Serotonin exhibits a vast repertoire of actions including cell proliferation and differentiation. It is a potent mitogenic factor and is involved in the remodeling of tissue (15,16). Several studies have demonstrated that serotonin receptors in the liver are upregulated after liver resection in mice and that treatment with serotonin receptor antagonists inhibits liver regeneration (9,17). However, clinical studies with patients undergoing partial hepatectomy show opposed results regarding the role of serotonin in the stimulation of liver regeneration (18,19).Beside the release of growth factors from platelet granules, the study of cell-cell interactions

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1between platelets and various liver cells are of great interest for researchers. It seems that direct interaction between platelets and liver cells is crucial for platelet-mediated stimulation of liver regeneration as demonstrated by an impaired platelet proliferative capacity when platelet-hepatocyte binding was blocked in vitro (12). Furthermore, Murata et al. demonstrated in vitro that platelet binding to liver endothelial cells (LSEC) and liver specific macrophages (Kupffer cells) is important for the release of pro-inflammatory cytokines in the liver. Those cytokines are crucial for the onset of liver regeneration in the early phase after partial liver resection (20,21)

Platelet RNAsAlthough platelets are anucleated, they contain miRNAs and mRNAs and it has been demonstrated that platelets contain the necessary molecular machinery to conduct translation (22,23). Historically, platelet RNA was recognized merely in platelet research and it has long been considered that the cytoplasmic platelet RNAs are residual transcripts of their forming cell, the megakaryocyte. Nowadays several studies challenge this assumption and support a more fluid role for platelet RNA in platelet function and disease development. Platelets can actively translate RNA to protein. In response to various physiologic stimuli, platelets are able to synthesize biologically relevant proteins de novo that are regulated at translational RNA level and does not require a nucleus (24). In addition to the capacity to synthesize proteins de novo, several independent research groups have demonstrated in the last years that platelets have the ability to transfer their endogenous cytoplasmic miRNAs and mRNAs to recipient cells (25-27). Previously RNA transfer between exosomes/ microvesicles and several recipient cells has been investigated and mentioned as novel mechanism of genetic exchange between cells (28-30). Moreover, it has been demonstrated that microvesicles derived from human liver stem cells were taken up by hepatocytes, resulting in transfer of mRNA (31). The transferred mRNA may result in accelerated hepatocyte proliferation and induced apoptosis resistance. Regarding this result, it seems conceivable that also platelet RNAs are involved in the stimulation of hepatocyte proliferation.

Aim of this thesisThe aim of this thesis is to investigate the molecular mechanism of platelet-mediated liver regeneration after partial hepatectomy. Better insight in the mechanisms of platelet-mediated liver regeneration is an essential step in the development of novel therapies that can be applied in patients with liver failure and insufficient liver regeneration. Until now, in those patients, a liver transplantation is the only lifesaving option.Chapter 2 is a letter in response to an article published in the Journal of Hepatology, which summarizes current knowledge on the role of blood platelets in liver regeneration and the role of platelet RNA. In Chapter 3 we investigated platelet-mediated stimulation of hepatocyte proliferation in vitro as a model for liver regeneration and gained novel insights into the role of platelet RNA in platelet-mediated liver regeneration. The study of the mechanism of platelet recruitment into the liver parenchyma after partial liver resection is the topic in Chapter 4. We test our hypothesis that platelet recruitment in the early phase after liver resection is essential for the regenerative process. Chapter 5 investigates growth factor levels in blood plasma and in platelets of patients undergoing hemihepatectomy or a pancreatico-duodenectomy (PPPD). In Chapter 6 we focus on liver regeneration in mice with non-alcoholic fatty liver disease (NAFLD) and the effect of partial liver resection on the progression of NAFLD. In Chapter 7, we present an “intermezzo” in this thesis. Recombinant factor VIIa (rFVIIa) has been recently shown to prevent spontaneous bleeding in inhibitor-complicated hemophilia when administered once daily. We propose in this study that redistribution of rFVIIa to the bone marrow compartment and uptake by megakaryocytes which results in production of platelets containing rFVIIa. Finally, in Chapter 8, all results are summarized and discussed, followed by a view on the future perspectives of platelet-mediated liver regeneration research and their therapeutic applications.

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1References

1. DeOliveira ML, Clavien PA, Kambakamba P. Advances in liver surgery for cholangiocarcinoma. Curr

Opin Gastroenterol 2013; 29(3): 293-8.

2. Melloul E, Lesurtel M, Carr BI, Clavien PA. Developments in liver transplantation for hepatocellular

carcinoma. Semin Oncol 2012; 39(4): 510-21.

3. Lavine JE, Schwimmer JB, Van Natta ML, Molleston JP, Murray KF, Rosenthal P, et al. Effect of vitamin

E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the

TONIC randomized controlled trial. JAMA. 2011;305: 1659-1668.

4. Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin

E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362: 1675-1685.

5. Bozza FA, Shah AM, Weyrich AS, Zimmerman GA. Amicus or adversary: platelets in lung biology,

acute injury, and inflammation. Am J Respir Cell Mol Biol. 2009;40(2):123-134.

6. Nurden AT. Platelets, inflammation and tissue regeneration. Thromb Haemost. 2011;105 Suppl

1:S13-33.

7. Smyth SS, McEver RP, Weyrich AS, et al. Platelet functions beyond hemostasis. J Thromb Haemost.

2009;7(11):1759-1766.

8. Nocito A, Georgiev P, Dahm F, et al. Platelets and platelet-derived serotonin promote tissue repair

after normothermic hepatic ischemia in mice. Hepatology. 2007;45(2):369-376.

9. Lesurtel M, Graf R, Aleil B, Walther DJ, Tian Y, Jochum W, Gachet C, et al. Platelet-derived serotonin

mediates liver regeneration. Science 2006; 312(5770): 104-107.

10. Myronovych A, Murata S, Chiba M, Matsuo R, Ikeda O, Watanabe M, Hisakura K, et al. Role of

platelets on liver regeneration after 90% hepatectomy in mice. J Hepatol 2008; 49(3): 363-372.

11. Alkozai EM, Nijsten MW, de Jong KP, et al. Immediate postoperative low platelet count is associated

with delayed liver function recovery after partial liver resection. Ann Surg. 2010;251(2):300-306.

12. Matsuo R, Ohkohchi N, Murata S, et al. Platelets Strongly Induce Hepatocyte Proliferation with

IGF-1 and HGF In Vitro. J Surg Res. 2008;145(2):279-286.

13. Michalopoulos GK and DeFrances MC. Liver regeneration. Science. 1997;276(5309):60-66.

14. Matsuo R, Ohkohchi N, Murata S, Ikeda O, Nakano Y, Watanabe M, Hisakura K, Myronovych A,

Kubota T, Narimatsu H, Ozaki M. Platelets strongly induce hepatocyte proliferation with IGF-1 and

HGF in vitro. J Surg Res. 2008 Apr;145(2):279-86.

15. de Abajo FJ. Effects of selective serotonin reuptake inhibitors on platelet function: mechanisms,

clinical outcomes and implications for use in elderly patients. Drugs Aging. 2011;28(5):345-67.



17. Balasubramanian S, Paulose CS. Induction of DNA synthesis in primary cultures of rat hepatocytes

by serotonin: Possible involvement of serotonin S2 receptor. Hepatology. 1998;27(1):62-66.

18. Starlinger P, Zikeli S, Fleischmann E, Brostjan C, Gruenberger T, Schauer D, et al. Evidence for

serotonin as a relevant inducer of liver regeneration after liver resection in humans. Hepatology

2014; 60(1): 257-66.

19. Alkozai EM, Lisman T, Porte RJ, van Faassen M, Kema IP. Evidence against a role of serotonin in liver

regeneration in humans. Hepatology 2015; 62(3).

20. Takahashi K, Kozuma Y, Suzuki H, Tamura T, Maruyama T, Fukunaga K, Murata S, Ohkohchi N. Human

platelets promote liver regeneration with kupffer cells in SCID mice. J Surg Res. 2013;180(1):62-72.

21. Selzner N, Selzner M, Odermatt B, et al. ICAM-1 triggers liver regeneration through leukocyte

recruitment and kupffer cell-dependent release of TNF-alpha/IL-6 in mice. Gastroenterology

2003; 124(3): 692-700.

22. Weyrich AS, Lindemann S, Tolley ND, et al. Change in protein phenotype without a nucleus:

translational control in platelets. Semin Thromb Hemost. 2004;30(4):491-498.

23. Rowley JW, Schwertz H, Weyrich AS. Platelet mRNA: The meaning behind the message. Curr Opin

Hematol 2012; 19(5): 385-391.

24. Weyrich AS, Zimmerman GA. Evaluating the relevance of the platelet transcriptome. Blood. 2003;

102:1550-1551

25. Risitano A, Beaulieu LM, Vitseva O, Freedman JE. Platelets and platelet-like particles mediate

intercellular RNA transfer. Blood. 2012;119(26):6288-6295.

26. Gidlöf O, van der Brug M, Ohman J, Gilje P, Olde B, Wahlestedt C, Erlinge D. Platelets activated

during myocardial infarction release functional miRNA, which can be taken up by endothelial cells

and regulate ICAM1 expression. Blood. 2013;121(19):3908-17.

27. Laffont Benoit B. Activated platelets can deliver mRNA regulatory Ago2microRNA complexes to

endothelial cells via microparticles. Blood 2013-7-11; 122(2): 253-61.

28. Mittelbrunn M, Gutierrez-Vazquez C, Villarroya-Beltri C, et al. Unidirectional transfer of microRNA-

loaded exosomes from T cells to antigen-presenting cells. Nat Commun. 2011;2:282.

29. Skog J, Wurdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA and proteins that

promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10(12):1470-

1476.

30. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of

mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol.

2007;9(6):654-659.

31. Herrera MB, Fonsato V, Gatti S, et al. Human liver stem cell-derived microvesicles accelerate

hepatic regeneration in hepatectomized rats. J Cell Mol Med. 2010;14(6B):1605-1618.

the role of platelets In lIver regeneratIon – what don’t we know?Ton Lismanmarc KirschbaumroberT J. PorTe

PubLished in JournaL of hePaToL.

2015;63(6):1537-8

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Chapter 2 The role of plaTeleTs in liver regeneraTion – whaT don’T we know?

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In an elegant review published in the Journal, Meyer and coworkers summarize current knowledge on the role of blood platelets in liver regeneration (1). Better insight in how platelets act in amplifying liver regeneration following a partial hepatectomy might have important clinical consequences. Currently, no strategies to enhance liver regeneration to treat or avoid the ‘small for size syndrome’ are clinically available. Platelets are an unexpected, but interesting new target for clinical intervention aimed at accelerating liver regeneration. Given the vast clinical experience with platelet-modulating drugs in treatment of platelet-associated bleeding disorders or arterial thrombosis, implementation of platelet-targeted therapy for stimulation of liver regeneration is a realistic scenario. Nevertheless, many questions of the mechanism by which platelets promote liver regeneration remain unsolved.In their review, Meyer and coworkers combine knowledge obtained from in vitro models with in vivo studies on liver regeneration after partial hepatectomy and in vivo studies on liver inflammation (notably lipopolysaccaride-induced liver injury). The authors propose that following partial hepatectomy, platelets are recruited to the liver sinusoids and the space of Disse by a yet unidentified mechanism after which they release molecules (notably proteins) that directly or indirectly stimulate liver regeneration. Internalization of platelets by liver endothelial cells or hepatocytes is proposed to contribute to platelet-mediated liver regeneration.Although we agree with these proposed mechanisms, we would like to stress that many of these proposed steps have not yet been shown to contribute to liver regeneration in vivo as they are extrapolated from either in vitro models or in vivo models of inflammation, in which regeneration was not a primary read-out. It has yet to be demonstrated that release of alpha and dense granule content (containing growth factors and serotonin, respectively) within the liver remnant is required for platelet-mediated liver regeneration in vivo. Although it appears plausible that platelets deliver liver-directed mitogens to support regeneration, alternative scenarios deserve attention. For example, a role for serotonin in liver regeneration has been clearly established (2), and it has been reported that serotonin levels within platelets decrease following a partial hepatectomy in humans, which supports the theory that platelet granule excretion drives liver regeneration (3). However, a study from our center found no evidence for serotonin consumption in this setting (4). In addition, as indicated by Meyer, the reported role of serotonin in liver regeneration in animal models may not only be explained by a direct mitogenic effect of serotonin on liver cells, but can also be explained by functional defects of serotonin deficient platelets. Importantly, platelet serotonin depletion by selective serotonin reuptake inhibitors has clinically relevant effects on platelet function resulting in an increased bleeding risk and a protection from arterial thrombosis (5).Thus, although increasing clinical and experimental evidence supports the stimulatory role of platelets in liver regeneration, the mechanisms remain incompletely identified. As the manuscript by Meyer and coworkers was under review, we have published a study

proposing an alternative scenario for the role of platelets in liver regeneration (6). Using in vitro models, we demonstrated that internalization of platelets by hepatocytes contributes significantly to platelet-mediated hepatocyte proliferation. In addition, we demonstrated that platelets internalized by hepatocytes transfer RNA to the hepatocyte. Transfer of RNA from platelets to hepatocytes contributed significantly to platelet-mediated hepatocyte proliferation. Platelets contain ~9500 mRNA and ~500 miRNA species (7), and we propose that functional transfer of either or both coding and regulatory RNA species from platelets to hepatocytes may be important drivers of platelet-mediated liver regeneration. It is conceivable that delivery of platelet-derived RNA to liver cells alters the phenotype of these cells to support the regenerative process, and an increasing literature on the role of miRNAs in liver regeneration supports this theory. Importantly, our studies also demonstrated platelet internalization in hepatocytes following a partial hepatectomy in mice, suggesting that RNA transfer also occurs during liver regeneration in vivo.We fully agree with Meyer and coworkers that we need to expand our knowledge on mechanisms of platelet-mediated liver regeneration. In designing future experiments we should acknowledge that we are as yet unsure whether factors secreted by platelets (proteins or RNA) are relevant for platelet-mediated liver regeneration or that other properties of platelets drive liver regeneration. We should take effort to design rigorous in vivo studies to validate data obtained in cell culture models. Finally, we should realize that the mechanism of platelet-mediated liver regeneration may be different in the various clinical scenarios in which liver regeneration occurs. Therapies aimed at simulation of liver regeneration are not only relevant in the context of a partial hepatectomy, but also in settings of acute liver failure, ischemia-reperfusion injury, and liver fibrosis. Future research should focus on the mechanisms of platelet-mediated liver regeneration in these distinct contexts.

Chapter 2 The role of plaTeleTs in liver regeneraTion – whaT don’T we know?

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References

1. Meyer J, Lejmi E, Fontana P, Morel P, Gonelle-Gispert C, Bühler L. A focus on the role of platelets

in liver regeneration: do platelet-endothelial cell interactions initiate the regenerative process? J

Hepatol. 2015, 63(5):1263-71.

2. Lesurtel M, Graf R, Aleil B, Walther DJ, Tian Y, Jochum W, et al. Platelet-derived serotonin mediates

liver regeneration. Science 2006;312(5770):104-107.

3. Starlinger P, Assinger A, Haegele S, Wanek D, Zikeli S, Schauer D, et al. Evidence for serotonin as a

relevant inducer of liver regeneration after liver resection in humans. Hepatology. 2014;60(1):257-66.

4. Alkozai EM, van Faassen M, Kema IP, Porte RJ, Lisman T. Evidence against a role of serotonin in liver

regeneration in humans. Hepatology. 2015, 62(3):983

5. de Abajo FJ. Effects of selective serotonin reuptake inhibitors on platelet function: mechanisms,

clinical outcomes and implications for use in elderly patients. Drugs Aging. 2011;28(5):345-67.

6. Kirschbaum M, Karimian G, Adelmeijer J, Giepmans BN, Porte RJ, Lisman T. Horizontal RNA transfer

mediates platelet-induced hepatocyte proliferation. Blood. 2015, 126(6):798-806.

7. Clancy L, Freedman JE. The role of circulating platelet transcripts. J Thromb Haemost. 2015;13(Suppl

1):S33-9.

horIzontal rna transfer medIates platelet-Induced hepatocyte prolIferatIonmarc Kirschbaum GoLnar KarimianJeLLe adeLmeiJerben n.G. GiePmansroberT J. PorTeTon Lisman

PubLished in bLood. 2015;126(6):798-806

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Chapter 3 Horizontal rna transfer mediates platelet-induced Hepatocyte proliferation

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Abstract

Liver regeneration is stimulated by blood platelets, but the molecular mechanisms involved are largely unexplored. Although platelets are anucleate they do contain coding or regulatory RNAs which can be functional within the platelet or, after transfer, in other cell types. Here we show that platelets and platelet-like particles (PLPs) derived from the megakaryoblastic cell line MEG-01 stimulate proliferation of HepG2 cells. Platelets or PLPs were internalized within one hour by HepG2 cells, and accumulated in the perinuclear region of the hepatocyte. Platelet internalization also occurred following a partial hepatectomy in mice. Annexin A5 blocked platelet internalization and HepG2 proliferation. We labeled total RNA of MEG-01 cells by incorporation of 5-ethynyl-uridine (EU) and added EU-labeled PLPs to HepG2 cells. PLP-derived RNA was detected in the cytoplasm of the HepG2 cell. We next generated PLPs containing GFP-tagged actin mRNA. PLPs did not synthesize GFP, but in co-culture with HepG2 cells, significant GFP protein synthesis was demonstrated. RNA-degrading enzymes partly blocked the stimulating effect of platelets on hepatocyte proliferation. Thus, platelets stimulate hepatocyte proliferation in a mechanism which is dependent on platelet internalization by hepatocytes followed by functional transfer of RNA stored in the anucleate platelet. This mechanism may contribute to platelet-mediated liver regeneration.

Introduction

Blood platelets have essential roles in hemostasis and thrombosis, inflammation, host defense, and wound healing (1-4). Emerging evidence from recent in vitro and in vivo studies suggests that platelets have a pivotal role in liver regeneration (5-8). In experimental animal models in which platelets were depleted or functionally impaired, liver regeneration after a partial liver resection was substantially delayed (6). Conversely, following a partial liver resection in animals with a drug-induced thrombocytosis, liver regeneration was accelerated (9, 10). In a clinical study, we showed that a low platelet count is an independent predictor of delayed postoperative liver function recovery following a partial liver resection, suggesting that platelets stimulate liver regeneration also in humans (11).The molecular mechanisms of platelet-mediated stimulation of liver regeneration are largely unexplored. Platelets contain two distinct types of storage organelles (the alpha and dense granules). The alpha granules contain among many proteins, a number of growth factors that have an established role in liver regeneration (platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF)) (8, 12). In addition, the dense granules contain serotonin, which is also an established mediator of liver regeneration (5, 6). It seems plausible that local release of these factors following stimulation of the platelets contributes to platelet-mediated stimulation of regeneration. Indeed, it has been demonstrated that platelets stimulate hepatocyte proliferation in vitro, and it has been suggested that direct contact of platelets and hepatocytes is required for this effect (8). Also, in vivo studies have demonstrated that platelets accumulate in the liver parenchyma following a partial liver resection (13). Recently, a novel mechanism by which platelets may communicate with their environment has been described, which involves de novo protein synthesis. Although platelets lack a nucleus, they do contain a wide array of (pre-)mRNAs, which may be translated to protein (14-17). Protein synthesis by platelets may occur in particular following stimulation of the platelet (14-16). In addition, it has been convincingly demonstrated that platelets are capable of transferring their mRNA to other cell types including monocytic and endothelial cells (17). It was shown that the recipient cell is capable of translating platelet-derived mRNA, which may have biologically relevant effects on the recipient cell. Platelet also contain micro RNAs (miRNAs) (18-19), and functional transfer of platelet miRNA to endothelial cells has recently been described (20).Here we studied the fate of platelets during platelet-mediated stimulation of hepatocyte proliferation in vitro. We observed that platelets were internalized by hepatocytes. Based on our observation that platelets are, after internalization, directed towards the hepatocyte nucleus, we hypothesized that platelets deliver their RNA content to the hepatocyte, and that this RNA transfer contributes to platelet-mediated hepatocyte proliferation.


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Material & Methods

Cell cultureHepG2 cells (ATCC, Georgetown, WA) were cultured in DMEM medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA). MEG-01 cells (Health Protection Agency: HPA Culture Collections, London, United Kingdom) were grown in suspension using Dulbecco’s modified RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 4.5% (v/v) L-glutamine and 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were cultured without the addition of antibiotics to the culture medium.MEG-01 cells were differentiated to mature megakaryocytes and stimulated to form platelet-like particles (PLPs) according to previously described methods with some modifications (17, 21). In short, for differentiation, cells were treated for at least 10 days with 5mM valproic acid (VPA) (Sigma, St. Louis, MO). Subsequently, PLP production was stimulated by treatment with recombinant human thrombopoietin (rTPO, Life Technologies, Carlsbad, CA, 100 ng/ml, diluted in culture medium) for 72h. Cell culture medium from differentiated, rTPO-stimulated MEG-01 cells was centrifuged at 100g for 10 minutes to remove nucleated cells from the medium. The supernatant was centrifuged at 1000g for 10 minutes and the pellet containing PLPs was resuspended in culture medium. The PLP preparation contained no detectable nucleated cells as evidenced by flow cytometry using the nuclear dye Draq5 (Thermo Fisher Scientific, Etten Leur, The Netherlands). PLPs stained positive for glycoprotein Ibα and integrin αIIbβ3 by flow cytometry using antibodies from BD Biosciences, Franklin Lakes, NJ (data not shown).In selected experiments we used MEG-01 cells that, following differentiation with VPA, were transfected with CellLight® Actin-GFP (Molecular Probes, Carlsbad, CA), a modified baculovirus containing an actin-GFP mRNA construct. After 48h of incubation, virus-containing medium was replaced by standard culture medium containing rTPO. PLPs were isolated 72h following rTPO addition. Actin-GFP expressing PLPs were co-cultured up to 48h with HepG2 cells in 96well plate to assess transfer of actin-GFP mRNA to HepG2 cells. GFP synthesis was quantified by measuring fluorescence intensity (emission and excitation wavelengths were 485nm and 535nm, respectively) using a Victor3 plate reader (Perkin Elmer, Waltham, MA). A standard curve of purified GFP (Cell Biolabs, San Diego, CA) was used to quantify the amount of GFP in the cells, and values were corrected for background fluorescence of cells without actin-GFP.In another set of experiments, PLPs or isolated human platelets (200.000/µl, see below in the section on platelet isolation) were treated with RNaseA (10 or 100 U/ml Sigma, St. Louis, MO) for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse (0.1-10 U/µl) for 30 minutes (Invitrogen, Carlsbad, CA). Platelet RNA was fully degraded by this procedure (Supplementary figure 1), but platelet functionality, as assessed by flow cytometry and flow-based platelet adhesion assays, was fully preserved

(Supplementary figure 2). PLPs were washed twice with culture medium after RNaseA and RNase-inhibitor treatment.

