1
Local and systemic complement activation and potential molecular mechanisms in three forms
of vascular disease:
Atherosclerosis, Graft Vascular Disease, and Uteroplacental Acute Atherosis
Ingrid Knutsdotter Fosheim
Supervisor: Prof. Annetine Staff, MD, PhD
Project thesis, Faculty of Medicine University of Oslo
2019
2 Table of Contents
Introduction ... 3
Abstract ... 4
Background ... 5
Methods... 7
Results ... 9
The complement system ... 9
Atherosclerosis ... 14
Graft vascular disease ... 17
Acute atherosis ... 18
Discussion ... 22
Possible future approaches for complement related research on uteroplacental acute atherosis ... 24
Literature ... 26
3 Introduction
This project thesis reviews the human complement system and its significance in three arterial lesions; uteroplacental acute atherosis of pregnancy, atherosclerosis, and graft vascular
disease. The project thesis is written as part of the medical student program at the Medical Faculty at the University of Oslo. The project thesis has two main aims – to review the literature on the field, and also to use these results to propose how complement related research could be used to make progress in pregnancy related uteroplacental acute atherosis research in the future.
My supervisor has been Annetine Staff (Oslo University Hospital and University of Oslo), who has given excellent guidance and great advice throughout the work with this project, and I am very grateful for her support and enthusiasm.
4 Abstract
Objectives: The objectives of this project thesis were to review the literature regarding the human complement system and its relation to three arterial lesions; atherosclerosis, graft vascular disease and uteroplacental acute atherosis, and to propose possible research approaches for the significance of complement activation in acute atherosis.
Methods: This project thesis is based on a non-systematic literature review, using relevant search terms and combinations in PubMed to identify research findings relevant for the complement system in atherosclerosis, graft vascular disease and acute atherosis. Review articles were selected from the literature search, and relevant original research papers in English were then selected from the reviews, based on abstract reading.
Results: The complement system is activated in atherosclerosis, graft vascular disease and acute atherosis. Activation pathways and extent of cascade activation differ between these arterial lesions. The classical, lectin, and alternative pathways are activated in both
atherosclerosis and graft vascular disease, and activation is extended to the terminal complex.
Activation of the classical pathway is indicated in acute atherosis, but contribution of the lectin and classical pathways and the extent of activation remain uncertain.
Conclusions: The complement system holds an important role in the pathophysiology of atherosclerosis and graft vascular disease, both on a local and systemic level. Although there is evidence for complement activation in acute atherosis, there is still need for further research regarding the role of complement in the formation and progression of acute atherosis, as well as for a possible contribution of systemic complement activation to the lesion.
5 Background
More than 100 years ago, the complement system was discovered and named due to its
“complementing” role in antibody bacteriolysis (1). It was later discovered to be a cascade system, and part of the innate immune system. The complement system consists of more than 40 known soluble and membrane bound proteins. Most of the complement proteins regulate the functions of other components of this system (2, 3). Activation of the complement system results in inflammation, phagocytosis, and/ or cell lysis by membrane attack (4), the system’s three main functions. The complement system works on its own as well as assisting other parts of the immune system (5).
Atherosclerosis is a cardiovascular disease in which a large artery hardens (“sclerosis” from Greek “to harden”), following the growth of atheromatous plaques in the vessel wall. It is the main cause of myocardial infarction, stroke, and gangrene (6). Atherosclerosis is largely considered an inflammatory disease that develops over the course of years, with many parts of the immune system being involved in the different stages of the disease (7-9).
Graft vascular disease is an arterial complication observed after allograft transplantation.
Graft vascular disease is also known as graft arterial disease, allograft vasculopathy,
transplant vasculopathy, and graft vascular sclerosis. This project thesis will use the term graft vascular disease. Graft vascular disease lesions are restricted to vessels of the transplanted organ, and do not affect host vessels. Development of graft vascular disease lesions has a significant immunological component, as the lesion occurs less often in immunosuppressed recipients than in non-immunosuppressed recipients (10).
