Inflammation and Complement Activation in Intracranial Artery Aneurysms

Abstract

Intracranial artery aneurysms (IAs) are estimated to be present in 2.3% of the population. A rupture of an IA causes subarachnoid hemorrhage, with up to 50% mortality. The annual low rupture risk of an IA indicates that most IAs never rupture. The current treatment options are invasive and somewhat risky. Thus rupture-prone IAs should be identified and this requires a better understanding of the IA wall pathobiology. Inflammatory cell infiltrations have been found to precede IA rupture, indicating the role of inflammation in IA wall degeneration and rupture. The complement system is a key mediator of inflammation and house-hold processing of injured tissue. This study aimed at identifying the role of complement activation in IA wall degeneration and the complement activators involved and determining how the complement system is regulated in the IA wall. In immunostainings, the end-product of complement activation, the terminal complement complex (TCC), was located mainly in the outer part of the IA wall, in areas that had also sustained loss of cells. In electron microscopy, the area of maximum TCC accumulation contained cellular debris and evidence of both apoptotic and necrotic cell death. Complement activation correlated with IA wall degeneration and rupture, de-endothelialization, and T-cell and CD163-positive macrophage infiltration. The complement system was found to become activated in all IAs by the classical pathway, with recruitment of alternative pathway amplification. Of the potential activators immunoglobulins G and M and oxidatively modified lipids were found in large areas. Lipid accumulation was observed to clearly colocalize with TCC and C-reactive protein. In the luminal parts of the IA wall, complement activation was limited by cellular expression of protectin (CD59) and extracellular matrix-bound inhibitors, C4b binding protein and factor H whereas the outer part of the wall lacked cells expressing protectin as well as matrix-bound factor H. In single nucleotide polymorphism-analysis, age-related macular degeneration-associated factor H Y402H polymorphism did not associate with the presence of IAs or their rupture The data suggest that complement activation and TCC formation are involved in IA wall degeneration and rupture. Complement seems to become activated by more than one specific activator. The association of complement with de-endothelialization and expression of several complement activators indicate a possible role of endothelial dysfunction and/or impaired clearance mechanisms. Impaired complement regulation seems to be associated with increased complement activation in IA walls. These results stress the role of chronic inflammation in IA wall pathobiology and the regulatory role of complement within this process. Imaging inflammation would possibly enhance the diagnostics of rupture-prone IAs, and targeting IA treatment to prevent chronic inflammation might improve IA treatment in the future. Aivovaltimoaneurysma (AA) on aivoverisuonen pullistuma, jonka kantajia arvioidaan olevan Suomessa 100 000. Vuosittain noin 1000 keskimäärin työikäistä henkilöä saa AA:n puhkeamisen aiheuttaman aivoverenvuodon, johon puolet potilaista kuolee ja kolmasosalle jää pysyvä haitta. AA:n puhkeamista ei osata ennustaa eikä siihen johtavia biologisia mekanismeja tunneta tarkasti, vaikkakin tulehdussolujen kertymisen sekä AA-seinämän haurastumisen tiedetään liittyvän puhkeamiseen. Vaikka todennäköisesti osa AA:sta jääkin puhkeamatta, hoitoa ei osata toistaiseksi kohdistaa puhkeamisriskissä oleviin AA:iin eikä AA:n puhkeamista osata estää ilman leikkausta. Tässä väitöskirjatyössä tutkittiin tulehdusta välittävän komplementtijärjestelmän aktivoitumista ja säätelyä AA-kudosnäytteistä ja sekä etsittiin AA:n puhkeamista selittäviä eroja. Komplementtijärjestelmä on ryhmä veressä ja kudoksissa olevia proteiineja, joista osa osallistuu komplementin kaskadimaiseen aktivoitumiseen ja osa estää sitä. Komplementin aktivoituminen välittää inflammaatiota (steriili tulehdus ilman mikrobeja), vaurioittaa terveitä soluja ja houkuttelee tulehdussoluja, joita AA:ssa on aiemmissa tutkimuksissa havaittu. Tulehdus puolestaan voi haurastuttaa AA:n seinämää. Tulosten perusteella komplementtijärjestelmä aktivoituu kaikissa AA:ssa ja aktivaatio tapahtuu ns. klassista tietä eli tiettyjen aktivaattoreiden aiheuttamana. AA:sta löydettiin kertymiä C-reaktiivista (tulehdus)proteiinia, vasta-aineita, hapettuneita LDL-kolesterolipartikkeleita sekä kuolleita soluja, jotka kaikki voivat aktivoida komplementtia. Komplementin täydellinen aktivaatio sijoittui vyöhykemäiselle vähäsoluiselle alueelle AA:n ulkoseinämässä. Alueen laajuus liittyi selkeästi seinämän sisäsolukerroksen (endoteeli) katoamiseen, seinämän haurastumiseen, AA:n puhkeamiseen ja tiettyjen tulehdussolujen kertymiseen. Elektronimikrokopiassa komplementtiaktivaation lopputuotteita löytyi kuolleiksi soluiksi sopivista rakenteista. AA:n sisäseinämässä komplementtiaktivaatiota näytti rajoittavan soluihin ja seinämän tukiaineeseen sitoutuneet inhibiittorit (protektiini sekä C4bp ja faktori H). Näistä protektiinia ei havaittu juurikaan AA:n ulkoseinämässä. Lisäksi tutkittiin faktori H:n geneettistä Y402H-polymorfiaa, jolle ei löydetty yhteyttä AA:iin tai AA:n puhkemiseen. Tulokset osoittavat komplementtiaktivaation edeltävän AA:n puhkeamista ja liittyvän AA-seinämän haurastumiseen ja puhkeamiseen. AA-seinämän komplementtiaktivaatio näyttäisi olevan mahdollisesti seurausta usean aktivaattorin kertymisestä AA-seinämään ja liittyvän vaillinaisen aktivoitumisen eston kautta seinämäsolujen kuolemiseen. Komplementtiaktivaation tarkempi analyysi viittaa AA:n pitkäkestoiseen inflammaatioon, mikä voi olla seurausta normaalien suoja- ja puhdistusmekanismien häiriintymisestä. Saadut tulokset tuovat merkittävää lisätietoa AA-seinämän heikkenemisen mekanismeista ja saattavat ohjata puhkeamisherkkien AA:n tunnistamista ja hoitoa tulevaisuudessa.