Partial hepatectomy in miceMale C57Bl6 mice (Harlan Laboratories, Venray, The Netherlands) of 8-10 weeks of age underwent a 70% partial hepatectomy according to published protocols (22). One hour after hepatectomy, mice were terminated by exsanguination. Livers were flushed with saline and were processed for transmission electron microscopy as indicated below.

Platelet isolation, activation and labelingBlood from healthy volunteers, who claimed not to have used aspirin or other non-steroidal anti-inflammatory drugs for the preceding 10 days, was drawn into one-tenth volume of 3.4% sodium citrate. The local institutional review board approved the study, and written informed consent was obtained from each blood donor. Washed platelets were isolated as described (23), finally resuspended in cell culture medium, and platelet count was adjusted to 200.000/μL. Platelet preparations contained <1% of CD45 positive cells as assessed by flow cytometry. Platelets were activated by addition of 15μg/ml adenosine diphosphate (ADP) (Stago BNL, Leiden, The Netherlands) and 15μg/ml Thrombin Receptor Activating Peptide (TRAP) (Bachem, Bubendorf, Switzerland) and were incubated for 30 minutes at 37°C. In selected experiments, isolated platelets or PLPs (200.000/μL) were fluorescently labeled with 2.5 µM CellTracker green CMFDA (Life Technologies, Carlsbad, CA). This dye was added to the platelet suspension for 30 minutes, during which it is taken up by the platelet spontaneously. Platelets were washed with cell culture medium and diluted to a platelet count of 200.000/μL.In a separate set of experiments, platelet microparticles were isolated from resting or activated platelet preparations as described (24). In short, platelet preparations were centrifuged thrice at 1250g for 15 minutes. The final supernatant contained only platelet microparticles as evidenced by size (<1 µm) and presence of glycoprotein Ibα and integrin αIIbβ3 by flow cytometry.

Quantification of cell proliferationThe proliferation rate of HepG2 cells was estimated by quantification of 5-bromo-2’-Deoxyuridine (BrdU) incorporation using a commercially available enzyme-linked immunosorbent assay (ELISA) (Cell proliferation ELISA, BrdU, Roche, Basel, Switzerland). HepG2 cells were plated in a 96-well plate at 15.000 cells/well and cultured overnight. Subsequently, cells were washed twice with serum-free culture medium and vehicle, platelets or PLPs (both at 200.000/µl) or platelet microparticles were added and incubated under serum-free conditions for 48 hours. Alternatively, platelets were removed at earlier time points by gentle washing with culture medium, and hepatocytes were incubated with serum-free medium alone. Platelet integrity after 48 hours was preserved as evidenced


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by only a marginal increase in phosphatidylserine exposure (5.7% ± 1.7% of platelets were Annexin A5 positive by flow cytometry at baseline, compared to 9.4% ± 1.9% after 48 hours). In selected experiments platelets were treated for 30 minutes at 37°C with 30mM AnnexinA5 (ebioscience, Santa Clara, CA), or O-sialyglycoprotein endopeptidase (OSE) (30µg/ml) (Cedarlane, Burlington, OT, Canada) as described previously (25) before adding them to the HepG2 cells. Alternatively, HepG2 cells were treated with 100µg/ml Asialofetuin (Asf) (Sigma, St. Louis, MO) for 30 minutes at 37°C prior to the addition of platelets as described previously (26). All compounds were diluted in cell culture medium.After 48h of incubation of HepG2 cells with platelets or PLPs, cell culture medium was replaced by fresh culture medium containing BrdU. After 2 hours, HepG2 cells were washed three times with PBS. Cells were subsequently fixed with 4% paraformaldehyde (PFA) diluted in distilled H2O, for 10 minutes after which BrdU incorporation was measured by ELISA. In these experiments, HepG2 cells cultured in medium containing 10% serum were used as positive control and HepG2 cells cultured in serum-free medium were used as negative control. In selected experiments we ascertained that the BrdU incorporation assay gives an accurate estimate of cell proliferation by comparing BrdU incorporation with manual cell counting. Whereas the number of cells per well clearly increased by platelets, PLPs, or FCS-containing medium, serum free conditions only led to a marginal increase in the number of cells in each well, confirming BrdU test results.

Labeling of total RNA of PLPsDifferentiated MEG-01 cells were incubated for 48h with 0.5mM 5-Ethynyl Uridine (EU) (Life Technologies, Carlsbad, CA) in combination with rTPO. This procedure results in production of PLPs that have incorporated EU (a modified form of uridine) in their RNA. Isolated PLPs were washed twice in RPMI 1640 medium (Lonza, Walkersville, MI). EU was visualised using the Click-iT® RNA Alexa Fluor® 488 Imaging Kit (Life technologies, Carlsbad, CA). EU incorporation was subsequently analyzed by flow cytometry on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ) or immunofluorescence microscopy. Flow cytometry data acquisition was performed using FlowJo X (TreeStar, Ashland, OR).

Immunofluorescence stainingFor immunofluorescence experiments, HepG2 cells were cultured overnight and were plated to obtain ~50% confluence on poly-L-lysine (0.1mg/ml) (Santa Cruz Biotechnology, Dallas, TX) coated glass coverslips (Thermo Scientific, Waltham, MA) in a 24 well plate (Corning, Corning, NY). Coverslips were treated with poly-L-lysine for 30 minutes at 37⁰C and were washed three times with PBS before HepG2 cells were seeded. After addition of CellTracker Green CMFDA-labeled platelets, the culture medium was removed after various time points and cells were washed twice with PBS to remove non-adherent platelets or PLPs. Cells were subsequently fixed with 4% paraformaldehyde (PFA) for 10 minutes.HepG2 cells were stained for actin with Alexa 594-labelled phalloidin (Life Technologies,

Carlsbad, CA) or with a rabbit anti-actin antibody (Sigma, St. Louis, MO) followed by an Alexa 594-labeled goat anti rabbit antibody (Life Technologies, Carlsbad, CA). Nuclei were identified using DNA staining with DAPI by using VECTASHIELD Hard-set mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Imaging was performed on a Leica SP2 AOBS confocal microscope (Leica Microsystems, Solms, Germany) and on a Leica DMI4000B LED. Images were captured using Leica Confocal Version 2.5 software or Leica Application Suite Advanced Fluorescence software (LAS AF). Image processing was performed using Imaris 7.1.1 (Bitplane Scientific Software, Zurich, Switzerland).

Electron microscopyFor ultrastructural analysis, HepG2 cells were co-cultured with platelets and were fixed with 2.5% glutaraldehyde and 1% PFA in 0.1M sodium cacodylate buffer (pH 7.4) for 24 hours. Pieces (~1 mm3) of remnant livers from mice that underwent a 70% partial hepatectomy were also fixed in this buffer. The fixed cells or tissue were then rinsed with 0.1M sodium cacodylate buffer and were postfixed with 2% osmium tetroxide for 1h at 4⁰C. Cells were washed three times in double distilled H2O, dehydrated with a graded series of ethanol (50%, 70% and 100%; 3 x 10 minutes each) followed by embedding in Epon and ultrathin sectioning. After uranyl acetate and lead citrate staining, ultrathin sections were examined by the transmission electron microscope FEI CM100 (Philips, Amsterdam, The Netherlands). Images were captured with an Advantage CCD camera using iTEM software (Olympus, Tokio, Japan).

Statistical analysisStatistical analysis was performed with the GraphPad Prism 5 (San Diego, CA) software package. Continuous variables were expressed as mean ± SD or median and range. Values are representative of at least 3 independent experiments performed in triplicate. Continuous data were tested for normality and analyzed by t-test, Mann-Whitney U-test or one-way ANOVA, as appropriate. A P value of less than 0.05 was considered statistically significant. Images are representative for at least 3 independent experiments.

Supplementary materials & methods

Platelet activation analysis by flow cytometryIsolated human platelets (200.000/µl) were activated by addition of 15µg/ml adenosine diphosphate (ADP) (Stago BNL, Leiden, The Netherlands) and 15µg/ml Thrombin Receptor Activating Peptide (TRAP) (Bachem, Bubendorf, Switzerland) and were incubated for 30 minutes at 37°C. Resting or activated platelets were treated with RNaseA (100 U/ml Sigma, St. Louis, MO) for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse (10 U/µl) for 30 minutes (Invitrogen, Carlsbad, CA). Platelets were


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stained with a phycoerythrin-labeled antibody to CD62-P (BD Biosciences, Franklin Lakes, NJ). CD62-P expression was subsequently analyzed by flow cytometry on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Flow cytometry data acquisition was performed using FlowJo X (TreeStar, Ashland, OR).

RNA integrityIsolated human platelets (200.000/µl) were treated with RNaseA (1, 10 or 100 U/ml Sigma, St. Louis, MO) for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse (10 U/µl) for 30 minutes (Invitrogen, Carlsbad, CA). Platelets were centrifuged at 500g for 15 minutes. For total RNA isolation, the pellet was lysed in Trizol reagent (Invitrogen, Carlsbad, CA) and processed according to the manufacturer´s instructions (Invitrogen, Carlsbad, CA). The integrity of RNA was analyzed by gel electrophoresis. The gel contained 1% agarose in TBE buffer (89 mM Tris, 89mM boric acid, 2mM EDTA) and 2 mg/ml ethidium bromide. RNA was diluted in nuclease-free water (Invitrogen, Carlsbad, CA) and the loading dye Orange G (Sigma, St. Louis, MO) was added. 500 ng RNA from each sample was loaded on the gel. Gels were run at 90V for 25 minutes. Images were captured using an ImageMaster VDS-CL (GE Healthcare Bio Sciences, Pittsburgh, PA).

Platelet Adhesion AssayRed blood cells and platelets were isolated from whole blood of healthy volunteers who had blood group O. Platelets were treated with RNaseA (100 U/ml Sigma, St. Louis, MO) for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse (10 U/µl) for 30 minutes (Invitrogen, Carlsbad, CA). Cells were mixed with patient plasma or plasma from healthy volunteers to obtain reconstituted blood with a hematocrit of 40% and a platelet count of 250,000/µl. Platelet adhesion in reconstituted blood samples was assessed using a cone and plate viscometer (Diamed Impact R, Turnhout, Belgium). Uncoated Diamed wells were perfused at shear rate of 1,800/second for 2 minutes according to the instructions of the manufacturer. Platelet adhesion was quantified using May-Grünwald staining followed by software-assisted morphometric analysis using the Diamed apparatus and software delivered by the manufacturer.

Results

Platelets stimulate hepatocyte proliferationTo investigate the proliferative effect of platelets on hepatocytes we co-cultured freshly isolated human platelets for 48h with HepG2 cells under serum-free conditions. Serum free conditions for 48h were well tolerated by HepG2 cells as evidenced by a lack of Trypan Blue uptake (not shown). Addition of resting platelets resulted in a 1.5-fold increase of BrdU incorporation into HepG2 cell DNA (Fig. 1A). When activated platelets were added to the HepG2 cells, a 2.2-fold increase in BrdU incorporation was seen. Subsequently, we added resting platelets to hepatocytes, removed platelets at various time points, and assessed hepatocyte proliferation at 48 hours. A maximal effect on hepatocyte proliferation was observed when platelets were present for at least 24h hours (Fig 1B). We next investigated whether platelet microparticles also stimulated HepG2 cell proliferation. Whereas resting or activated platelet preparations potently stimulated hepatocyte proliferation, microparticles isolated from these preparations had no stimulatory effects (Fig.1A).

Figure 1: Isolated platelets increase proliferation of hepatocytes

(A) Resting or activated platelets (plt) or microparticles (MP) isolated from these platelet preparations were added to HepG2 cells and incubated under serum free conditions. After 48 hours, cell proliferation rate was estimated by quantifying BrdU incorporation. Control groups represent HepG2 cells cultured in presence or absence of FCS. *P < 0.05 compared with -FCS. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.(B) Resting platelets were added to HepG2 cells and incubated under serum free conditions. After various time points, platelets were removed by gentle washing and replaced by serum free medium. After 48 hours, cell proliferation rate was estimated by quantifying BrdU incorporation. Control groups represent HepG2 cells cultured in presence or absence of FCS. *P < 0.05 compared with -FCS. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.


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Platelets are internalized during hepatocyte proliferationTo study whether platelets stimulate proliferation of HepG2 cells by direct contact, we added CellTracker green CMFDA-labeled platelets to HepG2 cells. After 30 minutes of co-incubation, platelets were present at the HepG2 cell membrane as shown by fluorescent imaging (Fig. 2A, B) and electron microscopy (Fig. 2B). After 1h platelets were observed inside the HepG2 cells (Fig. 2A). Confocal laser scanning microscopy (CLSM) confirmed that platelets were located inside the cell and not attached to the outer cell membrane (Fig. 2C). After one hour, platelets appeared predominantly located in the perinuclear region of the HepG2 cells, which was confirmed by electron microscopy as shown in Fig. 2D. Platelets were found within a few nanometers from the nucleus and were surrounded by endoplasmic reticulum. Internalized platelets were observed in >50% of HepG2 cells. Platelet internalization in hepatocytes was also demonstrated in mice that underwent a 70% partial hepatectomy. One hour after hepatectomy, platelets were found within hepatocytes (Fig. 2E).

Inhibition of platelet uptake reduces hepatocyte proliferation It has been well established that clustering of platelet glycoprotein Ibα is responsible for clearance of chilled platelets by hepatocytes in vivo, and that this process is mediated by the hepatocyte Ashwell-Morell receptor (27). Additionally, the Ashwell-Morell receptor is responsible for clearance of human platelets by porcine liver endothelial cells in a xenotransplantation context (28). Based on these data, we tested the involvement of platelet glycoprotein Ibα and the hepatocyte Ashwell-Morell receptor in platelet internalization by HepG2 cells. Treatment with O-sialoglycoprotein endopeptidase (OSE), which removes glycoprotein Ibα from the platelet surface, or asialofetuin (Asf), a competitive inhibitor of the Ashwell-Morell receptor did not reduce platelet uptake by HepG2 cells (Fig. 3A). In contrast, incubation of activated platelets with AnnexinA5 prior to 2h of co-culturing with HepG2 cells resulted in a significant reduction of platelet internalization by approximately 65% (Fig. 3A).We next investigated whether platelet uptake is required for platelet-mediated HepG2 cell proliferation. AnnexinA5-treated platelets were co-cultured for 48h with HepG2 cells, and cell proliferation was estimated by a BrdU incorporation assay. AnnexinA5-treated activated platelets showed a significantly reduced proliferation of HepG2 cells compared to untreated platelets (Fig. 3B/C). Treatment with OSE or Asf did not reduce HepG2 cell proliferation (Fig 3B).

Figure 2: Isolated platelets are internalized by hepatocytes

(A) Activated platelets (green) were labeled with CellTracker green CMFDA and were incubated under serum free conditions with HepG2 cells. Fluorescent images were taken after 5 min, 30 min, and 1h. HepG2 cells were stained for actin (red) and nuclei were stained with DAPI (blue). Original magnification 200x. Scale bar denotes 20µm.(B) (i) High magnification fluorescence image of single platelets (arrows) attached to the HepG2 cell membrane after 30 minutes of co-culturing. Original magnification 400x. Scale bare denotes 10µm. (ii) Electron micrograph of a single platelet attached to a HepG2 membrane. Original magnification 9700x, Scale bar denotes 1µm.(C) Confocal microscopy image of HepG2 cells that have been exposed to platelets. Images were captured after one hour incubation with CMFDA-labeled platelets (green). The HepG2 cells were stained for actin (red). The image shows a slice from the middle of the confocal stack. Original magnification 400x.(D) TEM imaging of hepatocytes that have been exposed to platelets.Lower magnification (9700x) image of a group of HepG2 cells showing platelets within hepatocytes indicated by arrows. Scale bar denotes 5µm.High magnification image of the same region (24500x) shows the internalized platelet (*) surrounded by ER and located close to the nucleus (#). The arrow indicates the plasma membrane of the HepG2 cell. Scale bar denotes 1µm. Images are representative for at least 3 independent experiments.(E) TEM imaging of mouse liver one hour after hepatectomyA section of liver tissue taken from a mouse that underwent a 70% hepatectomy. The arrow indicates a platelet within a hepatocyte. Original magnification 5800x. Scale bar denotes 10µm.High magnification image of the same region (33000x) shows the internalized platelet by the hepatocyte. Scale bar denotes 1µm.


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Figure 3: AnnexinA5 inhibits platelet uptake and platelet mediated hepatocyte proliferation

(A) Activated platelets were added to HepG2 cells in presence of Annexin A5 (AV), O-sialoglycoprotein endopeptidase (OSE) or asialofetuin (Asf). Internalized platelets were quantified based on fluorescence microscopy. Platelets were manually counted in at least 5 high-power fields and expressed as number of platelets/100 cells. ***P < 0.001. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.(B) Activated platelets were added to HepG2 cells in presence or absence of various inhibitors and incubated under serum free conditions. After 48 hours, cell proliferation rate was estimated by quantifying BrdU incorporation. Control groups represent HepG2 cells cultured in presence or absence of FCS. In addition, the effect of AnnexinA5 on BrdU incorporation in the absence of platelets, but in the presence of FCS is shown. *P < 0.05 compared with -FCS. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.(C) Representative fluorescence microscopy image from activated platelets incubated with HepG2 cells in absence (i) or presence (ii) of AnnexinA5 for 2 hours.HepG2 cells were stained for actin (red). Platelets were labeled with CMFDA (green). Original magnification 200x. Scale bar denotes 20µm.

Transfer of RNA from PLPs following internalization by hepatocytesWe next used platelet-like particles (PLPs) generated from the megakaryoblastic cell line MEG-01 to further assess the mechanism behind platelet-induced hepatocyte proliferation. PLPs had an identical potency to stimulate HepG2 proliferation compared to isolated platelets (Fig. 4A). CFMDA-labeled PLPs were also internalized by HepG2 cells and once internalized were located in the perinuclear region (Fig. 4B). To assess potential transfer of platelet RNA to the hepatocyte following uptake of platelets, we labeled RNA in MEG-01 cells by incorporation of 5-Ethynyl Uridine (EU), and generated PLPs from these labeled megakaryocytes. EU-modified PLPs were isolated 48h after rTPO stimulation from differentiated MEG-01 cells and were analyzed by flow cytometry using a fluorescently labeled probe that specifically binds EU. Approximately 37% of the PLPs were positive for EU (Fig. 4C). Next, EU-containing PLPs were co-cultured with HepG2 cells and were assessed by confocal microscopy. EU-positive PLPs were observed in the perinuclear region of HepG2 cells. EU staining was predominantly present in structures resembling PLPs. Importantly, EU accumulated over time in the cytoplasm of the recipient HepG2 cell (Fig. 4D), indicative of transfer of PLP-derived RNA to HepG2 cells.

Figure 4: Platelet-like particles stimulate proliferation of and are internalised by HepG2 cells

(A) Activated platelets or PLPs were added to HepG2 cells and incubated under serum free conditions. After 48 hours, cell proliferation rate was estimated by quantifying BrdU incorporation. *P < 0.05. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.(B) PLPs (green) were labeled with CMFDA and were incubated under serum free conditions for 4h with HepG2 cells. Original magnification 400x. Scale bar denotes 10µm.(C) Quantification of EU incorporation into PLP RNA by flow cytometry. PLPs were generated from MEG01 cells that had been exposed to EU. EU in PLPs was visualised using Click-iT® RNA Alexa Fluor® 488. Histogram plot overlay shows control PLPs (red line) and PLPs generated from EU-treated MEG01 cells (blue line).(D) EU labeled PLPs were incubated under serum free conditions for 2, 4, or 8h with HepG2 cells. Dashed lines represent the HepG2 cell membrane. Structures resembling PLPs (arrows) as well as parts of the cytoplasm of the HepG2 cell are EU positive (green, arrowheads). Original magnification 400x. Scale bar denotes 10µm.


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RNA from PLPs is translated by hepatocytes following PLP-to-hepatocyte RNA transferTo test whether RNA transferred by the platelet to the HepG2 cell may be translated to protein by the recipient cell, we transfected MEG-01 cells with a GFP-tagged actin mRNA construct. By flow cytometry and fluorescence microscopy, we demonstrated expression of actin-GFP protein in MEG-01 cells (Fig. 5A/B). Approximately 26% of all PLPs generated from these transfected MEG01 cells were GFP positive (Fig. 5B). Incubation of HepG2 cells with these GFP-tagged actin mRNA containing PLPs, resulted in GFP expression throughout the hepatocyte cytoskeleton, suggesting translation of PLP-derived mRNA by the hepatocyte (Fig. 5C). To confirm that the GFP-tagged actin is synthesized by the hepatocyte from platelet mRNA, we quantified GPF protein content of hepatocytes over time. As shown in Fig. 5D, actin-GFP protein increased over time in HepG2 cells coincubated with PLPs. Importantly, the GFP content of PLPs cultured in the absence of HepG2 cells did not increase over time, indicating that the GFP present in the PLPs was already synthesized by the megakaryocyte. Subsequently, we treated actin-GFP-containing PLPs with RNA-degrading enzymes. GFP protein production by these RNase-treated PLPs was almost fully blunted confirming that HepG2 cells translate the actin-GFP mRNA transferred by PLPs to protein.