Acute atherosis is a pregnancy vascular lesion affecting the spiral arteries of the decidua (endometrium of pregnancy). These uteroplacental spiral arteries perfuse the intervillous space and thereby the placenta villous tissue in pregnancy. The mechanisms by which acute atherosis lesions develop are not fully understood, but many consider the immune system as well as excessive inflammation to be of importance (11, 12).
In both atherosclerosis and graft vascular disease, activation of the complement system is of importance, especially in regards to endothelial dysfunction (13, 14), but this has not been
6
adequately explored in acute atherosis. Acute atherosis is morphologically similar to early lesions of atherosclerosis, with formation of intramural foam cells. There are also several other similarities between acute atherosis and graft vascular disease, as reviewed by my supervisor and coauthors previously (12). The aim of this student thesis was therefore to explore whether the complement activation patterns also are comparable between these three different arterial lesions.
7 Methods
This project thesis is based on a non-systematic literature research in PubMed in August 2018. To address the topic in question, articles focusing on complement activation in
atherosclerosis, graft vascular disease, and acute atherosis of the uteroplacental spiral arteries were included.
Search terms included “acute atherosis”, “preeclampsia”, “atherosclerosis”, “graft vascular disease”, “graft vessel disease”, “graft vasculopathy”, “allograft vascular disease”, “allograft vasculopathy”, and “transplant vasculopathy”. These terms were combined with the key words “complement activation”, “complement system”, and “complement”. Search results were filtered by “English language only”. For details regarding the search terms and combinations, please see Table 1.
Selection of articles for the project was then performed in two steps; first, all articles other than systematic reviews were filtered out. Reviews were then selected for further reading based on their abstracts. Due to the large yield in search results for atherosclerosis, a time filter (“published in the last five years”) was added to the search. Original research papers and additional interesting reviews were selected from reading the full-text of the reviews.
Secondly, for search results regarding graft vascular disease and acute atherosis, some original research papers were also selected from the search results based on reading their abstracts. This method was not applied to the search results for atherosclerosis due to the large yield in search results, even when filtering out papers older than five years. Although human studies were preferred for this project, some studies performed on animals were also included in the review.
Articles were selected in agreement with supervisor Annetine Staff, who has extensive background and competence in this field of research. She also provided some additional studies and reviews of particular relevance, adding expertise insight from the research frontline of acute atherosis of uteroplacental tissue in pregnancy.
8
Search term Combinations Yield Date
1. Complement activation 2. Complement system 3. Complement
4. Acute atherosis 5. Preeclampsia 6. Atherosclerosis 7. Graft vascular disease 8. Graft vessel disease 9. Graft vasculopathy 10. Allograft vascular
disease
11. Allograft vasculopathy 12. Transplant vasculopathy Filter: English language only
(1 OR 2 OR 3) AND 4 5 (of which 2 reviews)
08.08.18
(1 OR 2 OR 3) AND 6 795 (of which 229 reviews, 60 published in the last 5 years)
08.08.18
(1 OR 2 OR 3 ) AND (7 OR 8 OR 9 OR 10 OR 11 OR 12)
63 (of which 21 reviews)
08.08.18
(1 OR 2 OR 3) AND 5 295 (of which 65 reviews)
08.08.18
Table 1. Search terms and combinations used in this project thesis.
9 Results
The complement system
George Nuttall, Hans Buchner, Jules Bordet, and Paul Ehrlich were all central in the earliest discoveries of complement. Nuttall discovered in 1888 that blood serum from sheep contained a component with bactericidal properties, when he noted that the serum had the ability to inactivate the Bacillus anthracis, the bacteria that causes anthrax. This property disappeared when the sheep serum was heated, suggesting that the killing component was heat-labile (15, 16). This component was first named alexin, which is Greek for “to ward off”, by Buchner in 1891. Bordet, a supporter of this humoral immunity theory, proved that the bacteriolysis caused by the bactericidal component required both a heat-labile factor (alexin) and a heat- stable factor, which he named sensitizer. Paul Ehrlich renamed the heat-labile component alexin to complement and the heat stable sensitizer to amboceptor (today known as antibody) in 1899 after describing the side-chain theory of antibody formation and the mechanisms of antibody neutralization of toxins and bacteriolysis with complement (15-17). According to his theory, a wide range of receptors (antibodies) on all immune cells are able to recognize antigens, multiply, and be shed into the circulation. The theory also proposed that antibodies could bind to complement. Ehrlich further proposed that this antibody-complement complex formed an enzyme with cytolytic abilities. Ehrlich was awarded the Nobel Prize in 1908, along with Elie Metchnikoff, for this important work in immunology (17).