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Neurosurgery Research Group, Biomedicum Helsinki
Department of Bacteriology and Immunology, Haartman Institute
Helsinki University Central Hospital
University of Helsinki
Helsinki, Finland
Inflammation and Complement Activation
in Intracranial Artery Aneurysms
Riikka Tulamo
Academic Dissertation
To be presented, with the permission
of the Faculty of Medicine of the University of Helsinki,
for public discussion in Lecture Hall 1 of Töölö Hospital
on March 19th, 2010, at 12 noon.
Helsinki 2010

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Supervised by:
Professor Seppo Meri, M.D., Ph.D
Department of Bacteriology and Immunology
University of Helsinki
Helsinki, Finland
Associate Professor Mika Niemelä, M.D., Ph.D
Department of Neurosurgery
Helsinki University Central Hospital
Helsinki, Finland
Reviewed by:
Professor Juha Öhman, M.D., Ph.D
Department of Neurosciences and Rehabilitation
Tampere University Hospital
Tampere, Finland
Professor Olli Lassila, M.D., Ph.D
Department of Medical Microbiology and Immunology
University of Turku
Turku, Finland
Opponent:
Professor Peter Vajkoczy, M.D., Ph.D
Department of Neurosurgery
Charité Universitätsmedizin Berlin
Berlin, Germany
Riikka Tulamo, M.D.
Neurosurgery Research Group, Biomedicum Helsinki
riikka.tulamo@hus.fi
ISBN 978-952-92-7003-3 (paperback)
ISBN 978-952-10-6132-5 (PDF)
Helsinki University Print
Helsinki 2010