Platelet RNA plays a critical role in platelet-mediated stimulation of hepatocyte proliferationTo test whether transfer of platelet RNA to the HepG2 cell is required for platelet-mediated stimulation of HepG2 cell proliferation, we performed proliferation experiments in which we compared proliferative activity of intact and RNA-depleted platelets. Treatment of platelets with an RNA-degrading enzyme substantially and significantly decreased platelet-mediated stimulation of HepG2 proliferation (Fig 5E).

Figure 5: Actin-GFP mRNA from PLPs is transferred to and translated by HepG2 cells

(A) Representative fluorescence microscopy image from actin-GFP transfected MEG-01 cells, 48h after transfection. Shown is a bright field image combined with actin-GFP signal. Original magnification 100x. Scale bar denotes 25µm.(B) Representative flow cytometry analysis of isolated PLPs from actin-GFP transfected MEG-01 cells. Left panel: Histogram plot of actin-GFP expressing MEG-01 cells (green line) compared to control MEG-01 cells (black line). Right panel: Histogram plot of PLPs derived from actin-GFP expressing MEG-01 cells (green line) compared to control PLPs (black line).(C) PLPs from actin-GFP transfected MEG-01 cells (green, indicated by arrowheads) were co-cultured with HepG2 cells for 4h. Internalization was assessed by confocal microscopy. (i) Total actin inside the HepG2 cell is stained in red. (ii) Actin-GFP from PLPs (green) was localized throughout the cytoplasm of the HepG2 cell. (iii) Total actin in HepG2 cells that were not treated with PLPs is shown for comparison. Original magnification 400x. Scale bar denotes 5µm.(D) Actin-GFP expressing PLPs were cultured alone or in combination with HepG2 cells for up to 48h. PLPs that were co-cultured with HepG2 cells were pretreated with 100 U/ml RNaseA or vehicle prior to addition to the HepG2 cells. Actin-GFP expression was quantified at indicated time points. *P < 0.05. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.(E) Activated platelets treated with vehicle or different concentrations of RNaseA were added to HepG2 cells and incubated under serum free conditions. After 48 hours, cell proliferation rate was estimated by quantifying BrdU incorporation. Control groups represent HepG2 cells cultured in presence or absence of FCS. *P < 0.05. Data represent the mean of three independent triplicate experiments. Error bars indicate SD.


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Supplementary figure 1: Platelet RNA degradation by RNaseA treatment

(A) Platelets were treated with 1, 10 or 100 U/ml of RNaseA or vehicle for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse. Platelet RNA was isolated and RNA integrity was assessed by agarose gel electrophoresis.

Supplementary figure 2: Platelet functionality is preserved after RNaseA treatment

(A) Platelets were treated with 10 or 100 U/ml of RNaseA or vehicle for 1 hour at 37°C. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse. Platelets were added to isolated red blood cells and allowed to adhere to a plastic surface under conditions of flow. Aggregate size and surface coverage quantification are shown. Data represent the mean of three independent experiments performed in triplicate. Error bars indicate SD.(B) Quantification of CD62-P (P-selectin) expression on resting and activated platelets by flow cytometry. Resting or activated platelets were treated with 100 U/ml RNaseA or vehicle. Subsequently, RNaseA was inhibited by incubation with SUPERase In RNAse and platelets were stained for CD62-P. Histogram plot overlay shows control platelets (black line), RNaseA treated platelets (green line) activated platelets (red line) and activated RNaseA-treated platelets (blue line).

Discussion

This study shows that platelet-mediated stimulation of HepG2 cell proliferation requires platelet internalization by hepatocytes. Following this internalization, platelets transfer RNA to the hepatocyte, and we demonstrated protein synthesis from platelet-derived mRNA. Importantly, platelet RNA contributes substantially to platelet-mediated hepatocyte proliferation suggesting that transfer of platelet RNA to the hepatocyte with subsequent protein synthesis by the recipient cell is key in this process. Since we also demonstrated platelet internalization by hepatocytes following a partial hepatectomy in mice, it appears plausible that functional platelet RNA transfer is also relevant for platelet-mediated liver regeneration.The molecular mechanisms of platelet-mediated stimulation of liver regeneration are only poorly understood. Local release of growth factors such as PDGF, HGF, IGF, VEGF, and serotonin from platelet granules may explain the stimulatory effect of platelets on liver regeneration (6, 8, 12). Nevertheless, it has yet to be demonstrated that 1) local release of growth factors occurs within the liver parenchyma, and 2) that growth factor release from platelets is the major mechanism responsible for platelet-mediated liver regeneration. Our results indicate that transfer of platelet RNA to the hepatocyte cytoplasm may be a crucial mechanism for platelet-mediated liver regeneration in addition to release of growth factor proteins stored in platelet granules. As degradation of all platelet RNA did not completely reduce the proliferative effect on hepatocytes, it appears that both release of growth factors by the platelet and transfer of platelet RNA are important for platelet-mediated hepatocyte proliferation. Whether both mechanisms are also relevant for platelet-mediated liver regeneration in vivo remains to be established.Platelets contain about 8500 individual mRNA species and it has been convincingly demonstrated that several of those mRNAs can be translated by the platelet itself into protein as the platelet contains the full machinery required for protein synthesis (14, 16, 29). In line with previously published data we demonstrate that this platelet mRNA is transferred to and translated by nucleated cells (17). This ‘parasitic protein synthesis’ may be much more efficient compared to protein synthesis by the platelet itself. Platelets also contain ~500 miRNA species, and transfer of regulatory RNAs in addition to or instead of transfer of coding RNA could also be required for platelet-mediated hepatocyte proliferation (19). We demonstrate that transfer of platelet RNA to the nucleated hepatocyte has direct effects on the functional properties of the recipient cell, and it is tempting to speculate that transfer of platelet RNA to the recipient cell is directly responsible for the increased cellular proliferation. Which of the 8500 platelet mRNA or ~500 miRNA species are required for platelet-mediated hepatocyte proliferation remains to be determined, but may include mRNAs encoding growth factors, transcription factors, or cell-cycle genes, and/or regulatory RNAs.


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It has been previously shown that transfer of mRNA and miRNA between two types of nucleated cells occurs via vesicular transport (30-32). Importantly, mRNA transfer between two nucleated cells has functional consequences for the recipient cell. Moreover, it has been demonstrated that microvesicles derived from human liver stem cells were internalized by hepatocytes, resulting in transfer of mRNA. This vesicular RNA supported hepatocyte proliferation and induced apoptosis resistance (33).Given the small size of the anucleate platelet, platelet RNA transfer to nucleated cells resembles the RNA transport via microvesicles between two nucleated cells (32,34). Since vesicular RNA transport between two nucleated cells appears a common and widespread phenomenon, it is not unlikely that also transfer of platelet RNA to nucleated cells with resulting biological changes in the recipient cell is a common physiological or pathophysiological phenomenon. Interestingly, platelet-derived microparticles had no effect on hepatocyte proliferation, suggesting that platelet-specific functions are required for this platelet to hepatocyte communication. It has now been well established that platelets have biological functions that by far exceed their well-recognized role in thrombosis and hemostasis (2-4). Transfer of platelet RNA to nucleated cells resulting in parasitic protein synthesis with consequent changes in the recipient cell may be relevant in the role of platelets in processes such as inflammation, angiogenesis, and repair of tissue other than the liver.To our knowledge, we are the first to show that platelets that are taken up by cells preferentially accumulate at the nucleus. This peculiar phenomenon may be required for hepatocyte proliferation in a mechanism involving transfer of platelet pre-mRNA to the hepatocyte nucleus with subsequent nuclear splicing, export of mature mRNA to the cytosol and eventually protein synthesis. Earlier studies on platelet uptake by hepatocytes were performed in the context of removal of aged or dysfunctional platelets from circulation (26,35). Platelet uptake by hepatocytes in this context is assumed to result in platelet breakdown, presumably in lysosomes, although the fate of platelets after uptake by hepatocytes has not been studied in this context. Platelet clearance by hepatocytes has been shown to occur in a process dependent on platelet GPIb and the hepatocyte Ashwell-Morell receptor (26,36). The platelet uptake described in our studies was independent from these receptors. Rather platelets were taken up in a process dependent on negatively charged phospholipids, which may explain why activated platelets are better stimulators of HepG2 proliferation compared to resting platelets. The trafficking of the platelet toward the hepatocyte nucleus following platelet internalization appears a process that is distinct from platelet uptake in the context of platelet clearance. The mechanisms responsible for platelet uptake and translocation to the nucleus are yet unknown, but may resemble translocation of plasma membrane proteins such as EGF receptors, the insulin receptor, and the HGF receptor cMet to the nucleus of the hepatocyte (37-39).

In conclusion, our combined results demonstrate that platelets stimulate hepatocyte proliferation in a mechanism which is, at least in part, dependent on platelet internalization by hepatocytes followed by transfer of RNA stored in the anucleate platelet. Although in vivo confirmation of this mechanism is required, these studies provide fundamentally new insights in the stimulatory effects of platelets on liver regeneration. In addition, platelet communication with nucleated cells by transfer of RNA may be relevant in other processes in which platelets play key modulatory roles.


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Acknowledgements:We thank Susanne Veldhuis for expert technical assistance with the animal experiment. This work was supported in part by a grant from The Netherlands Organisation for Scientific Research (VIDI, 917.11.304 to T.L.).

Authorship and conflict of interest statementMK, GK, JA: performed experiments and interpreted data, BNGG: supervised microscopy experiments and interpreted data, RJP: interpreted data, TL: designed and supervised the study, interpreted data, obtained funding. MK and TL wrote the manuscript. All authors revised and approved the manuscript.None of the authors have a conflict of interest to report

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transIent von wIllebrand factor-medIated platelet Influx stImulates lIver regeneratIon after partIal hepatectomy In mIce marc KirschbaumcraiG n. JenneZwanida J. VeLdhuisKLaas a. sJoLLemaPeTer J. LenTinGben n.G. GiePmansroberT J. PorTePauL KubescéciLe V. denisTon Lisman

PubLished in LiVer inTernaTionaL 2017

10.1111/LiV.13386

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Chapter 4 TransienT von Willebrand facTor-mediaTed plaTeleT influx sTimulaTes liver regeneraTion afTer parTial hepaTecTomy in mice

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Abstract

Background & aims: In addition to their function in thrombosis and hemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx.Methods: We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry.Results: We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopenia was induced 2 hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. Conclusions: A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.

Introduction

The liver has a unique regenerative capacity following damage or surgical resection. Liver regeneration starts with a well-organized and complex series of signals which is generated by cytokines and growth factors (1). Accumulating evidence from in vitro and in vivo studies suggests that platelets have a pivotal role in liver regeneration (2-11). In animal models in which platelets were depleted or functionally impaired, liver regeneration was substantially delayed after a partial liver resection (2,3). Conversely, induction of thrombocytosis stimulated liver regeneration (3,4,12). In a clinical study, we showed that a decreased platelet count is an independent predictor of delayed postoperative liver function recovery after a partial liver resection (10). More recently, it was demonstrated that intraoperative platelet count and platelet transfusion were associated with faster liver regeneration in living donor transplant recipients (11). The molecular mechanisms of platelet-mediated stimulation of liver regeneration are, however, still largely unknown (13).Platelets bind to liver sinusoidal endothelial cells in vitro and this interaction stimulates hepatocyte proliferation (14). In addition, platelets were found to accumulate in the liver parenchyma after a partial liver resection in experimental animal models (3,9). A direct interaction between platelets and hepatocytes is crucial for platelet-mediated stimulation of hepatocyte proliferation in vitro (5) but the significance of these findings for liver regeneration in vivo are unclear (15). Release of growth factors that are stored within platelets may be responsible for platelet-mediated liver regeneration (5,16,17), but direct evidence from in vivo experiments for this is lacking (15,18). For example, release of serotonin from platelet dense granules has been suggested by some to mediate platelet-mediated liver regeneration in mice and humans (2,16). However, although mice lacking serotonin in their platelets have reduced regenerative capacity, this may be explained by a reduced functional capacity of serotonin-deficient platelets rather than by a specific defect in mitogenic activity of the platelets (19). Furthermore, human studies have provided additional evidence against a role of serotonin in liver regeneration in humans (20).An alternative scenario for platelet mediated liver regeneration involves transfer of RNA from platelets to hepatocytes as has been recently demonstrated in an in vitro model (9). Although platelet influx of the liver remnant shortly after partial hepatectomy has been well established in rodent models, it is yet unknown what triggers platelet accumulation and whether platelets persist in the liver remnant over time.It has been shown that levels of the platelet adhesive protein von Willebrand factor (VWF) are increased in plasma following a partial liver resection in rats and in mice (21,22). VWF protein is also highly upregulated in liver sinusoidal endothelial cells following a partial hepatectomy (21). Although the latter finding was interpreted as a potential role for VWF in tissue remodeling during liver regeneration, it may also be that VWF release from liver sinusoidal endothelial cells leads to platelet influx into the liver remnant following partial hepatectomy.In this study we tested the hypothesis that VWF is involved in platelet influx into the liver


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remnant. By use of intravital microscopy we studied the dynamics of platelet influx into the liver sinusoids upon partial liver resection in mice. We demonstrated rapid and transient platelet influx into the liver parenchyma after partial liver resection, which was dependent on VWF.

Material & Methods

Partial liver resection in miceMale C57Bl6 mice (Charles River, Leiden, The Netherlands) of 8-10 weeks of age or male mice deficient in VWF (VWF-/-, on a C57BL/6 background (23)) underwent a 70% partial liver resection according to published protocols (24). In sham surgeries, mice underwent an identical procedure with the exception of ligation and removal of the liver lobes. Surgical procedures were performed under isoflurane inhalation anesthesia (Abbott, Chicago, IL). Thrombocytopenia was induced by intravenous injection of a rat monoclonal antibody directed against mouse GpIbα (4µg/g body weight) (Emfret, Würzburg, Germany). In selected experiments, 50µg of the polyclonal rabbit anti-VWF antibody A0082 (DAKO, Glostrup, Denmark) was intravenously injected 30 minutes prior to resection. The Institutional Animal Care and Use Committee of the University of Groningen, The Netherlands approved these studies.Mice were terminated by exsanguination from the inferior vena cava after injection of 150µl 3.4% sodium citrate (Merck, Germany) diluted in NaCl (0.9%) in the spleen. Collected blood samples were centrifuged at 1400g for 10 minutes (without brake) to obtain plasma and were stored at -80°C. Livers were fixed in 4% formaldehyde or were snap frozen in liquid nitrogen for immunohistochemical analyses.

Confocal intravital microscopy Platelets were imaged in living mice shortly after the hepatectomy by intravital microscopy as described previously (25). Platelets were labeled in vivo by intravenous injection of 1.6µg phycoerthrin (PE)-conjugated hamster anti-mouse CD49b (clone HMα2) (BD Pharmigen, San Diego, CA) just prior to an imaging session as described (25). After partial liver resection or sham operation the animal was placed in a right lateral position on an adjustable microscope stage. Mouse body temperature was maintained at 37 °C. After the liver was exteriorized, it was placed on the inverted microscope, the liver surface was covered with a small piece of saline-soaked KimWipe (Kimberly-Clark, Roswell, GA) to keep the organ moist and hold the organ in position. Image acquisition was started as soon as possible and was performed for 1 hour. Alternatively, mice were examined after 4 hours, 1 day or 3 days after the hepatectomy by relaparotomy. We used two different confocal microscope set-ups in this study. The first set-up has been described previously (25). Another set of experiments was performed using an inverted Zeiss LSM 780 NLO microscope (Axio Observer.Z1; Carl Zeiss, Ulm, Germany) equipped with a temperature controlled incubator (XL S1 DARK; Pecon, Erbach, Germany). For these experiments a 20×PlnApo, 0.8 NA

objective was used. Images were captured using 488nm Argon laser and a gallium arsenide phosphide (GaAsP) spectral detector (Carl Zeiss, Ulm, Germany) at 508 nm to 561 nm for autofluorescence detection, revealing the vasculature, and 569 nm to 655 nm for PE-detection. Hardware control was via the ZEN Black acquisition software (Carl Zeiss, Ulm, Germany).

Intravital microscopy image processing and platelet aggregate analysisFor IVM data analysis, tif images were exported from the Volocity (Improvision Inc., Lexington, MA) acquisition software or from ZEN Black software. Images for platelet aggregate quantification were imported directly into ImageJ (version 1.45; US National Institutes of Health) and image contrast was set to maximum for sharp definition of the borders of each platelet aggregate. The same settings were applied to images from all treatment groups within a single experiment. Analysis of platelet aggregates was performed using the Analyze Particles function within ImageJ. Videos underwent contrast enhancement within the acquisition software package, adjusting the Black Point for each fluorescence channel. Again, the same settings were applied to the videos of all treatment groups within a given experiment. Videos were exported as .avi files and were converted to an appropriate size, resolution, and frame rate using Microsoft Movie Maker (Microsoft, San Jose, CA). A platelet aggregate was defined as a positive signal of 10 pixels (1 μm) or more.

ImmunohistochemistryDeparaffinized liver sections were subjected to antigen retrieval. Ki-67 sections were incubated for 20 minutes in boiling Tris/EDTA buffer, pH 9.0. Endogenous peroxidase was blocked by 3% H2O2 for 30 minutes. Sections were incubated with monoclonal rabbit anti-Ki67 antibody (1:200 in TBS + 1%BSA) (Abcam, Cambridge, UK) at 4°C for at least 16h. Next, a secondary peroxidase-conjugated goat-anti-rabbit antibody (1:100, DAKO, Glostrup, Denmark) and a tertiary peroxidase-conjugated rabbit-anti-goat antibody (1:100, DAKO, Glostrup, Denmark) were used. Prior to the incubation with the secondary and tertiary antibody normal rabbit serum (1:100) was added for 30 minutes. The peroxidase activity was visualized by a 10 minute incubation in 3,3-diaminobenzidine tetrachloride (Sigma, St.Louis, MO). Subsequently the sections were counterstained for 1 minute with haematoxylin and mounted with Kaiser’s glycerin gelatin. Ki-67-positive hepatocytes were manually counted in at least 5 high-power fields per mouse and expressed as percentage of all hepatocytes.

Statistical analysisStatistical analysis was performed with the GraphPad Prism 5 (San Diego, CA) software package. Continuous variables were expressed as mean ± SD or median and range. Values are representative of at least 3 independent experiments performed in triplicate. Continuous data were tested for normality and analyzed by t-test, Mann-Whitney U-test or one-way ANOVA, as appropriate. A P value of less than 0.05 was considered statistically significant.


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Results

Transient platelet influx into the liver remnant immediately after partial liver resectionWe studied platelet influx after partial liver resection in mice using intravital microscopy. Following partial liver resection, an immediate platelet influx in the remnant liver was observed (Fig. 1A). Platelets appeared to attach to the endothelial cells and formed both small and larger aggregates in the sinusoid, but the aggregates never became occlusive (Supplementary video 1). The aggregates were unstable as platelets or groups of platelets frequently detached from these aggregates, and continuous reattachment of new platelets was observed. In sham-operated mice some platelets transiently attached to the endothelial cells but the quantity and size of platelet aggregates was substantially less compared to the mice that underwent partial hepatectomy (Fig. 1A, supplementary video 2). Intravital imaging at 4h, 1 day, and 3 days after liver resection showed minimal platelet deposition compared to the early phase (<1h) after liver resection (Fig. 1B, supplementary video 3).

Figure 1: Platelet influx into the liver remnant immediately after partial liver resection (A) Representative intravital microscopy images of livers of c57/bl6 mice at 15 minutes after a partial liver resection or sham operation. Platelets were labeled in vivo with PE-conjugated anti-CD49 (red). The autofluorescence signal of the liver is displayed to visualize liver anatomy (green). Original magnification 200x. Scale bar denotes 50µm.(B) Quantification of the total number of platelet aggregates in the sinusoids of the liver remnant. Platelet aggregates were quantified in sham operated mice at 15 minutes after the sham surgery and at different time points after partial liver resection. *P < 0.05, **P < 0.01 versus sham. + P < 0.05 versus 30 min hepatectomy. Data represent the mean of three animals. Error bars indicate SD.

Transient platelet influx stimulates liver regenerationTo study whether the transient influx of platelets following a partial hepatectomy is sufficient to stimulate liver regeneration, we compared liver regeneration in mice that were rendered thrombocytopenic 2 hours prior to or two hours after a partial hepatectomy. Intravenous injection of platelet-depleting antibodies resulted in a >90% reduction in platelet count within 15 minutes (data not shown). Compared to mice with a normal platelet count, mice that were rendered thrombocytopenic prior to partial hepatectomy showed markedly impaired regeneration as evidenced by a reduced liver/body weight ratio (Fig. 2A) and a reduction in ki67-positive cells at day 3 after partial hepatectomy (Fig. 2B-C). In contrast, liver regeneration was only marginally impaired in mice that were rendered thrombocytopenic 2 hours after partial hepatectomy. Importantly, no excessive perioperative bleeding was observed in thrombocytopenic animals, nor was there evidence of bleeding observed during termination.

Figure 2: Transient platelet influx stimulates liver regeneration. (A) Quantification of liver to body weight ratio in mice 3 days after partial liver resection. Thrombocytopenia was induced either 2h prior to or 2h after partial liver resection (pHx) and compared to untreated (control) mice. *P < 0.05. Horizontal bars represent means. Error bars indicate SD.(B) Immunohistochemical staining of Ki-67 on liver paraffin sections. Mice were sacrificed 3 days after partial liver resection. (i, ii) Ki-67 staining on mice with a normal platelet count (i) original magnification 200x, (ii) original magnification 400x, (iii, iv) mice that were rendered thrombocytopenic two hours prior (iii) 200x, (iv) 400x and (v, vi) mice that were rendered thrombocytopenic two hours after partial liver resection (v) 200x, (vi) 400x. Images are representative for six animals.(C) Quantification of Ki-67-positive hepatocytes in groups represented in (B). ***P < 0.001. *P < 0.05. Data represent the mean of six animals. Error bars indicate SD.


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Platelet influx into the liver is mediated by VWFTo assess the role of VWF in platelet influx into the liver remnant, we blocked VWF function by a polyclonal antibody to VWF and performed intravital imaging of platelets immediately following partial hepatectomy. As shown in figure 3, VWF blockade substantially reduced platelet influx following partial hepatectomy to levels similar to that observed in sham operated mice (Fig. 3A). We observed a significant reduction of the number of platelet aggregates after partial liver resection when VWF was inhibited (Fig. 3B).