Soon after discovering complement, scientists realized that complement included more than just the two components, as it was in fact a broader system of proteins. The proteins of the system were named successively as they were discovered, instead of in their respective order of action within the pathways (5, 15), see Fig. 1 (Reproduced with permission from (5), Copyright Massachusetts Medical Society).
Scientists originally considered complement solely as a defense system against microbes, but the complement system in fact has a number of tasks. Some of its most important tasks include surveilling the host and identifying danger, assist antibody in phagocytosis by opsonizing antigens, cell lysis by formation of the membrane attack complex (MAC),
attracting leukocytes to a site of inflammation, and removal of apoptotic cells in the host (5) .
10
The first step of complement activation is activation of complement component 3 (C3) convertase. There are three pathways to this (Fig. 1); the classical pathway – named so because it was the first to be discovered – activated by antibody binding to antigen; the lectin pathway, activated by mannose binding lectin (MBL); and the alternative pathway, which is spontaneously and continuously activated at a low level due to hydrolysis of C3. C3
convertase cleaves C3 to C3a and C3b, in a self-amplifying manner. C3 convertase is composed of different complement components in the different pathways (Fig. 1). C3b amplifies the cascade as well as binding to microbial or apoptotic
surfaces
(opsonization). The next step in the cascade is activation of C5 convertase. C5 convertase is composed of C3 convertase with an additional C3b attached to it. C5 convertase cleaves C5 to C5a and C5b.
Both C3a and C5a are potent anaphylatoxins, and recruit immune cells and promote inflammation. C5b initiates the formation of the MAC, also known as the terminal complement complex. This is the
Figure 1. Overview of the complement system, with details from each activation pathway.
Reproduced with permission from (5), Copyright Massachusetts Medical Society.
11
terminal pathway of complement activation. C5b, C6, C7, C8, and C9 form the MAC, which inserts into the cell wall of the pathogen, allowing C9 to polymerize and create a pore in the pathogen. This exposes the pathogen’s insides to the host’s extracellular fluid, leading to cell lysis and death (1).
With a vast array of potent cytolytic and inflammatory enzymes, there is a definitive need for regulation. The regulators of the complement system are mainly inhibitory, with the exclusion of properdin. Properdin stabilizes C3 convertase in the alternative pathway and increases activation. Three complement inhibitors mentioned in this thesis are factor H (inhibits mainly the alternative pathway, but also the classical pathway), decay-accelerating factor (DAF,
inhibits all three pathways by increasing decay of C3 convertase, thus inactivating it), and C4 binding protein (binds C4 of the classical and lectin pathways and inactivates it) (1, 16).
The complement system may seem difficult to navigate, with many activation pathways and several steps to reach the common end goal, assembly and activation of the membrane attack complex. How do you tell which pathway(s) have been activated? How do you know how far the cascade has been activated?
These are central questions to ask when studying complement activation.
To simplify the understanding of the
complement system, one may think of it as a trident, as illustrated in Fig. 2. The three spikes symbolize each of the three activation pathways: the classical, lectin and alternative pathway. Where the spikes gather to form the handle is where you will find C3, the first
Figure 2. Summary of activation pathways of the complement system. CP = classical pathway. LP = lectin pathway. AP = alternative pathway. MAC = membrane attack complex.
12
component of the common activation pathway. Further progression of complement activation will in turn activate C5, which sits halfway down the spear. At the very end of the spear, the MAC is located, and may be activated by C5.