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TABLE OF CONTENTS
TABLE OF CONTENTS.......................................................................................... 3
LIST OF ORIGINAL PUBLICATIONS.................................................................... 5
ABBREVIATIONS................................................................................................... 6
ABSTRACT............................................................................................................. 8
IINTRODUCTION............................................................................................. 10
IIREVIEW OF THE LITERATURE .................................................................... 13
1 Intracranial artery aneurysm and subarachnoid hemorrhage.............. 13
1.1 Epidemology..................................................................................................... 13
1.2 Risk factors for intracranial aneurysm rupture................................................. 13
1.2.1 Aneurysm-related risk factors................................................................ 13
1.2.2 Patient-related risk factors...................................................................... 14
1.2.3 Environmental factors ............................................................................ 15
1.2.4 Family history......................................................................................... 15
1.3 Diagnosis, prevention of rupture, and treatment............................................... 15
1.4 Outcome............................................................................................................ 16
1.5 Histopathology of intracranial aneurysms........................................................ 16
1.5.1 Cerebral artery wall................................................................................ 17
1.5.2 Intracranial aneurysm formation............................................................ 17
1.5.3 Intracranial aneurysm wall..................................................................... 19
1.5.4 Mechanisms altering intracranial aneurysm wall structure.................... 21
1.5.5 Role of extra-aneurysmal factors ........................................................... 29
1.5.6 Role of intrinsic and acquired risk factors.............................................. 30
1.6 Molecular pathology of intracranial aneurysms ............................................... 32
2 Inflammation in the vascular wall ........................................................... 32
2.1 Inflammation and immunity............................................................................. 33
2.1.1 Acute and chronic inflammation............................................................ 33
2.2 Innate and adaptive immunity........................................................................... 34
2.2.1 Inflammatory cells.................................................................................. 35
2.3 Complement system.......................................................................................... 39
2.3.1 Complement activation........................................................................... 40
2.3.2 Regulation of complement activation..................................................... 44
2.3.3 Role of complement in clearance of cellular debris............................... 48
2.3.4 Role of terminal complement activation in cell fate .............................. 49
2.3.5 Complement system and coagulation..................................................... 50
2.4 Complement in the vascular wall...................................................................... 50
2.4.1 Complement in normal arteries.............................................................. 50
2.4.2 Complement in vascular diseases........................................................... 51
III AIMS OF THE STUDY .................................................................................... 54
IV PATIENTS AND METHODS........................................................................... 55
1 Patients and samples............................................................................... 55
2 Histological studies (I-III) ......................................................................... 55
2.1 Histological stainings (I-III) ............................................................................. 55
2.2 Immunohistochemistry and immunofluorescence stainings (I-III) .................. 56

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2.3 TUNEL assay (I)............................................................................................... 58
2.4 Electron microscopy and immunoelectron microscopy (I)............................... 58
2.5 Imaging............................................................................................................. 59
2.6 Histological evaluation..................................................................................... 59
3 Analysis of single-nucleotide polymorphisms (III)................................ 60
4 Statistical analysis.................................................................................... 61
RESULTS AND DISCUSSION........................................................................ 62
1 Complement activation in intracranial aneurysms................................ 62
1.1 Role of complement in intracranial aneurysm inflammation and vascular
damage.............................................................................................................. 62
1.1.1 Association with aneurysm wall degeneration and rupture (I, II).......... 63
1.1.2 Association with macrophage and T-cell infiltration (I)........................ 64
1.1.3 Endothelial dysfunction may potentiate complement activation and
accumulation in the aneurysm wall (I, II) .............................................. 65
1.1.4 Relationship between complement activation and cell death (I, II)....... 66
1.1.5 Complement activation and other clinical and histological parameters. 67
1.2 Complement activation pathways and potential complement activators.......... 67
1.2.1 Activation via the classical pathway and recruitment of alternative
pathway amplification (II)...................................................................... 67
1.2.2 Several known complement activators are present in the aneurysm
wall (II)................................................................................................... 68
1.2.3 Impaired clearance mechanisms and complement activation ................ 70
1.3 Complement regulation (III)............................................................................. 70
1.3.1 Factor H and C4bp protect the luminal part of the aneurysm wall from
complement membrane attack complex................................................. 70
1.3.2 CFH Y402H is not associated with intracranial aneurysms or
aneurysm rupture.................................................................................... 71
1.3.3 Protectin limits C5b-9 formation in the aneurysm wall......................... 72
2 Possible role of complement activation in intracranial aneurysms .... 74
3 Limitations of the study ........................................................................... 77
4 Future perspectives.................................................................................. 79
4.1 Imaging inflammation....................................................................................... 79
4.2 Improvements in treatment methods................................................................. 80
4.3 Systemic pharmacological therapy................................................................... 80
VI SUMMARY AND CONCLUSIONS.................................................................. 82
VII ACKNOWLEDGMENTS ................................................................................. 85
VIIIREFERENCES................................................................................................ 87
V

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LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, referred to in the text by their
Roman numerals:
I. Tulamo R, Frösen J, Junnikkala S, Paetau A, Pitkäniemi J, Kangasniemi M, Niemelä M,
Jääskeläinen J, Jokitalo E, Karatas A, Hernesniemi J, Meri S. Complement activation
associates with saccular cerebral artery aneurysm wall degeneration and rupture.
Neurosurgery 59:1069-76, 2006.
II. Tulamo R, Frösen J, Junnikkala S, Paetau A, Kangasniemi M, Peláez J, Hernesniemi J,
Niemelä M, Meri S. Complement system becomes activated by the classical pathway in
intracranial aneurysm walls. Lab Invest 90:168-79, 2010.
III. Tulamo R, Frösen J, Paetau A, Seitsonen S, Hernesniemi J, Niemelä M, Järvelä I, Meri
S. Lack of complement inhibitors from the outer intracranial artery aneurysm wall
predisposes it to complement terminal pathway activation. Submitted.
These articles have been reprinted with the kind permission of their copyright holders.