Figure 3: VWF mediates platelet influx into the liver (A) Representative intravital microscopy images of c57/bl6 control mice or mice treated with an inhibitory antibody to VWF. Platelets were labeled in vivo with PE-conjugated anti-CD49 (red). The autofluorescence signal of the liver is displayed to visualize liver anatomy (green). (i) 15 minutes after sham operation (ii) 15 minutes after partial liver resection (iii) or 15 minutes after partial liver resection in the presence of an inhibitory antibody to VWF. Original magnification 200x. Scale bar denotes 50µm.(B) Quantification of the total number of platelet aggregates in the sinusoids of the liver remnant. Platelet aggregates were analyzed in sham operated mice (n=3), mice that underwent partial liver resection (n=6) and mice that were treated with a blocking VWF antibody prior to partial liver resection (n=6). **P < 0.01 vs sham. ++ P < 0.01 vs liver resection. Error bars indicate SD.

VWF deficiency impairs liver regenerationAs VWF mediates transient platelet influx following partial hepatectomy and transient platelet influx stimulates liver regeneration, we next studied liver regeneration in VWF deficient mice. The liver/body weight ratio was similar in WT and VWF-/- mice at day 1 after partial hepatectomy, but was significantly higher in WT compared to VWF -/- mice at day 3 (Fig. 4A). In line with these results, the proportion of ki-67-positive hepatocytes was substantially higher in WT compared to VWF-/- at day 3 (Fig 4B-C). Importantly, no excessive perioperative bleeding was observed in VWF-/- mice, nor was there evidence of bleeding observed during termination.

Figure 4: Mice deficient in VWF show impaired liver regeneration

(A) Liver to body weight ratio of WT mice compared to VWF knock out mice at day 1 and day 3 after liver resection. ***P < 0.001. Horizontal bars indicate means. Error bars indicate SD.(B) Immunohistochemical staining of Ki-67 on liver sections from mice 3 days after partial liver resection. Representative images of Ki-67 staining of WT mice (i) original magnification 200x, (ii) original magnification 400x and of VWF knock out mice (iii) 200x, (iv) 400x.(C) Quantification of the percentage Ki-67-positive hepatocytes in WT mice and VWF knock out mice at day 3 after partial liver resection. ***P < 0.001. Data represent the mean of seven animals. Error bars indicate SD.


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Discussion

Using intravital imaging, we have demonstrated transient influx of platelets in the liver within 15 minutes after a partial hepatectomy in mice. Platelet accumulation within the liver was critically dependent on VWF. Early platelet accumulation within the liver sinusoids appears sufficient to support liver regeneration as removal of >95% of the circulating platelets 2 hours after partial hepatectomy had little effect on liver regeneration, whereas regeneration was substantially inhibited when platelets were depleted just prior to resection. In addition, decreased liver regeneration in VWF-deficient mice supports the notion that platelet recruitment to the liver remnant, mediated by VWF, is critical in platelet-mediated liver regeneration.Our study confirms that platelets accumulate in the sinusoids immediately after partial hepatectomy (3) and importantly, we show that within one hour after partial hepatectomy platelet aggregates have largely disappeared again. Using post-hepatectomy platelet depletion we show that platelets are only required in the early period after partial hepatectomy for stimulation of liver regeneration. These combined results suggest that platelets deliver mitogenic signals within the liver early after partial hepatectomy. Whether such signals involve growth factors secreted from platelet granules (5), RNA transfer (9), or a yet-to-be identified mitogenic stimulus remains to be determined.Our study also extends previous data on the role of VWF in liver regeneration (21). As it had been previously established that VWF levels in plasma and within the liver rapidly rise following a partial hepatectomy (21,22), we surmised that VWF may be involved in platelet accumulation in the regenerating liver. Indeed, both platelet accumulation and liver regeneration were strongly dependent on VWF, although we have not conclusively shown that LSEC (and not plasma or platelet VWF) mediates these processes. We have recently demonstrated that plasma VWF levels increase and levels of the VWF-cleaving protease ADAMTS13 decrease following partial hepatectomy in humans (26). Acute VWF release within the liver accompanied by inadequate or delayed removal of reactive VWF molecules by decreased ADAMTS13 levels may explain the transient VWF-dependent platelet accumulation after partial hepatectomy.The mechanisms leading to VWF release from liver sinusoidal endothelial cells are incompletely understood but may include hemodynamic effects as the blood flow through the liver is substantially increased by partial hepatectomy. Regardless of the mechanism involved, simulation of VWF release, for example by administration of 1-desamino-8-D-arginine vasopressin (DDAVP) might be an interesting clinical approach to stimulate liver regeneration. In conclusion, we find rapid and transient platelet influx into the liver remnant following a partial hepatectomy in mice in a process dependent on VWF. These results suggest that platelets deliver mitogenic cargo rapidly after liver resection, and knowledge of the mechanism behind this rapid and transient platelet influx may assist development of novel strategies to support liver regeneration.

Acknowledgements:This work was supported in part by a grant from The Netherlands Organisation for Scientific Research (VIDI, 917.11.304 to T.L.) and part of the work has been performed in the UMCG Microscopy and Imaging Center (UMIC), sponsored by a grant from The Netherlands Organisation for Scientific Research (175-010-2009-023).

Conflict of interest statementNone of the authors have a conflict of interest to report.


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vItamIn e attenuates the progressIon of non-alcoholIc fatty lIver dIsease caused by partIal hepatectomy In mIceGoLnar Karimianmarc KirschbaumZwanida J. VeLdhuisfernanda bomfaTiroberT J. PorTeTon Lisman

PubLished in PLos one. 2015;10(11):e0143121

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Chapter 5 Vitamin E attEnuatEs thE progrEssion of non-alcoholic fatty liVEr disEasE causEd by partial hEpatEctomy in micE

5

62 63

Abstract

Background and Aim: The progression of non-alcoholic fatty liver disease (NAFLD) likely involves a ‘multiple hit’ mechanism. We hypothesized that partial hepatectomy, a procedure performed frequently in patients with NAFLD, would accelerate the progression of disease.Methods: C57BL/6JolaHsd mice were fed a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA). Part of the mice in the CD-AA group received a diet enriched in vitamin E (~20 mg /day). Two weeks after the start of the diet, mice underwent a partial hepatectomy or a sham operation.Results: In the CD-AA group, NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P<0.05). In addition, TBARS, a measure for oxidative stress, in liver tissue of the CD-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham operated mice (P<0.05).Vitamin E therapy significantly reduced TBARS level at day 7 after partial hepatectomy compared to the CD-AA diet group (P< 0.05). Vitamin E suppletion reduced NAFLD activity score at day 7 after partial hepatectomy compared to the CD-AA group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05).Conclusion: Partial hepatectomy accelerates the progression of NAFLD. Disease progression induced by partial hepatectomy is substantially attenuated by vitamin E.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disorders worldwide (1). NAFLD includes a disease spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which may eventually progress to liver fibrosis and cirrhosis (2). Mechanisms of disease progression in NAFLD are incompletely understood, but likely a “multiple-hit” model is involved (2). In this model, the development of steatosis increases the sensitivity of the liver to other hits such as oxidative stress and cytokines, leading to hepatocyte damage and liver dysfunction (3, 4). As the number of individuals with mild to moderate liver steatosis is increasing, it is reasonable to assume that the number of patients with steatosis that require a partial hepatectomy is increasing (5, 6).Partial hepatectomy is performed to manage many types of malignant or benign liver pathologies (7-9). While a normal liver is capable to regenerate after a partial hepatectomy, severe steatosis and NASH impair liver regeneration and increase morbidity due to post-operative complications (5, 10). In contrast, several experimental and clinical studies indicate that mild steatosis does not impair liver regeneration (10-12). Although the effect of steatosis on liver regeneration has been studied extensively, the effect of partial hepatectomy on the progression of NAFLD, to our knowledge, has not yet been investigated.Liver regeneration after partial hepatectomy is associated with the activation of inflammatory signaling molecules and induction of oxidative stress (13, 14). Importantly, these factors are important contributors to the progression of NAFLD (3, 15). We hypothesized that partial hepatectomy would advance simple steatosis toward a progressive inflammatory phenotype and have investigated the effect of partial hepatectomy on the progression of the NAFLD in mice with mild steatosis. In the present study, we use a choline-deficient L-amino acid-defined (CD-AA) diet to induce mild steatosis in mice. We confirm previous studies that showed that mild steatosis does not impair liver regeneration. Importantly, we demonstrate that partial hepatectomy substantially accelerates the progression of NAFLD. In addition we show that anti-oxidant therapy with vitamin E attenuates the post-operative progression of NAFLD.


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Material & Methods

Animal studiesC57BL/6JolaHsd male mice (5-6 weeks old, Harlan Laboratories, NL) were fed either a CD-AA diet (mouse chow 518753, Dyets Inc., PA, USA) or a CS-AA (mouse chow 518754, Dyets Inc., PA, USA). Animals were caged in animal rooms with an alternating 12-hour light/dark period, and had free access to food and water for the duration of the experiment. The local Committee for Care and Use of laboratory animals of the University of Groningen approved the experiments of this study and all the experiments were performed in accordance with the guidelines of this Committee.

Experimental designTwo weeks after the start of the diet, mice were anesthetized using isoflurane/O2 and subjected to a midventral laparatomy with (PHx) or without (sham) a resection of two-thirds of the liver volume essentially as described previously (16). Mice remained on the CD-AA or CS-AA diet after the partial hepatectomy or sham operation until the end of the experiment. Six mice in each group were sacrificed at selected time points. In an independent experiment, mice were fed the CD-AA diet for two weeks. At the end of the second week all mice were subjected to a sham or a PHx operation. After surgery, the mice were fed the CD-AA diet or the CD-AA diet enriched with vitamin E for one week (Vitamin E was provided in the form of DL-Alpha Tocopheryl Acetate with concentration of 5 mg vitamin E/gr food, mouse chow 519557, Dyets Inc., PA, USA). Prior to the experiment, we determined that the mice (20-25 gr) eat 4-5 grams of CD-AA diet each day. Thus, the mice received at least 20 mg vitamin E per day by having free access to food. We selected this dosage of vitamin E based on similar published experiments in rats with the goal to substantially improve the antioxidant status by feeding a high dosage of vitamin E to mice (17). Nine mice in each group were sacrificed at day 7 after the operation.At the time of sacrifice in all experiments, mice were weighed and anesthetized. Blood was harvested from the inferior vena cava after the injection of an anticoagulant solution containing 3.4% sodium citrate in 0.9% saline (8 µl/g) in the inferior vena cava. The remaining liver lobes were removed, weighed, and processed for biochemical or histological analyses.

Liver histology and immunohistochemical stainingParaffin-embedded liver sections (5-µm thick) were stained with standard hematoxylin and eosin (H&E) staining. These H&E sections were graded using the NAFLD scoring system (18). At least 10 random high power fields (200X) were analyzed by two independent researchers. The NAFLD activity score was defined as an unweighted sum of scores for liver steatosis (<5 percent = 0, 5 to 33 percent = 1, >33 to 66 percent = 2, >66 percent = 3), lobular inflammation (no foci = 0, <2 foci per 200 X field = 1, 2 to 4 foci per 200 X field = 2, >4 foci per 200 X field = 3) and hepatocyte ballooning (none = 0, few balloon cells = 1, many cells/prominent ballooning = 2) (18). Paraffin-embedded sections were also stained using Masson’s trichrome

stain (Polysciences Inc., PA, USA) to assess presence of fibrosis. Hepatocyte proliferation was assessed by immunohistochemical staining of ki-67 on paraffin-embedded liver sections using a rabbit polyclonal antibody (abcam, Cambridge, UK). Proliferating hepatocytes were quantified by counting the number of nuclei positive for Ki-67, and were expressed relative to the total nuclei stained with hematoxylin per 200X field (hepatocyte proliferation index). At least six random high power fields were counted for each mouse.

Biochemical studies in plasma and liver tissuePlasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed using standard biochemical methods. Thiobarbituric acid-reactive substances (TBARS) were measured in liver homogenates as a marker for oxidative stress. Samples were homogenized in cold 1.15% KCl buffer and TBARS were determined using malondialdehyde as a standard as previously described (19). Protein concentration in liver homogenates was determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, IL, USA) using bovine serum albumin as a standard.

Statistical analysisGraphPad Prism (version 5.00) was used to perform all the analyses. Results are presented as the mean ± SD. A student t-test or a two-way analysis of variance (ANOVA) was used to determine the significance of differences between experimental groups. A P-value of less than 0.05 (P < 0.05) was considered statistically significant.


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Results

The CD-AA diet induces mild steatosis in mice by 3 weeksThere are several models to induce liver steatosis in experimental animals. However, models such as a high fat diet and ob/ob mice show minimal steatohepatitis and liver fibrosis and therefore are not suitable models to study the progression of NAFLD (20). In order to investigate the effect of partial hepatectomy on the progression of NAFLD in mild steatotic livers, we used the CD-AA diet model. The CD-AA diet induces mild steatosis by 2-3 weeks in mice. Consequently, the NAFLD progresses over time and histopathological features of steatohepatitis such as lobular inflammation, hepatocyte ballooning and fibrosis appear in the liver (21). In accordance with previous studies, the CD-AA diet induced mild steatosis by 3 weeks as was demonstrated by histological examination (Fig. 1a) and an elevated NAFLD activity score (1.8 ± 0.7). The NAFLD activity score in the mice fed the CS-AA diet was 0 in all mice. In addition, the liver-to-body weight ratio was significantly higher in the CD-AA group compared to the CS-AA group (P <0.05, Fig. 1b). Histological examination showed no liver fibrosis by 3 weeks in any of the mice (data not shown). Mice in the CD-AA group had significantly higher levels of ALT and AST in plasma compared to the CS-AA group by 3 weeks (Fig 1c and 1d, P <0.05). Altogether, our data indicate that the mice on the CD-AA diet had mild steatosis by 3 weeks.

Mild steatosis does not impair liver regeneration after partial hepatectomyIn order to investigate the effect of mild steatosis on the regenerative capacity of the liver, we performed a two-thirds partial hepatectomy on the mild steatotic livers of mice on the CD-AA diet or on the non-steatotic livers of mice on the CS-AA diet. We compared the liver mass restoration and hepatocyte proliferation rate at several time points after partial hepatectomy over a period of 7 days in both of the diet groups. Liver mass restoration over time was not different between the CD-AA and CS-AA groups (Fig. 2a). The hepatocyte proliferation rate, as assessed by ki-67 staining, was similar in both steatotic and non-steatotic livers when assessed at day 3 after the hepatectomy (Fig. 2b). In addition, aminotransferase levels in plasma increased significantly (P <0.05) after partial hepatectomy in both the CD-AA and CS-AA groups and returned to basal levels over time (Fig. 2c and 2d). Aminotransferase levels were similar in the CD-AA and CS-AA group at all time points examined. Altogether, our data indicate that mild steatosis does not impair liver regeneration after partial hepatectomy.

Figure 1. The choline-deficient L-amino acid-defined diet (CD-AA) induces mild steatosis by 3 weeks.

(A) Hematoxylin and eosin staining of paraffin-embedded liver sections of mice on a CD-AA diet (right panel) and control diet (CS-AA, left panel) by 3 weeks (Magnification, X 100).(B) Liver-to-body weight ratios of mice on a CD-AA diet or a CS-AA diet by 3 weeks (n = 9). (C) Plasma levels of ALT and (D) AST in mice on a CD-AA diet or a CS-AA diet by 3 weeks (n=9).


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Figure 2. Mild steatosis does not impair liver regeneration after partial hepatectomy.

(A) Graph represents the restoration of liver mass over the 7-days duration after partial hepatectomy.(B) Immunohistochemical staining of Ki-67 (yellow arrows show ki-67 positive cells) in mice on a CD-AA diet (right panel) or a CS-AA diet (left panel ) at day 3 after hepatectomy (Magnification, X 200). Graph represents the number of nuclei positive for Ki-67 relative to the total nuclei stained with hematoxylin at day 3 after hepatectomy. At least six random high power fields were counted for each mouse.(C) Plasma levels of ALT and (D) AST in mice on a CD-AA diet or a CS-AA diet at different time points after hepatectomy (n = 6 per time point).

Partial hepatectomy accelerates the progression of NAFLDTo investigate the effect of a partial hepatectomy on the progression of NAFLD, we compared the histological scores of steatotic livers of mice on a CD-AA diet at 7 days after a partial hepatectomy or a sham operation (Fig. 3a). In the CD-AA group, the NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P < 0.05; Fig. 3b). Liver fibrosis was not present in both sham and hepatectomy groups at all the time points that were analyzed in our study (data not shown). The livers of mice on the CS-AA diet did not have steatosis, lobular inflammation, ballooning or fibrosis at 7 days after the hepatectomy or sham operation (data not shown). Altogether, our results indicate that partial hepatectomy accelerates the progression of NAFLD in mild steatotic livers.

Figure 3. Partial hepatectomy accelerates the progression of NAFLD.

(A) Hematoxylin and eosin staining of paraffin embedded-liver sections of mice on a CD-AA diet at day 7 after a partial hepatectomy (right panel) or a sham operation (left panel) (Magnification, X 200).(B) Graph represents the NAFLD activity scores in mice on a CD-AA diet at day 7 after a partial hepatectomy or a sham operation (n=6 per group).(C) Graph represents the TBARS levels in the liver tissue at different time points after a partial hepatectomy (n=6 per group) or a sham operation (n=3 per group) in mice on CD-AA diet and CS-AA diet. Malondialdehyde (MDA) was used as standard. * P < 0.05 for CD-AA vs. CS-AA at day 1, day 3 and day 7 after partial hepatectomy, respectively.


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The level of lipid peroxidation increases significantly in steatotic livers after partial hepatectomySeveral experimental and human studies indicate that oxidative stress-mediated lipid peroxidation particularly contributes to the progression of NAFLD and acts as a “second-hit” (4, 22, 23). Therefore, we measured TBARS to estimate the extent of lipid peroxidation in liver homogenates at several time points after partial hepatectomy. TBARS in liver tissue of the CD-AA group but not of the CS-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham mice (Fig. 3c, P <0.05). These data suggest that oxidative stress level after partial hepatectomy increases particularly in steatotic livers.Vitamin E therapy after partial hepatectomy significantly reduces the oxidative stress level and attenuates the progression of NAFLD

Figure 4. Vitamin E attenuates the progression of NAFLD after partial hepatectomy.

(A) Hematoxylin and eosin staining of paraffin embedded-liver sections of mice on a CD-AA diet (right panel) or vitamin E enriched CD-AA diet (left panel) at day 7 after a partial hepatectomy (Magnification, X 200). (B) Graph represents the NAFLD activity scores in mice on a CD-AA diet or vitamin E enriched CD-AA diet at day 7 after a partial hepatectomy (n = 9 per group). (C) Graph represents the TBARS levels in the liver tissue at 7 days after a partial hepatectomy or a sham operation (n = 9 per group) in mice on CD-AA diet and vitamin E enriched CD-AA diet. Malondialdehyde (MDA) was used as standard. * P < 0.05 for CD-AA vs. vitamin E enriched CD-AA at day 7 after operation. (D) Graph represents the mRNA expression level of hemoxygenase-1 (HO-1) in the liver tissue at day 7 after a partial hepatectomy or a sham operation in mice on CS-AA diet (n = 6), CD-AA diet (n = 15) and vitamin E enriched CD-AA diet (n = 9). P< 0.05 for CD-AA vs Vitamin E enriched CD-AA at day 7 after operation.

A number of studies have suggested antioxidant therapy, particularly vitamin E, as an effective therapy against the progression of NAFLD (17, 24, 25). Therefore, in an independent experiment, we treated the steatotic mice for one week after partial hepatectomy with high dosage of vitamin E to increase the antioxidant status of the liver. We compared the histological scores of steatotic livers of mice on a CD-AA diet and vitamin E enriched CD-AA diet at 7 days after a partial hepatectomy or a sham operation (Fig. 4a). In this independent experiment, we confirm that partial hepatectomy accelerates disease progression in mice fed the CD-AA diet. In the vitamin E enriched CD-AA group, the NAFLD activity scores at 7

Supplementary figure 1: Triglycerde and total cholesterol levels in liver at day 7 after partial hepatectomy.

Triglycerde (A) and total cholesterol (B) in liver at day 7 after partial hepatectomy in CS-AA group, CD-AA group and CD-AA+ vitamin E group. Panels C,D,E represent the mRNA expression levels of CD-68, MCP-1 and nrf2 in liver at day 7 after partial hepatectomy.


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days after partial hepatectomy were significantly lower compared to the CD-AA diet group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05; Fig. 4b). In addition, vitamin E therapy significantly reduced the TBARS level in liver tissue at day 7 after partial hepatectomy (P<0.05, Fig. 4c) without delaying the liver regeneration (data not shown).We measured HO-1 (hemoxygenase -1) mRNA expression levels as a marker for oxidative stress at 7 days after the hepatectomy in CD-AA group and CD-AA Vitamin E group. We observed a significant decrease in HO-1 mRNA expression levels in Vitamin E treated group compared to the control group at day 7 after hepatectomy (Fig 4d). These data confirm our previously observed TBARS measurements.

We measured the triglyceride and cholesterol content in the liver tissue at day 7 after partial hepatectomy in CS-AA group, CD-AA group and vitamin E enriched CD-AA group. We observed a significant difference in the level of triglycerides and cholesterol between the CS-AA diet group and the other two groups. The triglyceride content at day seven after partial hepatectomy, however, was not significantly higher than sham operated mice (S1 Fig). Interestingly, the cholesterol content increased after the partial hepatectomy. These findings are in accordance with previous reports in the literature that partial hepatectomy does not affect the triglyceride content but cholesterol synthesis increases after the partial hepatectomy (27). Vitamin E treatment did not affect the lipid content after partial hepatectomy. These results reinforce the notion that the increase in NAFLD activity score following partial hepatectomy and the inhibition of this effect by vitamin E does not reflect direct alterations of fat content of the liver, but rather reflects effects on intrahepatic inflammatory responses. We also measured the mRNA expression level of CD68 and MCP-1 in CD-AA group and Vitamin E enriched CD-AA group at day 7 after partial hepatectomy. We did not observe any significant difference in the expression levels of these markers at these time points (S1 Fig).