Classical pathway Lectin pathway Alternative pathway Antibodies (IgG, IgM) X
C-reactive protein X
C1q X
C1r X
C4 X X
C2 X X
C4b, C2a X X
C4a, C2b X X
C4d X X
Mannose-binding lectin X
Ficolins X
Factor B, Bb, Ba X
Factor D X
Properdin (reg.) X
Factor H (reg.) X X
Table 2. Important components of the complement system, arranged by pathways of action. Reg. = regulatory protein. C3, C3a, C3b/C3d, and decay-accelerating factor (DAF, reg.) are not specific to any one activation pathway. C5, C5a, C5b, C6, C7, C8, C9, MAC, are part of the terminal activation pathway (1, 16).
The classical pathway may be activated by IgM or IgG. Only antibodies bound to antigens may activate C1. Thus, circulating, free IgM or IgG are not able to activate the complement cascade. C-reactive protein (CRP) is also able to activate the classical pathway (4, 16).
Activation of the classical pathway can be measured by deposition of IgM or IgG, by circulating or deposited C1 (or components thereof, such as C1q) or CRP.
The lectin pathway is activated by MBL binding to mannose on the surface of bacteria.
Additionally, ficolins (a group of pattern recognition receptors) can activate the lectin
13
pathway by binding to foreign structures in the body (4). Activation of this pathway is thus confirmed in the presence of MBL or ficolins.
The alternative pathway is, as previously mentioned, spontaneously and continuously
activated at a low level, but only up to the point of hydrolysated C3, which can bind to factor B. When in presence of factor D, the newly formed C3/B-compound acts as a C3 convertase and produces C3b. If C3b is formed in close vicinity to a foreign surface (such as the
membrane of a bacterium), C3b will bind to this surface and propagate further complement activation. Additionally, properdin is also exclusive to the alternative pathway (4, 16).
Presence of factor B, factor D, or properdin indicate activation of the alternative pathway.
The complement components C2, C3, C4, C5 and C6-9 (MAC) are not exclusive to any one pathway, but may give information on the extent of complement activation, i.e. how “far down” the complement cascade is activated (see Figs. 1 and 2 and Table 2). C2 and C4 are only found in the classical and lectin pathways. Presence of C3d (a stabilized and inactivated version of C3b) indicates that the C3 convertase has been formed.
Complement activation can be studied in several ways, and appropriate methods differ between studying local and systemic complement activation. Frequently used methods in studying local complement activation include immunohistochemistry and
immunofluorescence, in which the researcher aims to study the presence of a certain antigen (i.e. a peptide, protein, or hormone) in a tissue sample. By applying an antibody known to bind the antigen as well as a stain (immunofluorescent or chromatic), the researcher will be able to visualize the antigen in question. Presence of color or immunofluorescence in the prepared tissue indicates presence of antigen. One can also use gene expression techniques on tissue samples, for instance quantitative reverse transcription polymerase chain reaction, which identifies and quantifies a specific messenger RNA in a sample after extracting messenger RNA from the sample.
Enzyme-linked immunosorbent assay (ELISA) is a method that is suitable for evaluating systemic complement activation. ELISA is used to identify a certain peptide, protein or antibody in the circulation, typically blood serum. Serum is first applied to a plate with wells,
14
which contain different concentrations (titers) of an antigen known to bind the molecule the researcher aims to detect. If the molecule in question (called the specific antibody) is present, it will bind to the antigen. Another antibody, specific to (for example) human antibody, is then applied. The second antibody is bound to an enzyme that will generate a specific color. A plate reader will then electronically read the intensity of the color. Different titers of the specific antibody will generate different color intensities. Gene expression techniques can also be used for evaluating systemic complement activation, but this implies to extract messenger RNA from blood samples instead of from tissue.
Atherosclerosis
Atherosclerosis is the primary underlying cause of the cardiovascular diseases such as coronary artery disease, cerebrovascular disease, hypertension and more. These diseases are the leading causes of death and disability globally, according to the World Health
Organization, and are estimated to account for up to one-third of deaths annually.