Altogether, in accordance with previous studies (17,25,26), our data suggests that vitamin E acts as an antioxidant and attenuates the progression of NAFLD after partial hepatectomy.

Discussion

In this study, we report that mild liver steatosis does not impair liver regeneration in mice. However, partial hepatectomy substantially accelerates the progression of NAFLD. In addition, we provide evidence that enhanced oxidative stress following partial hepatectomy contributes to the progression of NAFLD and show that antioxidant therapy with vitamin E attenuates the progression of NAFLD.The clinical significance of liver steatosis on liver regeneration after hepatic resection depends upon the extent of lipid accumulation in hepatocytes and the presence of steatohepatitis (5, 6). Several human studies have indicated that mild steatosis does not impair liver regeneration (10, 11). However, in experimental models it is difficult to distinguish between the effects of simple steatosis and the effects of a progressive phenotype of NAFLD on liver regeneration since different models show different histopathological features of NAFLD, and rodents have high tolerance before they display inflammatory phenotypes even in a severe steatotic status (20). Therefore, data emerging from experimental studies on the effect of steatosis and steatohepatitis on regeneration are inconsistent (12, 26). In our study, we used the CD-AA diet model, which is suggested to be a model for simple steatosis with the ability to advance toward steatohepatitis over time, mimicking the natural history of NAFLD/NASH in humans (21). In accordance with previous studies (10-12), we found that mild steatosis does not impair liver regeneration after partial hepatectomy.Nevertheless, the development of liver steatosis sensitizes the liver to other hits (2). In our study, we found that a mild steatotic phenotype could advance to a progressive inflammatory phenotype of NAFLD after partial hepatectomy. In addition, the level of lipid peroxidation products following partial hepatectomy was particularly higher in steatotic livers. Many experimental and human studies have reported a positive association between increased levels of the oxidative stress-mediated lipid peroxidation and the presence of progressive inflammatory phenotypes of NAFLD (3, 22, 27). Several mechanisms have been suggested for the role of oxidative stress in the pathogenesis of NASH. Peroxidation of the mitochondrial/plasma membrane directly leads to necrosis/apoptosis of liver cells (28). ROS-induced Fas-ligand expression on hepatocytes may lead to hepatocyte apoptosis given that isolated hepatocytes from liver biopsies of NASH patients express higher levels of Fas (29). Covalent binding of the aldehyde products of lipid peroxidation (such as MDA) with hepatic proteins may lead to injurious immune responses in the liver (3). Activation of the NF-κB cascade by products of lipid peroxidation can increase transcription of inflammatory cytokines and death ligands by liver cells (30). Although we have not studied these mechanisms, they potentially explain why an increased level of lipid peroxidation after partial hepatectomy particularly contributes to the progression of NAFLD following a partial hepatectomy.These findings are important for clinical practice because they implicate that simple hepatic steatosis, which is a benign condition and non-progressive in the majority of patients, may advance toward a more progressive liver disease after hepatic resection. This may have


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adverse clinical consequences for humans with simple steatosis that may need a partial hepatectomy and studies to examine whether acceleration of disease progression also occurs in humans with appreciable steatosis who undergo a partial hepatectomy are therefore warranted. Currently, few therapies are available to prevent the progression of NAFLD (1). The prominent role of oxidative stress in NAFLD pathogenesis has prompted the use of antioxidants, particularly vitamin E, as an effective therapy in patients with progressive phenotypes of NAFLD. It has been reported that vitamin E improves all the histological features of NAFLD (except for fibrosis) and decreases the NAFLD activity score when compared with placebo (24, 25). We also showed in the present study, that vitamin E is a beneficial therapy to reduce the level of oxidative stress after partial hepatectomy and to prevent the progression of NAFLD. Would our findings be confirmed in humans, it may be of interest to examine whether patients with simple steatosis benefit from interventions such as vitamin E administration after partial hepatectomy.In summary, our findings indicated that despite the intact regenerative capacity of mild steatotic livers, these livers are predisposed to advance to a more progressive inflammatory phenotype of NAFLD after partial hepatectomy. In addition, oxidative stress plays a pivotal role in disease progression and vitamin E may be a beneficial therapy in preventing the advance of steatosis to steatohepatitis after hepatic resection.

Acknowledgements:This work was supported in part by a grant from The Netherlands Organisation for Scientific Research (VIDI, 917.11.304 to T.L.) Author ContributionsConceived and designed the experiments: GK TL. Performed the experiments: GK ZJV MK FB. Analyzed the data: GK. Contributed reagents/materials/analysis tools: GK TL RJP. Wrote the paper: GK MK ZJV FB TL RJP.


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evIdence agaInst a role for platelet-derIved molecules In lIver regeneratIon after partIal hepatectomy In humansmarc KirschbaumJeLLe adeLmeiJeredris aLKoZairoberT J. PorTeTon Lisman

PubLished in JournaL of cLinicaL and

TransLaTionaL research 2016; 2(3): 97-106

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Chapter 6 EvidEncE against a rolE for platElEt-dErivEd molEculEs in livEr rEgEnEration aftEr partial hEpatEctomy in humans

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Abstract

Blood platelets have been shown to stimulate liver regeneration after partial hepatectomy in animal models and humans, but the molecular mechanisms involved are unclear. It has been proposed that growth factors and angiogenic molecules stored within platelets drive platelet-mediated liver regeneration, but little direct evidence in support of this mechanism is available. We assessed levels of relevant platelet-derived proteins (vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, platelet-derived growth factor, thrombospondin, and endostatin) in platelet-rich and platelet-poor plasma taken at various perioperative time points from patients undergoing a (extended) right partial hepatectomy (n=17) or a pylorus-preserving pancreatico-duodenectomy (n=10). In addition, we collected intraoperative samples from the efferent and afferent liver veins prior to and after completion of liver resection. Twenty-four healthy controls were included to establish reference ranges for the various tests. Although we demonstrate perioperative changes in platelet and plasma levels of the proteins assessed, the changes observed in patients undergoing partial hepatectomy largely mirror the changes observed in patients undergoing a pylorus-preserving pancreatico-duodenectomy. In addition, no change in the growth factor levels in platelet-rich plasma between afferent and efferent liver veins was observed. Thus, the absence of an intra- or postoperative consumption of platelet-derived proteins in patients undergoing partial hepatectomy argues against a role of release of these molecules in stimulation of liver regeneration.

Introduction

Platelets are well known for their functions in thrombosis and hemostasis. In addition, platelets have roles beyond physiological or pathological thrombus formation (1-3). Animal models and clinical studies have suggested a pivotal role for platelets in stimulation of liver regeneration (4-10). Although there is broad consensus that platelets stimulate liver regeneration in animal models and likely also in humans, there is ongoing debate on the molecular mechanisms underlying platelet-mediated stimulation of liver regeneration (11, 12). Some studies suggest that local release of growth factors or angiogenic molecules stored within platelet alpha and dense granules are responsible for platelet-mediated liver regeneration (4, 13-16). Even though it has been reported that platelet-derived growth factors stimulate hepatocyte proliferation in vitro (13, 14, 17), it has not yet been established whether platelet-derived growth factors also drive platelet-mediated liver regeneration in vivo (12). Animal studies and a single study in humans have demonstrated accumulation of platelets within the liver remnant rapidly following a partial liver resection (5, 16, 18). Nevertheless, it has not been unequivocally demonstrated that platelet accumulation results in (selective) release of liver-directed growth factors, nor has it been demonstrated that platelet derived growth factors indeed drive platelet-mediated liver regeneration.For example, animal studies have suggested that platelet-derived serotonin drive platelet-mediated liver regeneration (4, 19). Indeed, mice that lack serotonin within platelet dense granules have a clearly delayed regeneration (4). However, as serotonin is a relevant platelet activator (20), the decreased functional capacity of serotonin deficient platelets, rather than a direct mitogenic effect of serotonin could explain the delay in liver regeneration in these mice. In addition, studies in humans have suggested consumption of platelet-derived serotonin (15) and release of other molecules (notably vascular endothelial growth factor) potentially driving platelet-mediated liver regeneration from platelets following a liver regeneration in humans (16). Unfortunately, these studies did not control for effects of a major surgical procedure, and in our opinion these data thus not convincingly demonstrate a role for platelet-derived proteins in liver regeneration in humans (21).Thus, even though there is consensus that platelets stimulate liver regeneration, the exact mechanisms involved have not been thoroughly studied. Besides release of growth factors from platelets, alternative scenarios that may underlie platelet-mediated liver regeneration have been postulated (11). Such mechanisms include transfer of RNA from platelets to hepatocytes as we have recently described (18), and platelet-mediated influx of inflammatory cells to the regenerating liver (22). The latter mechanism is yet unexplored in literature although it is well known that inflammatory cells drive liver regeneration and the interactions between platelets and inflammatory cells have been well established (23-25).Here we studied the levels of multiple relevant platelet-derived growth factors and angiogenic proteins in patients undergoing a partial liver resection and compared the levels to patients undergoing a pylorus-preserving pancreatico-duodenectomy (PPPD) and healthy controls.


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Material & Methods

PatientsSeventeen adult patients who underwent a right (segments 5-8, n = 15) or extended right (segments 4-9, n = 2) partial hepatectomy were included in this study. The control group consisted of ten patients who underwent a PPPD. Twenty-four adult healthy volunteers were included to establish reference values for the various tests performed. Details of patients and controls have been previously reported elsewhere (26, 27). In short, all patients were included in the University Medical Center Groningen, the Netherlands. Exclusion criteria were age younger than 18 years, pre-existing coagulation disorders, pre-operative anti-coagulation, and use of non-steroidal anti-inflammatory drugs or aspirin 1 week before surgery. Routine surgical and anesthesiological procedures were adopted. The study protocol was approved by the local medical ethical committee and informed consent was obtained from each subject before inclusion in the study.

Plasma samplesIn both patient groups, plasma samples for analyses were drawn at the following time points: after induction of anesthesia (baseline), at the end of surgery, and on post-operative days 1, 3, 5, 7 and 30. In addition we collected intraoperative blood samples in the patients undergoing partial hepatectomy. We took blood samples from the portal vein and from the hepatic vein of the liver remnant just before the start and just after completion of anatomical transection. Following surgery, all patients received standard thromboprophylaxis with (once-daily) low molecular weight heparin (LMWH) and at each post-operative day blood was drawn just prior to the administration of the LMWH. Blood samples from each subject were drawn by venipuncture and collected into vacuum tubes containing 3.8% trisodium citrate as an anti-coagulant, at a blood to anti-coagulant ratio of 9:1. Platelet counts in whole blood were determined by the diagnostic laboratory of our hospital. Platelet-rich plasma (PRP) was prepared by centrifugation at 250 g for 10 min. at room temperature. The platelet count in the PRP was determined by the diagnostic laboratory of our hospital and platelets were lysed by repeated free–thaw cycles and stored at −80°C until use. Platelet-poor plasma (PPP) was prepared by centrifugation of whole blood at 2000 g for 10 min at room temperature, with subsequent recentrifugation of the plasma fraction at 10.000 g for 10 min. at room temperature. Plasma was aliquoted, snap-frozen and stored at -80°C until use.

Assays for platelet-derived proteins Levels of platelet-derived proteins were measured in PRP lysates and in PPP. Specifically, levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), thrombospondin 1 (TSP1), and endostatin were quantified using commercially available enzyme-linked immunosorbent assays (ELISAs). For all proteins DuoSet ELISA Development Systems (R&D Systems, Oxon, United Kingdom)

were used according to the manufacturer´s instructions. Platelet residues in the PRP lysates were spun down prior to the assays. The concentration of growth factors within platelets was calculated by subtracting values obtained in PRP from those in corresponding PPP samples after which values were divided by the platelet count in the PRP sample.

StatisticsValues are expressed using box plots with medians, interquartile ranges, and minimal and maximal values indicated. Differences between values at a single time point between patients undergoing partial hepatectomy and patients undergoing a PPPD were assessed by the Mann–Whitney U-test. Differences between samples taken from the efferent and afferent liver veins were assessed using a paired t-test or the Wilcoxon matched pairs test as appropriate. Differences between patient values and levels measured in healthy controls were compared using one-way analysis of variance (with the Friedman test). P-values of 0.05 or less were considered statistically significant.


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Results

Patient characteristicsSeventeen patients who underwent a right (n = 15) or extended right (n = 2) partial hepatectomy, 10 patients who underwent a PPPD, and twenty-four controls were included in the study. The main characteristics of the study population have been reported elsewhere (26,27). The most common indication for liver resection was liver metastases from colorectal cancer and the most common indication for PPPD was pancreatic cancer.

Figure 1. Circulating platelet count prior to and after partial liver resection or PPPD.

(A) Partial liver resection (B) PPPD. Pre-OP, pre-operative, End-OP, end of surgery; POD, post-operative day.

Platelet counts during and after partial hepatectomy or PPPDPlatelet counts were slightly lower at post-operative day 1 compared to baseline values in both patient groups (Fig. 1). In the PPPD group, platelet counts were substantially elevated at post-operative day 7, while no clear increase in platelet count was observed in the partial hepatectomy group.

Levels of growth factors and angiogenic proteins in platelet rich plasma taken from the afferent and efferent liver veins during partial hepatectomyWe measured levels of various growth factors and angiogenic molecules in PRP taken from the afferent and efferent liver veins just before the start and just after completion of the parenchymal transection in patients undergoing a partial hepatectomy. As we were only able to draw a limited amount of blood from afferent and efferent veins, we only processed blood to PRP, and did not assess levels in PPP.Prior to and just after completion of the parenchymal transection levels of all proteins assessed were not different between afferent and efferent liver veins (Fig. 2). Also, levels of all proteins were similar between both time points. Platelet counts measured in platelet rich plasma did not differ between the afferent and efferent liver veins both prior to and after completion of the parenchymal transection (p=0.47 and p=0.31, respectively, Fig. 2G)

Figure 2. Levels of growth factors and angiogenic molecules in afferent and efferent veins prior to and just after anatomical transection in patients undergoing partial hepatectomy.

Levels of (A) VEGF, (B) HGF, (C) bFGF, (D) PDGF, (F) TSP1, (E) endostatin, and the platelet count in PRP were measured prior to (pre) and just after (post) parynchymal transection in samples taken from the portal vein (PV) or hepatic vein (HV) during partial hepatectomy.


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Figure 3. Levels of growth factors and angiogenic molecules in PRP taken at various perioperative time points from patients undergoing partial liver resection or PPPD

Levels of (A) VEGF, (B) HGF, (C) bFGF, (D) PDGF, (F) TSP1 and (E) endostatin were measured in lysates of PRP in samples taken from healthy controls (white boxes), patients undergoing partial hepatectomy (dark grey boxes), and patients undergoing PPPD (light gray boxes). *P < 0.05, **P < 0.01, ***P < 0.001, versus pre-op (Friedman’s test). + P < 0.05, partial hepatectomy versus PPPD (Mann-Whitney’s U test). Pre-OP, pre-operative, End-OP, end of surgery; POD, post-operative day.

Figure 4. Levels of growth factors and angiogenic molecules in PPP taken at various perioperative time points from patients undergoing partial liver resection or PPPD

Plasma levels of (A) VEGF, (B) HGF, (C) bFGF, (D) PDGF, (F) TSP1 and (E) endostatin in samples taken from healthy controls (white boxes), patients undergoing partial hepatectomy (dark grey boxes), and patients undergoing PPPD (light gray boxes).. *P < 0.05, **P < 0.01, ***P < 0.001, versus pre-op (Friedman’s test). + P < 0.05, ++ P < 0.01, hemihepatectomy versus PPPD (Mann-Whitney’s U test). Pre-OP, pre-operative, End-OP, end of surgery; POD, post-operative day.


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Figure 5. Levels of growth factors and angiogenic molecules in platelets taken at various perioperative time points from patients undergoing partial liver resection or PPPD

Calculated intraplatelet levels of (A) VEGF, (B) HGF, (C) bFGF, (D) PDGF, (F) TSP1 and (E) endostatin in samples taken from healthy controls (white boxes), patients undergoing partial hepatectomy (dark grey boxes), and patients undergoing PPPD (light gray boxes).. *P < 0.05, **P < 0.01, ***P < 0.001, versus pre-op (Friedman’s test). + P < 0.05, hemihepatectomy versus PPPD (Mann-Whitney’s U test). Pre-OP, pre-operative, End-OP, end of surgery; POD, post-operative day.

Levels of growth factors and angiogenic molecules in plasma and platelets taken at various perioperative time points from patients undergoing partial hepatectomy or PPPDWe measured levels of various growth factors and angiogenic proteins in PRP (Fig. 3) and PPP (Fig. 4), and calculated the amount of protein within platelets (Fig. 5) in patients undergoing partial hepatectomy and PPPD and in healthy controls. Levels of VEGF within platelets were slightly higher in both patient groups compared to controls, increased at post-operative day 3, and normalized again at post-operative day 30. VEGF levels showed similar changes in the partial hepatectomy and PPPD groups. Plasma levels of VEGF did not appreciably change, except for an increase in the PPPD group at day 30. HGF levels in plasma and in platelets increased from the end of surgery up to day 1, after which levels gradually decreased towards baseline. No differences between the partial hepatectomy and PPPD groups were detected, except for slightly higher levels within platelets in the partial hepatectomy group at days 5 and 7. bFGF levels within platelets were slightly elevated compared to the control group at baseline, whereas plasma levels were not different between patients and controls. No changes in platelet or plasma bFGF levels occurred over time, and levels were not different between partial hepatectomy and PPPD patients. PDGF levels in platelets and plasma did not significantly change over time, nor were levels different between the two patient groups. Thrombospondin 1 (TSP1) levels within platelets were consistently higher in the PPPD group compared to the partial hepatectomy group, but no changes in platelet thrombospondin 1 levels occurred over time. Plasma levels of thrombospondin 1 did not change over time, and were similar between the PPPD and partial hepatectomy groups, except for an increase in the PPPD group on day 7. Plasma levels of endostatin were lower in the patients undergoing partial hepatectomy between post-operative day 1 and 7, but levels within platelets were similar between the groups at all time points.


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Discussion

In this study we found no difference between the circulating levels of six molecules that play important roles in liver regeneration between samples taken from the afferent and efferent liver veins immediately after a partial liver resection in humans. In addition, we found no clear differences in post-operative levels of these six proteins in platelets or plasma between patients that underwent a partial liver resection and patients that underwent a PPPD. Although levels of particularly VEGF and HGF changed over time, changes were similar between the partial hepatectomy and PPPD groups. Collectively, these results do not provide support for the hypothesis that selective release and consumption of proteins stored within platelets drives platelet-mediated liver regeneration.Our results contradict recent clinical studies that have suggested consumption of serotonin and VEGF following a partial hepatectomy in humans (15, 16). Importantly, these studies did not control for the effects of major abdominal oncological surgery on levels of these molecules in platelets and/or plasma. Although it has been suggested that differences in sample processing may explain the divergent results obtained in our lab and the laboratory of Starlinger and coworkers (28, 29), we feel there are other explanations for the divergent conclusions between our studies. First, the conclusion that consumption of VEGF and serotonin occurs following a partial hepatectomy in humans is not supported by our data in which we compared levels in PRP and PPP samples that have been processed in an identical fashion from patients undergoing partial liver resection and a PPPD. Although we did found changes in VEGF levels over time, these changes were very similar between the partial liver resection and PPPD groups. Similarly, we have recently reported that circulating serotonin levels similarly decrease in patients undergoing a partial liver resection and a PPPD (30). Second, Starlinger and coworkers have reported accumulation of platelets in the liver remnant immediately after parenchymal transection which was accompanied by platelet activation and release of VEGF and thrombospondin (16). In this and a previous study, we detected no changes in levels of serotonin (30) and the six proteins assayed in our present study between the afferent and efferent liver veins immediately following parenchymal transection, and found no evidence of a reduction in platelet count across the liver. We anticipate that surgical and anesthesiological differences, and differences in patient characteristics rather than differences in sample processing explain the apparent differences in secretion of platelet proteins within the liver between our studies.It has been shown that VEGF and HGF plasma levels increase following a partial liver resection in humans (16, 31), and these increases may directly contribute to liver regeneration. The regenerating liver synthesizes VEGF and HGF, presumably to aid its own regeneration (32, 33). It is therefore unclear why VEGF and HGF similarly increase in patients that underwent a PPPD. Our results, however, are in accordance with previously published data in which identical VEGF and HGF serum levels were shown at several time points following partial hepatectomy and pancreas resection (34). These authors concluded that the increases

in VEGF and HGF reflect wound healing and systemic inflammation as a result of surgical trauma. Similar conclusions were obtained in a study in which growth factor levels were compared between patients that underwent a partial liver resection and patients that underwent a laparotomy but did not undergo a partial hepatectomy as the tumor was found unresectable during surgery and in a study on VEGF levels following major abdominal surgery (35, 36).Baseline platelet VEGF, HGF, bFGF, and endostatin levels were higher in both patient groups compared to the controls, whereas plasma levels were very similar. These findings are in accordance with previous work that showed an increase in the levels of angiogenic proteins in platelets but not in plasma in both mouse models of cancer and in patients with cancer (37, 38). The consistently lower postoperative levels of the anti-angiogenic endostatin in patients undergoing partial hepatectomy as compared to the PPPD patients is of interest and may reflect the requirement for angiogenesis in the partial hepatectomy group.Several limitations of our work must be acknowledged. First, the sample size of our study was small and variation in some of the analytes between patients was high. Consequently our conclusion that levels of the proteins assayed generally do not differ between patients undergoing a partial liver resection and a PPPD should be taken with caution. Secondly, it has been well established that plasma levels of platelet-derived proteins are very sensitive for pre-analytical variation. Although we had a standardized protocol to process blood, our protocol results in increased ex-vivo platelet activation compared to other published protocols (39). Plasma levels of some of the analytes thus may be much higher than true ‘in vivo’ levels, although these falsely high values are likely present to a similar extent in all samples. Nevertheless, the extent of ex-vivo platelet activation does not lead to a substantial redistribution of proteins from platelet granules to plasma. For example, plasma thrombospondin levels are (according to published data and data obtained in our laboratory) ~10 fold higher using our blood processing protocol compared to the protocol recommended by Starlinger and coworkers (39). However, the majority of thrombospondin is still within platelets (When comparing levels in PRP with that in PPP ~12.5% of total thrombospondin is within platelets in our study, this level is ~1.25% using our methodology in the Starlinger study (39), and ~0.125% using the optimized methodology when comparing PPP versus serum levels as reported in that study). As we aimed to test the hypothesis that platelets (selectively) released proteins in patients undergoing liver (but not pancreas) resection, we feel that our final conclusion is justified despite these methodological issues. We do acknowledge that additional, larger studies with appropriate control groups are required to obtain definitive conclusions. Thirdly, one could argue that patients undergoing a PPPD are not ideal controls. We chose these patients as we then could compare two groups that both had major abdominal surgery for cancer with liver regeneration occurring in just one of the two groups. However, it has been suggested that the pancreas is also able to regenerate (40, 41), and as platelets may potentially play a role in this process, comparison of liver- and pancreas resection may not be straightforward. On the other hand, there is no consensus


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in literature that pancreas regeneration occurs in humans (42, 43). Finally, we have only taken samples right after completion of parenchymal transection and at day 1. Possible consumption of analytes assessed in the first few hours after surgery, during which liver regeneration is already initiated, may therefore have been missed.In conclusion, our data do not support the hypothesis that growth factors and angiogenic molecules in platelets are selectively released and consumed during liver regeneration after partial hepatectomy in humans. Although emerging evidence from clinical and experimental animal studies provides continuing support for the role of platelets in liver regeneration, the mechanism by which platelets stimulate this process may not involve a simple release of growth factors or angiogenic mediators from platelets.

AcknowledgementsThis study was funded in part by grants a grant from The Netherlands Organisation for Scientific Research (VIDI, 917.11.304 to T.L. and Mozaiek, 017.007.115 to E.M.A.)

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41. Berrocal T, Luque AA, Pinilla I, Lassaletta L. Pancreatic regeneration after near-total pancreatectomy

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Intermezzo: In vItro uptake of recombInant factor vIIa by megakaryocytes wIth subsequent productIon of platelets contaInIng hemostatIcally actIve drugmarc Kirschbaumanne marieKe schuTJeLLe adeLmeiJerPhiLiP G. de GrooTTon Lisman

PubLished in abbreViaTed form in

briTish JournaL of haemaToLoGy

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Chapter 7 Intermezzo: In vItro uptake of reCombInant faCtor vIIa by megakaryoCytes wIth subsequent produCtIon of platelets ContaInIng hemostatICally aCtIve drug

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Abstract

A once-daily prophylactic administration of recombinant factor VIIa (rFVIIa) has been shown to reduce the number of bleeding events in patients with inhibitor-complicated hemophilia, which is difficult to explain given its plasma half-life of 2 hours. Redistribution of rFVIIa into the extravascular space, including the accumulation of rFVIIa in megakaryocytes has been previously demonstrated, which may explain the prophylactic effect of once-daily rFVIIa in hemophilia. Here we show that rFVIIa is taken up by cultured megakaryocytes (MEG-01 cells), and rFVIIa endocytosis was critically dependent on the endothelial protein C receptor. When stimulated, these megakaryocytes produce rFVIIa-containing platelet-like particles (PLPs), whereas rFVIIa is no longer detected in the remnant megakaryocytes. The MEG-01 cell-derived PLPs contain relevant quantities of hemostatically active rFVIIa, as shown by accelerated lag times and increased thrombin peaks in thrombin generation assays using factor VII or VIII depleted plasma when compared to MEG-01 cell-derived PLPs that had not been exposed to rFVIIa. We propose that also in vivo redistribution of rFVIIa to the bone marrow compartment results in production of platelets containing rFVIIa. The (delayed) generation of rFVIIa-containing platelets may (partly) explain the efficacy of once-daily rFVIIa prophylaxis in patients with inhibitor-complicated hemophilia.

Introduction

Recombinant activated factor VII (rFVIIa) has been recently shown to prevent spontaneous bleeding in inhibitor-complicated hemophilia when administered once daily (1,2). The prohemostatic effect of rFVIIa prophylaxis is difficult to explain given its plasma half-life of 2 hours. In the literature, four mechanisms explaining the prophy-lactic efficacy of once-daily rFVIIa administration have beenproposed, including the requirement of much lower doses ofrFVIIa to prevent than to treat bleeding, improvement ofendothelial cell permeability, uptake of rFVIIa by platelets,and redistribution of rFVIIa into the extravascular space (3). Accumulation of rFVIIa in boneand joints may explain the prophylactic activity of rFVIIa.One study suggested that rFVIIa was taken up by megakary-ocytes within the bone marrow (4).The present study tested the hypothesis that megakaryocytes that have taken up rFVIIa will produce rFVIIa-containing platelets, which may explain the prolonged haemostatic effect of rFVIIa in a prophylactic setting.


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Materials & Methods

Cell culture The megakaryoblastic cell line MEG-01 was cultured and differentiated by valproic acid as described earlier (5). After at least 10 days of differentiation, different concentrations of rFVIIa were added to the culture medium and after 2 hours, MEG-01 cells were either harvested or stimulated to produce platelet-like particles (PLPs) by addition of 100 ng/ml recombinant human thrombopoietin (Life Technologies, Carlsbad, CA) for 3 days. Cells or PLPs were harvested by centrifugation, washed, and lysed by freeze-thawing the samples twice.

Microtitre plate clotting assay to estimate rFVIIa levels in MEG-01 and PLP lysatesFVIIa activity in MEG-01 and PLPs lysates was studied by a microtitre plate clotting assay as described earlier (6). A calibration curve of rFVIIa was used to convert clotting times to rFVIIa concentrations. rFVIIa levels were normalized for total protein content of the samples to correct for enumeration differences between samples using the Pierce BCA protein assay kit (Thermo Scientific, IL, USA).

Flow cytometryDifferentiated MEG-01 cells were detached with 5mM (w/v) EDTA in phosphate-buffered saline (PBS) and stained for endothelial protein C receptor (EPCR) or glycoprotein Ibα using 10 µg/ml R-Phycoerythrin-labeled rat anti-human EPCR antibody or 156 µg/ml R-Phycoerythrin-labeled monoclonal mouse anti-human CD42b (GPIb, clone AN51) for 20 min at 37°C.Isolated PLPs were fixed with 2% (v/v) formaldehyde in 0.9% NaCl for 10 min at room temperature. In selected experiments, PLPs were permeabilized with 0.1% (v/v) triton X-100 in PBS for 10 min at room temperature. After washing, samples were stained for rFVIIa using 10 µg/ml monoclonal mouse anti-human FVII (ABIN951602, clone AA-3, antibodies-online, Germany) for 1 h at room temperature followed by 10 µg/ml Alexa Fluor 488-labeled goat anti-mouse antibody (A-11001, Life Technologies, Carlsbad, CA, USA) for 30 min at room temperature. Samples were analysed by flow cytometry.

Thrombin generation assay PLPs were counted using flow cytometry and added to FVII- or FVIII deficient plasma (Haematologic Technologies, Inc, Essex Junction, VT, USA) at a final concentration of 10.000 PLPs/µl plasma. Calibrated automated thrombography using the manufacturer’s reagents (Thrombinoscope PRP reagent) and protocols (Thrombinoscope BV, Maastricht, the Netherlands) was performed.

Immunofluorescence stainingMEG-01 cells or isolated PLPs were permeabilized with 0.1% (v/v) triton X-100 in PBS for 10 min at room temperature, and stained with 5 mg/ml monoclonal mouse anti-human FVII followed by 5 mg/ml Alexa Fluor 488 goat anti-mouse antibody. MEG-01 cell samples were counterstained with 4 units/ml Alexa Fluor 594 phalloidin (A12381, Life Technologies, Carlsbad, CA). All samples were fixed using Vectashield Hardset mounting medium with DAPI (Vector Laboratories Inc., CA, USA).

Statistical analysisTo evaluate differences in parameters derived from thrombin generation tests, paired t-tests were used. To evaluate differences in rFVIIa content of MEG-01 cells in absence or presence of blocking agents, one-way analyses of variance with Dunnett’s post-test test was used. P values ≤ 0.05 were considered statistically significant. Statistical analyses were performed using the GraphPad Prism 5.1 Software package (La Jolla, CA, USA).


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Results and Discussion

We used the megakaryoblastic cell line MEG-01, which was cultured and differentiated by valproic acid as described earlier (5). After at least 10 days of differentiation, different concentrations of rFVIIa were added to the culture medium and after 2 hours, MEG-01 cells were either harvested or stimulated to produce platelet-like particles(PLPs) by addition of 100 ng/ml recombinant human thrombopoietin (rTHPO; Life Technologies, Carlsbad, CA, USA) for 3 days. Cells or PLPs were harvested by centrifugation, washed and lysed by freeze-thawing the samples twice.We first showed that rFVIIa was dose-dependently taken up by MEG-01 cells (Fig. 1A). After 3 days, hardly any rFVIIa was detected in the MEG-01 cells (Fig. 1B). In contrast, the PLPs produced from these MEG-01 cells contained appreciable amounts of rFVIIa (Fig. 1C), which suggests megakaryocytes selectively transfer endocytosed rFVIIa to PLPs. Active selection of agents to be transferred to platelets in the process of megakaryocyte maturation has been previously demonstrated, as megakaryocytes transfer some, but not all, mRNA species examined to platelets (7).

To confirm that rFVIIa is taken up by MEG-01 cells (and not just associates with the cell), we performed fluorescence microscopy which showed ubiquitous presence of rFVIIa within the cell in a punctuate pattern 2 hours after addition of rFVIIa (Fig. 1D i), whereas no staining was detected in cells that had not been exposed to rFVIIa (Fig. 1D ii). After 3 days, no rFVIIa staining was detected in the MEG-01 cells (Fig. 1D iii), but the PLPs harvested at day 3 did stain positive for rFVIIa (Fig. 1D iv). We also used flow cytometry for rFVIIa in presence or absence of cell permeabilisation to confirm the presence of rFVIIa within PLPs. Indeed, rFVIIa was predominantly localized within the PLP as 55.6 % ± 11.3 % [mean ± SD, n = 3] of permeabilized cells stained positive for rFVIIa, as compared to 19.9 % ± 5.1 % [mean ± SD, n = 3] of non-permeabilised cells (Fig. 1E). rFVIIa within PLPs is haemostatically active, as levels were determined in PLP lysates with a functional coagulation assay. To determine whether this functionally active rFVIIa contributes to haemostasis in a plasma environment, we performed in vitro thrombin generation measurements using plasma to which intact PLPs were added. Addition of rFVIIa-containing PLPs resulted in a profound shortening of the lag time of the thrombin generation curve in normal, FVII-, and FVIII-depleted plasma, compared to addition of PLPs generated from MEG-01 cells that had not been exposed to rFVIIa (Fig 1F). rFVIIa-containing PLPs also had a slight but significantly increased peak thrombin generation compared to control PLPs (Fig 1G). Whether rFVIIa in PLPs does not encounter inhibitors (such as tissue factor pathway inhibitor and antithrombin) or whether the rFVIIa measured in our assay only represents a fraction of total rFVIIa in PLPs, with the remainder being in complex with an inhibitor, requires further study.

Figure 1: Dose-dependent uptake of rFVIIa by MEG-01 cells with subsequent production of PLPs containing hemostatically active rFVIIa.

(A-C) rFVIIa was added in different concentrations (0-100 nM) to valproic acid-stimulated MEG-01 cells, which were harvested after 2 hours (A), or stimulated with 100 ng/ml recombinant human thrombopoietin to produce PLPs. After 3 days, MEG-01 cells (B) or PLPs (C) were isolated by centrifugation. Lysates of MEG-01 cells and PLPs were tested for rFVIIa content using a microtitre plate-based clotting assay in factor VII-depleted plasma. Shown are means of 3 independent experiments, error bars indicate standard error of mean (SEM). (D) Immunofluorescent stainings of MEG-01 cells (i-iii) or PLPs (iv) using an antibody to factor VII(a). Shown are typical examples of MEG-1 cells that were exposed to 100 nM rFVIIa for 2 hours (i) or 3 days (iii) or cells that were exposed for 2 hours to vehicle (ii). In addition, PLPs derived from MEG-01 cells exposed to 100 nM of rFVIIa harvested at day 3 are shown. rFVIIa is stained in green, nuclei in blue, and the cell membrane is represented by the dotted line. Scale bars represent 20 µm (MEG-01 cells) or 10 µm (PLPs). Original magnification 1000x. (E) Flow cytometry analysis of PLPs derived from MEG-01 cells that have been exposed to 100 nM of rFVIIa using permeabilized (green line) or non-permeabilized (blue line) cells. The black line represents PLPs generated from MEG-01 cells that not have been exposed to rFVIIa. Data shown is representative of three independent experiments. (F,G) PLPs derived from MEG-01 cells exposed to 100 nM rFVIIa or vehicle were added to factor VII or factor VIII depleted plasma, which was tested by calibrated automated thrombography using the PRP reagent. Shown are lag time (E) and peak (F) values derived from thrombin generation curves. Data represent the mean of three independent experiments. Error bars indicate standard deviation. * p<0.05, ** P<0.01, *** P<0.001.


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Figure 2: rFVIIa uptake by MEG-01 cells is mediated by EPCR.

(A,B) MEG-01 cells were stained for glycoprotein Ibα (A) or EPCR (B) and analyzed by flow cytometry. The black lines represent the background fluorescence of the MEG-01 cells, and the blue lines correspond to appropriate isotype controls. Data shown is representative of three independent experiments. (C) MEG-01 cells were incubated with 100 nM of rFVIIa for 2 hours in presence or absence of 30 mg/ml Annexin V (AnnV) or 45 mg/ml rat monoclonal anti-EPCR antibody, and rFVIIa content of cell lysates was determined by a microtitre plate-based clotting assay using factor VII-depleted plasma. Shown is the quantity of rFVIIa in cell lysates expressed as percentage of control. Data represents the mean with error bars indicating SEM (n=3). *P ≤ 0.01, compared to control (-). (D) Immunofluorescent stainings of MEG-01 cells under the conditions of the experiment shown in panel C. rFVIIa is stained in green, nuclei in blue, and the cell membrane is represented by the dotted line. Scale bars indicate 20 µm. Original magnification 630x.

We next assessed the mode of uptake of rFVIIa by MEG-01 cells. We hypothesized a role for negatively charged phospholipids, GPIbα, or EPCR, which are all known binding partners for rFVIIa (8-10). GPIbα was hardly detected on the surface of differentiated MEG-01 cells; 2.5% ± 0.6% ([mean ± SD, n = 3] of cells stained positive for GPIbα (Fig. 2A). In contrast, EPCR was abundantly present on MEG-01 cells with positive staining on 73.2% ± 11.8% ([mean ± SD, n =3] of cells (Fig. 2B), which has to our knowledge not been reported before. Uptake of rFVIIa was not affected by Annexin A5 excluding a role for negatively charged phospholipids in rFVIIa uptake, but a 76.0 ± 8.7% [mean ± SD] reduction of rFVIIa uptake was observed in the presence of an antibody to EPCR (Fig. 2C). Immunofluorescent staining of rFVIIa confirmed these results (Fig. 2D). EPCR thus appears to fulfill multiple critical features in the mode of action of rFVIIa, including enhancement of hemostatic activity in the intravascular space (8), improvement of endothelial barrier function (9), transport of rFVIIa to extravascular sites (10), and uptake by megakaryocytes in the bone marrow (this study).Taken together, we demonstrate EPCR-dependent uptake of rFVIIa by megakaryocytes with subsequent production of rFVIIa-containing ‘prohemostatic’ platelets. Whether this mechanism acts in vivo requires further study, but delayed generation of rFVIIa-containing platelets appears a plausible mechanism to partly explain the efficacy of once-daily rFVIIa prophylaxis in inhibitor-complicated hemophilia.


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Author contributionsA.M. Schut and M. Kirschbaum participated in the design of the study, performed experiments, analysed and interpreted data and wrote the manuscript. J. Adelmeijer performed experiments, analysed and interpreted data. P.G. de Groot interpreted data. T. Lisman participated in the design of and supervised the study, interpreted data and wrote the manu-script. All authors revised and approved the manuscript.

Financial supportThis study was supported in part by an unrestricted educational grant from Novo Nordisk and by a grant from the Netherlands Organisation of Scientific Research (VIDI,917.11.304 to T.L.).

Conflict of interestThis study was supported by an unrestricted educational grant from Novo Nordisk, whose product is studied in thbe manuscript.

References

1. Young, G., Auerswald, G., Jimenez-Yuste, V., Lam-bert, T., Morfini, M., Santagostino, E. & Blanchette,V.,

PRO-PACT: Retrospective observational study on the prophylactic use of recombinant factor VIIa in

hemophilia patients with inhibitors. Thromb Research. 2012,130,864–870.

2. Konkle, B.A., Ebbesen, L.S., Erhardtsen, E., Bianco,R.P., Lissitchkov, T., Rusen, L. & Serban, M.A.

Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in

hemophilia patients with inhibitors. Journal of Thrombosis and Haemostasis 2007, 5, 1904–1913.

3. Lisman, T. & de Groot, P.G. The role of cell surfaces and cellular receptors in the modeof action of

recombinant factor VIIa. Blood Reviews. 2015, 29;223– 229.

4. Gopalakrishnan R, Hedner U, Clark C, Pendurthi UR, Rao LV. rFVIIa transported from the blood stream

into tissues is functionally active. J Thromb Haemost. 2010;8(10):2318-2321.

5. Kirschbaum M , Lisman T, Giepmans BN, Adelmeijer J, Karimian G, Porte RJ, Kirschbaum M. Horizontal

RNA transfer mediates platelet-induced hepatocyte proliferation. Blood 2015;126(6): 798-806.

6. Schut AM, Hyseni A, Adelmeijer J, Meijers JC, De Groot PG, Lisman T. Sustained pro-haemostatic

activity of rFVIIa in plasma and platelets in non-bleeding pigs may explain the efficacy of a once-daily

prophylaxis in humans. Thromb Haemost. 2014;112(2):304-310.

7. Cecchetti, L., Tolley, N.D., Michetti, N., Bury, L.,Weyrich, A.S. & Gresele, P. Megakary-ocytes

differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a

mechanism for regulating synthetic events. Blood 2011, 118;1903–1911.

8. Pavani G, Ivanciu L, Faella A, Marcos-Contreras OA, Margaritis P. The endothelial protein C receptor

enhances hemostasis of FVIIa administration in hemophilic mice in vivo. Blood. 2014;124(7):1157-

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9. Sundaram J, Keshava S, Gopalakrishnan R, Esmon CT, Pendurthi UR, Rao LV. Factor VIIa binding to

endothelial cell protein C receptor protects vascular barrier integrity in vivo. J Thromb Haemost.

2014;12(5):690-700.

10. Clark CA, Vatsyayan R, Hedner U, Esmon CT, Pendurthi UR, Rao LV. Endothelial cell protein C

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2012;10(11):2383-2391.

summary and dIscussIon8

Chapter 8 Summary and diSCuSSion

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Summary

Chapter 1 is a general introduction to this thesis, including the aims of each chapter. We provide a brief overview on liver resection, regeneration, and the role of platelets in this process. The subsequent Chapter 2 is a letter in response to a review article published in the Journal of Hepatology, which summarized the current knowledge on the role of platelets in liver regeneration. We pointed out that platelet-mediated liver regeneration has to be investigated more intensively and described what we know and don´t know about platelet-mediated liver regeneration.

In Chapter 3 we provide in vitro evidence for a potential novel mechanism of platelet-mediated liver regeneration. In our model we used HepG2 cells and freshly isolated human blood platelets. We demonstrated that platelet-mediated stimulation of HepG2 cell proliferation requires platelet internalization by hepatocytes. Following this internalization, platelets transfer their RNA to the hepatocyte and we demonstrated protein synthesis from platelet-derived mRNA by the hepatocyte. Importantly, platelet RNA contributed substantially to platelet-mediated hepatocyte proliferation. These findings suggest that transfer of platelet RNA to the hepatocyte with subsequent protein synthesis by the recipient cell is key in this process. In addition we also demonstrated in vivo platelet internalization by hepatocytes following a partial hepatectomy in mice. It appears plausible that functional platelet RNA transfer is also relevant for platelet-mediated liver regeneration.

In Chapter 4 we investigated the mechanism of platelet recruitment into the liver parenchyma after partial liver resection. We observed immediately after partial liver resection a temporary platelet infiltration and platelet aggregate formation in the remnant liver. By inducing thrombocytopenia 2 hours pre or 2 hours post partial liver resection, we were able to show that the presence of platelets in the very first hours after partial liver resection is essential for the progression of liver regeneration. In mice that were rendered thrombocytopenic 2hours prior to partial liver resection liver regeneration was significantly reduced compared to mice in which platelets were present until two hours after partial liver resection. In addition we provided evidence that the temporary platelet influx into the liver parenchyma depends on VWF expression. Importantly, deficiency in VWF resulted in impaired in liver regeneration. These results indicate that the need for platelet to stimulate liver regeneration is within the first hours after partial liver resection. Furthermore, the expression of VWF by liver sinusoidal endothelial cells is key in the temporary platelet influx after partial liver resection. Chapter 5 aimed to investigate in a prospective study, the levels of platelet related growth factors in patients undergoing a partial liver resection. Although platelet growth factors were subject of several in vitro studies in animal experiments, it has never been proven

that growth factors stored in platelets are actually responsible for platelet mediated liver regeneration in vivo, for example in patients undergoing partial hepatectomy (1). Our hypothesis was that platelet-derived growth factors are consumed during and after partial liver resection. We examined the levels of various growth factors (VEGF, HGF, FGF, PDGF, PF4, TSPI and endostatin) in plasma and in platelets and compared the levels obtained in patients undergoing partial hepatectomy to patients undergoing a Pylorus-preserving Pancreaticoduodenectomy (PPPD) and healthy controls. Our results showed no substantial differences between platelet and plasma levels of growth factors which were measured during surgery and at several post-operative days in patients undergoing partial hepatectomy, compared to patients undergoing a PPPD. These results suggest that platelet-derived growth factors are less important than generally assumed for platelet-mediated liver regeneration in humans.

In Chapter 6 we investigated whether partial liver resection accelerates the progression of NAFLD in mice and whether treatment with vitamin E reduces this progression. We fed mice with choline-deficient L-amino acid-defined diet to induce mild steatosis and performed partial liver resection. In our studies, mild liver steatosis did not impair liver regeneration in mice. However, partial hepatectomy substantially accelerated the progression of NAFLD. In addition, we provided evidence that enhanced oxidative stress following partial liver resection contributes to the progression of NAFLD and showed that antioxidant therapy with vitamin E attenuates the progression of NAFLD. These findings may have clinical relevance as increasingly partial hepatectomies are performed in patients with steatosis.

We present in Chapter 7 an intermezzo, investigating a novel mechanism which may partly explain the prophylactic properties of once-daily rFVIIa injections in patients with inhibitor-complicated hemophilia. First we showed that rFVIIa was dose-dependently taken up by the megakaryoblastic cell line MEG-01. Secondly, we were able to demonstrate that MEG-01 cells selectively transfer endocytosed rFVIIa to PLPs. Importantly, rFVIIa in PLPs was hemostatically active. Finally we demonstrated that rFVIIa uptake by megakaryocytes is dependent on the EPCR receptor. Taken together, rFVIIa-containing platelets appear a plausible mechanism to partly explain the efficacy of once-daily rFVIIa prophylaxis in inhibitor-complicated hemophilia.


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Discussion

The notion that blood platelets are involved in the stimulation of liver regeneration has been firmly established in the last years (1-4). The therapeutic options are very limited in patients with acute liver insufficiency and the mortality and morbidity rates are high. Therefore, platelets may be an exciting new target for the development of strategies to prevent liver damage and accelerate liver regeneration in patients with liver insufficiency. Nevertheless, we do not understand the molecular mechanisms of platelet-mediated liver regeneration very well. For example, the role of platelet-derived growth factors in the process of liver regeneration has only been poorly investigated and it has still to be demonstrated that delivery of platelet-derived growth factors to the regenerating liver indeed occurs in the process of liver regeneration (1). Furthermore, platelet communication with the liver environment after partial hepatectomy has also been insufficient investigated. The role of platelet RNA in liver regeneration has yet to be investigated. By yet unexplored mechanisms, platelets contribute to the repair of damaged liver tissue by stimulating liver cell proliferation. In this thesis, multiple aspects of platelet-mediated liver regeneration have been investigated in vitro and in vivo as well as in a clinical study with patients undergoing a partial liver resection.

Chapter 3: The role of platelet RNA in liver regeneration

We have described a novel mechanism of platelet-mediated stimulation of liver regeneration in vitro, in which platelets transfer their RNA content to the recipient hepatocyte. We show in Chapter 3, that following platelet uptake by the hepatocytes, platelet-derived mRNA is translated and protein is synthesized by the hepatocyte itself. We have shown in a set of various experiments that both the uptake of platelets by the hepatocyte and the platelet RNA content is essential for the proliferative response of hepatocytes to platelets.

In the last few years, there is growing evidence that platelets have a complex RNA signature, including a rich ensemble of mRNAs and miRNAs which contribute to various biological and pathological processes, such as inflammation and the progression of cancer (5-9). When compared to leukocytes or other cells, the quantity of mRNA in platelets is considerably less (10, 11). Therefore platelets have developed unique mechanisms to translate mRNAs more efficiently (12, 13). Numerous platelet mRNAs have a significant extended poly(A)-tail compared to mRNA from nucleated cells, leading to a prolonged half-life time of the platelet mRNA molecule and a higher translation efficiency (12). Also the 3’-UTRs of platelet mRNAs tend to be longer and more complex when compared to mRNAs of nucleated cells (13). As a result, more protein can be synthesized from platelet mRNA than from “nucleated mRNA” and a transferred platelet mRNA molecule would be much more efficient in stimulating protein synthesis than the “host” mRNA of a nucleated cell. From this point of view it seems

logical for both parties, the platelet as well as the hepatocyte, to transfer platelet mRNA to the hepatocyte. In addition to these unique platelet mRNA modifications, also the repertoire of platelet mRNAs and miRNAs is unique compared to that of nucleated cells (14). It seems plausible that platelets transfer platelet-specific mRNAs and miRNAs to the hepatocytes which are absent in the RNA repertoire of the recipient hepatocyte. Subsequently “foreign proteins” can be synthesized by the recipient cell from platelet mRNA. Platelet miRNAs, 19 to 24-nucleotide non-coding RNAs, can regulate several targets at once and thus can have a profound effect on the recipient cell with minimal material. The transfer of platelet miRNA to recipient cells presents a unique opportunity for platelets to have a large and broad effect on other cells. Nevertheless, the physiological relevance of platelet RNA transfer has only been poorly investigated. In the last years several different labs have investigated platelet mRNA and miRNA transfer to vascular/endothelial cells, resulting in 3 separate publications on the topic (8, 15, 16). Gidlöf et al. investigated platelet miRNA transfer and demonstrated that miRNA released from platelets during myocardial infarction can regulate ICAM1 expression in endothelial cells (15). The study by Risitano et al. has demonstrated that also platelets are capable of transferring mRNAs. They showed that labeled RNA from platelet-like particles is transferred to monocytes and endothelial cells, and this transfer alters gene expression patterns in the target cells (8). However, to our knowledge we were the first to demonstrate a functional change in the recipient cells caused by platelet RNA transfer. We demonstrated translation of platelet-derived mRNA into protein by the recipient hepatocyte, using an actin-GFP encoding RNA as a model gene. Future experiments will need to investigate which coding or regulatory RNA species are responsible for the stimulation of hepatocyte proliferation. At this moment we can only just speculate. Given the complexity and the abundance of around 8500 mRNA and 500 miRNA transcripts in platelets the pool of candidates is large (14). Nevertheless there are several indications that platelet-derived miRNAs are involved in the stimulation of liver regeneration. For example miRNA-21 is present in platelets and it has been demonstrated that increased levels of miRNA-21 stimulates the onset of liver regeneration and the cell-cycle progression of proliferating hepatocytes (17-21). Whether platelet derived miRNA-21 is the source for the elevated levels after partial liver resection or other sources are involved has to be the scope of future studies.

Until now, platelet internalization and transfer of platelet RNA to a recipient nucleated cell has been described for hepatocytes, monocytes, and endothelial cells (8, 15, 22). I think that it is plausible that this communication mechanism between platelets and nucleated cells is not limited to those three cell types and more recipient cells will be discovered. We are just beginning to understand the importance of RNAs in platelets and how the expression of mRNAs and miRNAs in platelets influence platelet functions and the processes in which platelets are involved. The limited studies performed have focused on specific miRNAs or mRNAs and used highly manipulated in vitro systems to investigate platelet RNA


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transfer. It is likely that many transcripts are transferred and will alter the recipient cell in unclear and unknown ways. Also unclear is, whether platelets can transfer a specific group of RNAs or only their entire RNA to the recipient cell. It has been previously demonstrated that megakaryocytes selectively transfer genetic material to platelets, and it is therefore plausible that also platelets are able to selectively transfer RNA species (23). In addition, the mechanism of RNA transfer and the efficiency of platelet-derived mRNA translation into proteins remain unclear and may be variable in different recipient cells. All these question will be subject of future studies to fully understand the mechanism of platelet RNA transfer and the impact on the recipient cells.

In the last decades, also the pharmaceutical industry has become interested in RNA as a novel therapeutic target and drug. Several barriers had to be broken, because RNA is inherently unstable, potentially immunogenic, and requires in most cases a delivery vehicle for efficient transport to the target cell (24, 25). However a number of RNA-based therapeutics were investigated successfully in the last years and are currently under clinical investigation for diseases ranging from genetic disorders to HIV infection to thrombosis and bleeding disorders (25-28). It is conceivable that RNA-based therapies represent an option to develop novel strategies to prevent liver damage and accelerate liver regeneration. Therefore the platelet transcriptome and the specific role of individual platelet RNAs, including mRNAs and miRNAs, in stimulating liver regeneration has to be investigated more intensively in the next years.

Chapter 4: Platelet infiltration after liver resection or the begin of an inflammatory response?!

Several independent studies have demonstrated impaired regeneration in mice that were thrombocytopenic or treated with platelet inhibitors (29-31). The recruitment of platelets in the liver after partial liver resection seems crucial for the onset of liver regeneration. It is, however, unclear how platelets infiltrate the liver remnant. Therefore, we aimed to investigate in Chapter 4 the mechanism of platelet recruitment into the liver parenchyma after partial liver resection in mice. In this study we show, to our knowledge for the first time, that VWF is an essential mediator in the onset of liver regeneration after partial liver resection in mice. Although our study is limited to the investigation of the mechanism behind platelet recruitment into the liver remnant after partial liver resection, we provide clear evidence that platelets infiltrating the liver remnant in the early phase after liver regeneration are responsible for platelet-mediated stimulation of hepatic regeneration. Future experiments have to investigate the molecular mechanism underlying the stimulating effect of platelets in the early phase after liver resection.We described in chapter 3 that direct contact between platelets and hepatocytes as well as platelet internalization is essential for platelet-mediated hepatocyte proliferation. In

addition, we showed in mice immediately after performing partial liver resection, platelet translocation from the sinusoidal space to the space of Disse and uptake by the hepatocyte. Whether this observation is part of a communication mechanism between platelets and hepatocytes in vivo is unknown. It is also unsure if this mechanism/observation is responsible for subsequent induction of the initiation of hepatocyte proliferation and the onset of liver regeneration. In vitro we demonstrated that platelet uptake and transfer of platelet RNA to the hepatocyte is in part responsible for platelet-mediated hepatocyte proliferation, but it is still the question whether this mechanism also takes place in vivo. Similarly, it has never been conclusively demonstrated that platelet-derived growth factors stimulate liver regeneration in vivo (1). Perhaps both mechanisms, platelet RNA transfer and the delivery of platelet-derived growth factors to the hepatic vasculature are irrelevant in vivo and have only indirect effects on liver regeneration.

We have shown in this chapter that impaired liver regeneration, such as in VWF deficient mice, is associated with reduced platelet infiltration and aggregation in the vasculature of the liver remnant. Multiple studies have investigated the interaction between platelets and (liver) endothelial cells (2, 32, 33). A study by Lalor et al. has demonstrated in vitro that platelets stimulate liver endothelial cell activation which induces the secretion of cytokines and chemokines form the endothelial cells (33). Subsequently immune cells such as neutrophils and leukocytes are attracted and bind to the liver endothelial cells. Lalor and co-workers were able to demonstrate the functional relevance of platelet binding by experiments that show a markedly reduced amount of attracted neutrophils and leukocytes in the absence of platelets. A direct association between leukocyte recruitment and the outcome of liver regeneration is described by Selzner and coworkers. They have shown in mice, deficient in the intercellular adhesion molecule 1 (ICAM-1), reduced leukocyte recruitment after partial liver resection and also a decreased hepatocyte proliferation activity (32). Moreover, several studies have demonstrated that pro-inflammatory cytokines, secreted from neutrophils and leukocytes, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF) are essential in the onset of hepatocyte proliferation (34-36).Based on our observations and the results from the before mentioned studies, I postulate an alternative hypothesis to explain the role of platelets in stimulation of liver regeneration. Perhaps platelets do not stimulate liver regeneration by direct mitogenic effects but by initiating the recruitment of immune cells into the hepatic vasculature after partial liver resection. Liver regeneration is a highly orchestrated process and is part of a local and systemic inflammatory response. I would like to take a look at the evolutionary of platelets to support my alternative hypothesis on platelet-mediated liver regeneration.


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The hemocyte: platelets ancestor

Limulidae polyphemus, the horseshoe crab, is the last surviving member of the class merostomata, which includes marine spiders (37). Horseshoe crabs have only one circulating blood cell in their circulation (hemolymph) which is called the Limulus amebocyte or hemocyte (38). Probably the Limulus hemocyte has been the most intensively investigated invertebrate blood cell involved in thrombosis and hemostasis. In contrast to mammalian platelets, the hemocytes are nucleated and their size range between 10 and 20 µm (38). Like platelets, after activation the discoid shaped hemocytes change their shape, form pseudopodia, and release the content of their granules (39). Large hemocyte aggregates are generated by cross-linking of soluble proteins with receptors on the surface of hemocytes, which is comparable to the formation of a platelet thrombus (40).It is obvious that hemocytes, as the only circulating cell in the hemolymph of invertebrates, have to be involved in more processes than only thrombosis and hemostasis. Like mammalian blood cells, hemocytes are also responsible for innate and adaptive immune responses and for the defense against pathogens (41). They can recognize pattern recognition receptors and scavenger receptors, which are crucial for host defense (42, 43). If hemocytes become activated, a subset of hemocytes are able to degranulate very rapidly, thereby releasing a battery of antimicrobial peptides, such as defensin, tachyplesins, and anti-LPS factor into the hemolymph (39, 44-46). Consequently, pathogens are killed and become phagocytosed by the hemocyte which is comparable to the function of macrophages in higher vertebrates. Hemocyte biology represents us the evolutionary link between platelet functions in thrombosis, hemostasis, and immunity. In general, coagulation is initiated by tissue damage and the exposure of collagen to the blood. In invertebrates, pathogen associated molecules, including LPS are also able to trigger coagulation. Hemocytes detect those pathogen associated molecules by their surface receptors and start aggregating with other hemocytes (44, 47). The “hemocyte clot” functions as a trap and kills the pathogens. This process clearly demonstrates functional overlap between hemostasis and immunity. It seems that both processes share the same evolutionary roots and despite more than 400 million years of evolution, mammalian platelets retain many of the immune functions which are still required in a variety of biological processes, such as sepsis, cancer and also liver regeneration (9, 48, 49). In summary, platelets are more than cellular fragments needed for blood clotting. Platelets are immune cells! I propose that this immune cell function for platelets is also vital for liver regeneration. Our results have demonstrated that the need for platelets after partial liver resection is limited in time. Platelets have to be present in the liver remnant merely in the first two hours after liver resection in order to initiate the full regenerative response. I propose that during this period platelets initiate the pro-inflammatory reaction cascade which is needed for successful liver regeneration. Immediately after liver resection, platelets

infiltrate the hepatic vasculature of the liver remnant, bind to the liver endothelial cells and facilitate the recruitment of neutrophils and leukocytes. Neutrophils and leukocytes and associated cytokines/chemokines are crucial for the successful progression of liver regeneration (32, 35). Without this platelet-mediated attraction of neutrophils and leucocytes, liver regeneration is impaired due to the absence of an inflammatory reaction (32). This alternative explanation for the mechanism behind platelet-mediated liver regeneration deserves attention in future studies.

Chapter 5: Platelet-derived growth factors in liver regeneration: not as important as generally assumed?

In Chapter 5 we investigated the levels of growth factors stored within platelet alpha granules in patients undergoing a partial hepatectomy or PPPD. We showed in this clinical study that growth factors levels in platelets and in plasma were comparable between the two procedures, and found no evidence for immediate or delayed consumption of platelet-derived growth factors by the regenerating liver. We thus did not find evidence for a role of platelet-derived growth factors in stimulation of liver regeneration in humans.

A single animal study has suggested a role for a specific platelet derived growth factor, serotonin, in platelet-mediated liver regeneration (2, 30, 50). However, the results of this study may also be explained by the platelet stimulating effect of serotonin instead of its mitogenic effect (Lisman, Kirschbaum 2015). Starlinger et al. published in 2014 that serotonin is a relevant inducer of liver regeneration after major partial liver resection in humans, a finding which was not confirmed by a study from our group (51, 52). Importantly, Starlinger and colleagues compared post-operative serotonin levels only to pre-operative levels and not to control patients, and were not able to conclude whether changes in platelet serotonin content related to liver regeneration or to effects of major surgery. We decided to compare growth factor levels of patients undergoing partial liver resection to a group of patients undergoing a PPPD, a major abdominal surgery of similar extent and in a patient group with cancer but without the requirement for liver regeneration.The findings from this clinical study indicate that platelet-derived growth factors and also serotonin may be less relevant in platelet-mediated liver regeneration in humans than previously assumed. Alternative options including RNA transfer and platelet-mediated initiation of inflammation should be explored also in the human setting. We are currently performing RNA sequencing of platelets isolated from the patients described in this chapter to investigate whether hepatectomy-specific changes in the platelet transcriptome can be detected.


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Chapter 6: Partial liver resection in mice and the progression of non-alcoholic fatty liver disease

The previous chapters discussed partial liver resection with focus on the mechanisms of platelet-mediated liver regeneration. In Chapter 6 we studied liver regeneration in mice with mildly steatotic livers and unexpectedly detected that liver resection markedly accelerated the progression of non-alcoholic fatty liver disease (NAFLD). We found that mild steatosis in mice does not impair liver regeneration after performing partial liver resection but accelerates the progression of a mild steatosis towards steatohepatitis and to a more progressive inflammatory phenotype of NAFLD. In addition we were able to show that antioxidant therapy with vitamin E attenuates the progression of NAFLD.

A number of studies have suggested that oxidative stress and cytokines play an important role in the pathogenesis of NAFLD (53-55). The stimulation of inflammatory responses is one mechanism by which oxidative stress triggers the progression of mild steatosis towards NAFLD (56). Here we demonstrated that partial liver resection and consequently liver regeneration is such a trigger to stimulate the progression of NAFLD. Interestingly, and perhaps an alternative explanation for our findings, platelets have been previously shown to accelerate the progression of NAFLD (57). It would therefore be of great interest if thrombocytopenic mice show similar progression patterns of a mild hepatic steatosis towards NAFLD after performing partial liver resection. It is conceivable, that a reduced platelet count decelerates the inflammatory response after partial liver resection, which consequently lowers the level of oxidative stress and the progression of NAFLD. Several studies have indicated in patients with chronic diseases, that anti-platelet therapy such as clopidogrel, results in a significant reduction of inflammatory and oxidative stress markers (58-60).

The results in chapter 6 show that the progression of a mild liver steatosis towards NAFLD is accelerated by major partial liver resection in mice. In Western countries, NAFLD is the most common hepatic parenchymal disorder, affecting 6% to 11% of individuals in the general population (61). In the last decades partial liver resection has become a common surgical procedure and is still the only potentially curative therapy for primary and metastatic tumors of the liver and biliary tract (62-64). While extensive partial liver resection (up to 70%) can be safely performed on patients with healthy livers, the risk of such resections in patients with underlying hepatic disease, such as NAFLD are elevated (65, 66). Whether progression of NAFLD also occurs in humans is currently unknown, but if this would be the case, the clinical implications are substantial. Given the success of vitamin E in delaying NAFLD progression, administration of vitamin E during and after a liver resection to avoid resection-induced acceleration of disease would be a realistic clinical scenario.

Taken together, the studies described in this thesis give new insights on the mechanism underlying platelet-mediated stimulation of liver regeneration. Some of the concepts outlined may have direct clinical relevance, as it may result in therapeutic strategies aimed at supporting liver regeneration, for example in patients at risk for the small for size syndrome. Nevertheless, future studies aimed at validating some of the concepts in animal models or humans are required. These studies include animal model and human studies aimed at testing the relevance of platelet RNA transfer in liver regeneration, exploration of alternative strategies to promote liver regeneration by enhancing the local inflammatory response required for liver regeneration, and assessment of the effect of liver resection in humans with NAFLD on disease progression.


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De lever heeft een enorm regeneratief vermogen. Rond 70% van de lever van een mens of proefdier kan veilig verwijderd worden door middel van een partiële leverresectie, waarna het resterende deel weer uitgroeit tot nagenoeg het oorspronkelijke volume.. Ook kan bij een levende donor levertransplantatie een deel van de lever gedoneerd worden waarna regeneratie in zowel donor als ontvanger plaatsvindt. In de mens groeit de lever weer tot nagenoeg het oorspronkelijke volume aan binnen enkele maanden. Leverregeneratie vindt niet alleen plaats nadat een deel van de lever verwijderd is maar kan ook plaats vinden nadat schade door toxines of virussen is ontstaan. Dit resulteert in veel gevallen in acute of chronische leverziektes. Terwijl de lever in sommige gevallen dankzij het regeneratief vermogen kan herstellen, zijn er patiënten die uiteindelijk aan leverinsufficiëntie gaan leiden. Voor patiënten met leverinsufficiëntie bestaan momenteel beperkte therapeutische opties en een levertransplantatie is uiteindelijk de enige behandeloptie. In de afgelopen jaren is door verschillende studies in proefdier en mens aangetoond dat bloedplaatjes betrokken zijn bij de stimulatie van leverregeneratie. Deze bloedplaatjes zouden een aangrijpingspunt kunnen zijn om nieuwe strategieën die leverschade kunnen voorkomen of het proces van leverregeneratie in patiënten met lever insufficiëntie kunnen versnellen. Helaas hebben wij nog te weinig inzicht in de moleculaire mechanismen die de stimulerende werking van bloedplaatjes op leverregeneratie verklaren. Als voorbeeld is de rol van groeifactoren opgeslagen in bloedplaatjes bij leverregeneratie te noemen. Hoewel wordt aangenomen dat deze groeifactoren een belangrijke rol spelen in bloedplaatjes-gemedieerde leverregeneratie moet nog steeds aangetoond worden, dat dit mechanisme inderdaad betrokken is bij bloedplaatjes-gemedieerde leverregeneratie. Andere mechanismen die het stimulerende effect van bloedplaatjes op leverregeneratie zouden kunnen verklaren, zoals de mogelijkheid dat bloedplaatjes hun RNA aan andere cellen overdragen, zijn nog niet in in vivo modellen onderzocht. In dit proefschrift zijn mogelijke mechanismen van bloedplaatjes-gemedieerde leverregeneratie onderzocht in in vitro en in vivo modellen, en in een klinische studie onderzocht.

Hoofstuk 1 is een algemene inleiding van dit proefschrift waarin het doel van elk hoofdstuk wordt beschreven. Er wordt een kort overzicht gegeven over leverresectie, regeneratie en de rol van bloedplaatjes in kader van dit proefschrift. Het volgende Hoofdstuk 2 is een brief in reactie op een review artikel gepubliceerd in Journal of Hepatology, die de huidige kennis samenvat op het gebied van de rol van bloedplaatjes in leverregeneratie. Aandachtspunt van deze brief is dat bloedplaatjes-gemedieerde leverregeneratie meer intensief onderzocht moet worden en beschrijft de bestaande en nog onbekende kennis op dit onderzoeksgebied.

In Hoofdstuk 3 wordt door middel van een aantal in vitro experimenten een potentieel nieuw mechanisme voor bloedplaatjes-gemedieerde leverregeneratie aangetoond. In het model wordt er gebruik gemaakt van HepG2 cellen en vers geïsoleerde humane bloedplaatjes. Er

wordt aangetoond dat voor bloedplaatjes-gemedieerde stimulatie van HepG2 cel-proliferatie de opname (internalisatie) van bloedplaatjes door hepatocyten vereist is. Na internalisatie dragen bloedplaatjes hun RNA over aan de hepatocyt. Vervolgens vindt eiwitsynthese vanaf het mRNA afkomstig van bloedplaatjes door de hepatocyt plaats. Nog belangrijker is dat bloedplaatjes RNA substantieel bijdraagt aan bloedplaatjes-gemedieerde hepatocyten proliferatie. Deze bevindingen suggereren dat transport van bloedplaatjes RNA naar de HepG2 cel en vervolgens eiwitsynthese door de ontvangende cel belangrijk is voor HepG2 celproliferatie. Daarnaast is er in vivo aangetoond dat bloedplaatjes internalisatie door hepatocyten plaatsvindt na partiële hepatectomie in muizen. Het lijkt aannemelijk dat functionele bloedplaatjes RNA overdracht ook relevant is voor bloedplaatjes-gemedieerde stimulatie van leverregeneratie.

In Hoofdstuk 4 is het mechanisme betrokken bij de aantrekking van bloedplaatjes door het leverparenchym na partiële leverresectie onderzocht. Onmiddelijk na partiële leverresectie werd er een tijdelijke bloedplaatjes infiltratie en bloedplaatjes aggregaatvorming in de leverrest gezien. Door het induceren van trombocytopenie 2 uur voor of 2 uur na partiële leverresectie, kon er aangetoond worden dat de aanwezigheid van bloedplaatjes in de eerste uren na partiële leverresectie essentieel is voor het stimuleren van leverregeneratie door de bloedplaatjes. Bij de muizen die trombocytopeen werden gemaakt 2 uur voorafgaand aan de partiële leverresectie, werd er een significant verminderde leverregeneratie aangetoond in vergelijking met muizen waarbij bloedplaatjes aanwezig waren tot 2 uur na partiële leverresectie. Daarnaast werd er aangetoond dat de tijdelijke bloedplaatjes instroom in het leverparenchym afhankelijk is van Von Willebrand Factor (VWF). Belangrijker nog, een deficiëntie in VWF resulteerde in een verminderde leverregeneratie. Deze resultaten laten het belang van de instroom van bloedplaatjes in het leverparenchym binnen de eerste uren na een partiele leverresectie zien. Of het vrijkomen van VWF uit de sinusoïdale lever endotheelcellen of de bloedplaatjes, of het plasma VWF belangrijk is voor de tijdelijke bloedplaatjes instroom na partiële leverresectie dient nog nader onderzocht te worden..

Hoofdstuk 5 laat de resultaten van een prospectieve studie zien, waarin de plasmaspiegels van bloedplaatjes-gerelateerde groeifactoren onderzocht zijn bij patiënten die een partiële leverresectie ondergingen. Alhoewel bloedplaatjes afkomstige groeifactoren bestudeerd zijn in verschillende in vitro studies en in dierexperimenten, is het nooit bewezen dat deze groeifactoren in bloedplaatjes daadwerkelijk verantwoordelijk zijn voor bloedplaatjes-gemedieerde leverregeneratie in vivo, bijvoorbeeld bij de patiënten die een partiële hepatectomie hebben ondergaan. Onze hypothese was dat bloedplaatjes-gerelateerde groeifactoren verbruikt worden tijdens en na partiële leverresectie. De plasmaconcentraties en spiegels in bloedplaatjes van verschillende bloedplaatjes-gerelateerde groeifactoren waarvan bekend is dat ze opgeslagen zijn in bloedplaatjes en die een rol spelen in

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leverregeneratie (VEGF, HGF, FGF, PDGF, PF4, TSPI en endostatine) werden onderzocht bij patiënten die een partiële hepatectomie of pylorus-preserving pancreaticoduodenectomy (PPPD) ondergingen en werden vergeleken met de concentraties van gezonde vrijwilligers. Er waren geen substantiële verschillen tussen bloedplaatjes en plasmaconcentratiess van bloedplaatjes afkomstige groeifactoren tussen patiënten die een partiele leverresectie of een PPPD ondergingen op verschillende tijdstippen na de ingreep. Deze resultaten suggereren dat groeifactoren opgeslagen in bloedplaatjes minder belangrijk zijn voor bloedplaatjes-gemedieerde leverregeneratie bij mensen dan tot nu toe aangenomen werd.

In Hoofdstuk 6 is er onderzocht of partiële leverresectie in muizen het proces van niet-alcoholische leververvetting (non-alcoholic fatty liver disease; NAFLD) versnelt en of behandeling met vitamine E dit proces reduceert. De muizen kregen een “choline-deficient L-amino acid-defined” dieet om milde leversteatose te induceren. Daarna werd er een partiële leverresectie uitgevoerd. In deze studie had milde leversteatose geen nadelig effect op de leverregeneratie. Echter, partiële hepatectomie versnelde de progressie van NAFLD aanzienlijk. Bovendien is er bewijs geleverd dat oxidatieve stress na partiële leverresectie bijdraagt aan de progressie van NAFLD. Ook verminderde behandeling met met de antioxidant vitamine E de progressie van NAFLD. Deze bevindingen kunnen klinische relevantie hebben omdat er steeds meer partiële hepatectomieen uitgevoerd worden bij patiënten met steatose.

Hoofdstuk 7 is een intermezzo, hierin wordt het onderzoek beschreven van een nieuw mechanisme wat deels het profylactische effect zou kunnen verklaren van een dagelijkse injectie met recombinant stollingsfactor VIIa (rFVIIa) bij patiënten met hemofilie die remmende antistoffen tegen factor VIII hebben ontwikkeld. Ten eerste wedr aangetoond dat rFVIIa dosisafhankelijk wordt opgenomen door de megakaryoblastische cellijn MEG-01. Ten tweede werd aangetoond dat de MEG-01 cellen rFVIIa selectief overbrengen naar plaatjes-achtige celfragmenten (platelet-like particles, PLPs) geproduceerd door deze cellen. Belangrijker nog, rFVIIa in PLPs is hemostatisch actief. Tenslotte is er gevonden dat rFVIIa opname door megakaryocyten afhankelijk is van de endotheliale proteïne C receptor.

Alle resultaten van hierboven beschreven studies zijn samengevat en worden bediscussierd in Hoofdstuk 8. De in dit proefschrift beschreven studies hebben nieuwe inzichten gecreëerd in verschillende mechanismes van bloedplaatjes-gemedieerde leverregeneratie. Enkele concepten, die in dit proefschrift gepresenteerd worden, zoals de betrokkenheid van VWF in de vroege fase na partiële leverresectie, zouden directe klinische relevantie kunnen hebben en in nieuwe therapeutische opties kunnen resulteren om leverregeneratie te stimuleren. Andere concepten zijn op dit moment in een experimentele fase en vereisen nog uitgebreid vervolgonderzoek. Transfer van bloedplaatjes RNA zou in toekomstige studies zowel in vitro als ook in dierexperimenten verder onderzocht moeten worden om de daadwerkelijke relevatie van dit nieuwe mechanisme voor het proces van leverregeneratie vast te stellen.

author affiliationsa

Author AffiliAtions Author AffiliAtions

a

132 133

Ton Lisman1,2

Robert J. Porte2

Golnar Karimian1

Jelle Adelmeijer1

Ben N.G. Giepmans3

Craig N. Jenne4

Paul Kubes4

Zwanida J. Veldhuis1

Klaas A. Sjollema3

Peter J. Lenting5

Cécile V. Denis5

Fernanda Bomfati1

Edris Alkozai1

Anne Marieke Schut1

Philip G. de Groot6

1. Surgical Research Laboratory Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

2. Section of Hepatobiliairy Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

3. Department of Cell Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

4. Calvin, Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

5. Insitut National de la Santé et de la Recherche Médical Unit 770. Le Kremlin-Bicêtre, France6. Department of Clinical Chemistry and Haematology, University of Utrecht, University

Medical Center Utrecht, Utrecht, the Netherlands

dankwoord

d

dankwoord dankwoord

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Prof. dr. Ton Lisman, beste Ton, in begin 2011 hebben zich de wegen van ons gekruisd. Jij had net je VIDI grant binnen gehaald en was op zoek naar een promovendus. Ik was net afgestudeerd aan de Radboud Universiteit Nijmegen en solliciteerde bij jou als promovendus. Het einde hiervan is bekend. Zonder jou keuze van toen, stond ik vandaag niet hier. De eerste periode van het promotietraject was vooral gekenmerkt door mijn enthousiasme en de vrijheid die jij mij hebt gegeven. Bloedplaatjes onderzoeken op het gebied van leverregeneratie was nog maar matig onderzocht. De creatieve ruimte om met dit onderzoek alle kanten op te gaan was erg groot. Jij hebt mij al deze ruimte gegeven en in mij geïnvesteerd met als highlight een bezoek bij Prof. dr. Paul Kubes in Calgary, specialist op het gebied van intravital imaging. Hiervoor ben ik jou erg dankbaar. Met een microscoop in een levend dier/orgaan te kijken is en blijft een fascinatie voor mij. Maar je moest mij ook dwingen om begonnen projecten af te maken en niet steeds weer met nieuwe ideeën te komen. Dit was niet altijd makkelijk voor mij maar ook hievoor wil ik jou mijn dank uitspreken. Ik kan mij geen betere promotor voorstellen als het gaat om wetenschappelijk onderzoek vooral m.b.t het schrijven van wetenschappelijke artikelen. Jouw revisies van manuscripten waren altijd super snel, erg goed en voor mij uitdagend. Hierbij heb ik in jou een ware leermeester gevonden. Dank je voor al je hulp in de afgelopen jaren dat ik bij jou mocht promoveren.

Prof. dr. Robert Porte, beste Robert, je was een goede co-promotor voor mij. Je hebt altijd objectief en ook kritsch naar mijn onderzoek gekeken. Het heeft mij erbij geholpen om de goede weg in mijn onderzoek te vinden. Dank je voor de betrokkenheid bij al mijn projecten en voor je steun.

Mijn bijzonder dank gaat uit naar de beoordelingscomissie, Prof. dr. K.N. Faber, Prof. dr. S.C.D. van IJzendoorn en Prof. dr. J.C.M. Meijers voor het beoordelen van dit proefschrift. Prof. dr. Henri Leuvenink, beste Henry, jouw gezelschap op het lab heeft mij altijd een welkom gevoel gegeven. Dank je voor je humor en de grapjes die wij samen over Duitsers en Nederlanders hebben gemaakt.

Dr. Peter Lenting, beste Peter, heel erg bedankt voor de goede samenwerking met jou en de mooie tijd die ik dankzij jou in Paris had.

Prof. dr. Klaas Nico Faber, Prof. dr. Han Moshage, Dr. Peter Olinga en Prof. dr. Sven van IJzendoorn, jullie heel erg bedankt voor de de inspirerende gesprekken tijdens verschillende lever congressen. De Dutch Liver Retreat (DLR) in Spier was voor mij altijd een hoogtepunt van het jaar. Ik kijk met veel plezier hierop terug.

Medewerkers en analisten van het Chirurgisch Onderzoekslaboratorium Jelle Adelmeijer, Susanne Veldhuis, Jacco Zwaagstra, Petra Ottens, Janneke Wiersema-Buist en Renée Gras, heel erg bedankt voor al jullie ondersteuning en hulp bij de verschillende projecten. Ik heb jullie altijd ontzettend gewaardeerd. Een bijzonder dank gaat aan Jelle en Suus. Jelle je hebt mij geleerd hoe ik met bloedplaatjes moet omgaan en mij bij een aantal experimenten geholpen. Heel erg bedankt voor de leuke tijd op het lab. Suus, jij hebt in de afgelopen jaren heel veel voor mijn onderzoek betekend m.b.t. proefdierwerk. Samen hebben wij bij honderden muizen partiële leverresecties uitgevoerd en dat niet alleen hier in Groningen. Samen zijn wij door de wereld gereisd en hebben onze trucjes in Calgary, Canada en in Paris uitgevoerd. In deze tijd hebben wij elkaar goed leren kennen en leren waarderen. Dank hiervoor.

Medewerkers van het University Medical Imaging Center (UMIC) Klaas Sjollema, Jeroen Kuipers en Prof. dr. Ben Giepmans. Sinds het begin van mijn promotietraject is imaging een van mijn grote passies. Voor het oog ontzichtbare dingen zichtbaar maken. In het bijzonder bedank ik Klaas en Jeroen. Klaas, je hebt uren met mij in donkere kamers doorgebracht om mooie plaatjes van levercellen en bloedplaatjes te maken. Gezamelijk hebben wij met groot succes intravital liver imaging in muizen opgezet. Het was mij in al die jaren een groot plezier om met jou samen te werken. Jeroen, jij hebt mij het vak van de electronen microscopie geleerd. Echt een fascinerende wereld van de microscopie waar ik dankzij jou en beste Ben mocht induiken. Een bijzonder dank gaat hier ook aan Prof. dr. Ben Giepmans, zonder de ondersteuning was het niet mogelijk om deze projecten te realiseren.

Mede-promovendi van het Chirurgisch Onderzoekslaboratorium, Anne Marieke Schut, Dafna Groeneveld, Laura Burlage, Edris Alkozai, Alix Matton, Greg Hugenholtz, Michael Sutton, Sanna op den Dries, Andrie Westerkamp, Wilma Potze, Welmoet Westendorp, Jeffrey Damman, Anne Koning, Rolando Rebollede, Leon van Dullemen, Rick Mencke, Anne Koning, Maximila Hottenrott, Deborah van Dijk, Freha Arshad, Negin en Golnar Karimian. Met jullie het lab, de AIO-kamer en/of de gang delen was bijzonder aangenaam; van Duitse themafeestjes tot samen pipetteren tot vrijdagmiddagborrels met rondvliegende borrelnootjes, het is me een genot geweest! Zonder jullie zou het onderzoek een stuk ongezelliger geweest zijn.

Mede-promovendi van het MDL lab, Esther Verhaag, Mark Hoekstra, Anouk Regeling, Brenda Hijmans, Floris Haijer, Andrea Schreuder, Tim van Zutphen, Marleen Schonewille, Nienke Volker-Touw, Weilin Liu, Marjolein Tiebosch en Vera Nies, bedankt voor jullie leuke gezelschap.

Het celkweek clubje: Valerie, Peter, Djoke, Iris, Jurien, Douwe, Hans, Edwin en Wijnand bedankt voor de vele uren achter de flowkasten en het delen van frustraties en successen, wat soms zo dicht bij elkaar kan liggen.

dankwoord dankwoord

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Medewerkers van de Centrale Dienst Proefdieren (CDP), in het bijzonder Michel Weij, Arie Nijmeijer en Annemiek Smit-van Oosten, alsook proefdierdeskundige Dr. Catriene Thuring. Bedankt voor jullie hulp, inzet en flexibiliteit tijdens de proefdierexperimenten.

PRA Healthscience collega´s, Astrid Leegte, Martine Broekema, Barry van der Strate, Janet Stegehuis, Chris den Hengst, Anniek Visser, Riejanne Bax-Siegers, Daphne Ferwerda, Arjen Dijkstra, Kees Veenstra, Nanda Gruben, Kees Mulder, Agnes Geerts, Elinda Bruin, Henko Tadema, Marie Geerlings, Sijranke Post, Edwin Hooijschuur en Peter Ketelaar. Jullie zijn geweldige collega’s. Betere kan ik mij op dit moment niet voorstellen. Een bijzondere dank gaat naar Astrid voor al je steun en motivatie.

Bram Prins, bedankt voor de leuke borrels in “der Witz”.

Dane Hoeksma, wat hebben wij avonden en weekenden samen in het lab en in het triade gebouw gezeten. Met elkaar hebben wij de frustratie zowel ook het succes van ons onderzoek gedeeld. Maar ook buiten het lab hebben wij de afgelopen jaren een geweldige tijd gehad. Dank je voor al je steun. Je bent een goede vriend!

Anne van Erp, dank je voor al de leuke gesprekken die wij met elkaar hadden. Jou enthousiasme heeft mij altijd erg gemotiveerd.

Lian Molema & Martijn Gunnewijk samen met jullie is het altijd gezellig. Bedankt voor de leuke dagen en avonden die wij met elkaar hebben en dat wij nog heel vaak Risk gaan spelen.

Michael de Vet & Marieke Vredeveld, wat begon met het kopen van een keuken, eindigde in een goede vriendschap met jullie twee. Zonder jullie was de Sushi consumptie in de afgelopen jaren significant lager geweest. Ik ben blij dat jullie er zijn!

Jan Hofmann, zusammen begonnen wir das Studium der medizinischen Biologie 2006 in Nijmegen. Auch wenn sich unsere Wege rasch wieder trennten, ist mir in Dir ein sehr guter Freund erhalten geblieben. Ich blicke immer wieder gerne auf die Zeit zurück die wir zusammen verbracht haben. Auch ein Dank an dein Freundin Lisa. Zusammen seit ihr Spitze!

Sandy & SennaOns twee prachtige honden! Wat ben ik er toch gek op! Jullie zijn er altijd en geven mij onvoorwaardelijke liefde.

FoX Barista is meer dan een hobby, het is een passie voor mij. Koffie zetten is wetenschap, maar vooral geeft het mij heel veel energie. Energie die ook voor dit proefschrift belangrijk

was. Iedereen bedankt die eraan heeft bijgedragen om FoX Barista op te starten.“We have created a space and designed a lifestyle that lets me escape from the reality”.

Familie de Vos, Maarten & Marlise, Jolande & Hilko, Annerieke & Erik en alle neefjes en nichtjes. Heel erg bedankt voor jullie motivatie in de afgelopen jaren. Ik ben blij dat jullie er zijn.

Ger & ElsJullie zijn geweldige schoonouders en ik ben heel erg gelukkig dat jullie er zijn. Ook jullie hebben een aandeel aan dit proefschrift. Had je mij een aantal jaren geleden gevraagd of ik ooit een keer bij mijn schoonouders zou gaan wonen, had ik die persoon echt voor gek verklaard. Maar het was nodig om ons huidig huis te verbouwen. Een heel bijzondere tijd waarvoor ik jullie heel dankbaar ben. Voor al jullie ondersteuning in de afgelopen jaren wil ik jullie heel erg bedanken. Ik kan mij vooral geen betere schoonouders voorstellen.

Pascal, mein Bruder, auch an Dich geht ein ganz besonderer Dank. Fast 2 Jahre lang hast du mit mir auf der Noorderdwarsstraat 6 in Groningen gewohnt. Eine ganze besonder Zeit, in der wir sehr viel zusammen erlebt haben. Ich denke häufig an die Abenden auf diversen Bowlingbahnen in Assen, Groningen und Leek zurück. Danke für tolle Zeit die wir zusammen in Groningen hatten.

Mama & Papa, meine lieben Eltern. Mit meiner Doktorarbeit findet ein langer Weg sein Ende. Es ist ein Kapitel in meinem Leben welches ich mit disem Buch abschließe. Hieran habt auch Ihr einen großen Anteil. Mit stets großem Interesse habt ihr meine Forschungsarbeiten in den vergangenen Jahren verfolgt und mit mir zusammen Höhen und Tiefen mitgemacht. In teils stundenlangen Gesprächen habe ich euch so gut möglichst probiert zu erklären was ich genau erforsche. Das war zugegeben nicht immer einfach, hat aber manchmal auch mir geholfen. Für all eure Unterstützung in den vergangenen Jahren bin ich euch sehr dankbar. Ohne diese würde ich wahrscheinlich heute hier nicht stehen. Danke Mama & Papa.

Corline, mijn allerliefste. Wij hebben elkaar ontmoet toen ik in het derde jaar van mijn promotie traject was. Het was geen makkelijke tijd voor mij. Te aantrekkelijk waren andere dingen in het leven naast mijn promotie. Gelukkig heb ik ook dankzij jou mijn hoofd weer de goede kant op gekregen. Jij wist precies wat ik nodig had om mijn doel niet uit mijn ogen te verliezen en wist mij erg goed te motiveren. Maar vooral had je heel veel geduld met mij. Je was en bent nog steeds de bron voor heel veel energie voor mij. Samen zijn wij een geweldig team, dat niemand kan verslaan. Dank je voor al je steun, motivatie en liefde in de afgelopen jaren. Ik ben gek op jou!

about the authora

about the author about the author

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Marc Kirschbaum was born on the 24th of June 1987 in Tönisvorst, Germany. He graduated in 2006 from the episcopal Maria-Montessori School in Krefeld. During his last year in school he decided to move from Germany to the Netherlands. In the summer of 2006 he enrolled in the Biomedical school at the Radboud University of Nijmegen and obtained in 2009 the Bachelor degree and in 2011 the Master degree in Biomedical Sciences. In the last two years as Master student at the University of Nijmegen, Marc focused in his internships on different aspects of breast cancer metastasis and investigated new potential therapeutic options for patients with triple-negative breast cancer.In early 2011, Marc joined as a PhD student the research group from Prof. dr. Ton Lisman and Prof. dr. Robert J. Porte at the University Medical Centre Groningen. In the upcoming 4 years, he focused on the investigation of platelet-mediated liver regeneration and the underlying molecular mechanisms.Since 2015, Marc is Project Manager at PRA Health Sciences and responsible for the conduct of bioanalytical studies.

Documents

University of Groningen Molecular mechanisms of platelet … · 2017-09-13 · ISBN: 978-94-92679-10-9 Cover design: Marc en Corline Kirschbaum Lay out by Sinds1961 Printed by Print