Cardiovascular diseases are in large preventable by means of reduction in use of tobacco, alcohol, and by increased physical activity (18).
The pathogenesis of atherosclerosis begins with formation of atheromatous plaque (Fig. 3).
Plaque formation takes place when circulating lipoproteins (mainly low-density lipoprotein, LDL) accumulate in the tunica intima (the innermost layer of the vessel wall) and become oxidized, which in turn damages the endothelium. When damaged, the endothelium may become dysfunctional and more permeable, by mechanisms such as an altered production of proinflammatory cytokines and upregulation of adhesion molecules, among other possible pathways (6, 19). Circulating monocytes will migrate to areas of such lipoprotein aggregates, transmigrate across the endothelium, and consume the lipoproteins. The monocytes-turned- macrophages are at this point lipid-rich and referred to as ‘foam cells’. Accumulations of subendothelial foam cells are called ‘fatty streaks’, and make up the earliest lesions of the atherosclerotic development (Fig. 3A). As the lesion advances, smooth muscle cells from the tunica media receive signals that promote growth and proliferation, as well as migration to the intima. There, the SMCs secrete extracellular matrix, which forms a ‘fibrous cap’ around the fatty streak (19, 20). This makes up the atherosclerotic plaque. If the fibrous cap ruptures, the contents of the plaque will be exposed to blood and cause thrombus formation. The thrombus
15
may cause occlusion of the vessel (or travel with the circulation to occlude another vessel), downstream cellular injury, and even death by tissue ischemia or infarction (7, 19).
In a review from 2012, LDL was described as “the spark that ignites complement activation in
atherosclerosis” (23). This statement is supported by several original papers that demonstrate that
enzymatically modified low density lipoprotein (E- LDL) is able to spontaneously activate the alternative complement pathway (24), as well as the classical pathway in presence of CRP, and that oxidized LDL (ox-LDL) and native LDL do not possess these properties (24-26). However, a later report showed that ox-LDL too is able to activate the classical pathway, when bound to C1q (27). Presence of CRP ensured that only initial complement activation took place, and by some mechanism inhibited terminal complement activation (26). Bhakdi et al. (26) also showed that CRP and E-LDL co-localize in early atherosclerotic lesions in the intimal layer, together with deposition of large amounts of C3d and smaller amounts of C5b-9 (membrane attack complex).
C1q’s presence in human atherosclerotic lesions has been confirmed by several reports (28, 29). Fraser and Tenner (27) showed that in addition to the ability of C1q to bind ox-LDL and activate the classical pathway, C1q also increased monocytes’ ability to ingest and clear ox-LDL in vitro.
They also found that MBL, similarly to C1q, was able to increase clearance of ox-LDL. In a study on patients with rheumatoid arthritis, serum levels of MBL were found to be associated with intima-media thickness (a surrogate for severity of atherosclerosis) (30). Patients with rheumatoid arthritis have increased risk of atherosclerosis. Both too high and too low serum levels of MBL were associated with increased intima-media thickness (more severe
Figure 3. Examples of A) early atherosclerotic lesion (reproduced with permission from (21), copyright 2007 American Society for Investigative Pathology), B) graft vascular disease reproduced with permission from (22), copyright 2010 College of American
Pathologists) and C) acute atherosis (own data).
C
16
atherosclerosis). The population-based HUNT2 study from Norway found that MBL- genotypes causing low levels of serum MBL were associated with increased risk of myocardial infarction (31).
Speidl et al. showed (32) that C5a is present in human atherosclerotic lesions, and that C5a in vivo was able to upregulate the activity of matrix metalloproteinases. The same group had previously found increased serum levels of C5a to represent a marker for adverse
cardiovascular events (e.g. myocardial infarction, stroke, death) in patients with advanced atherosclerosis (33). De Hoog et al. (34) also found increased serum levels of C5a to be a marker of acute coronary syndrome.
Helske et al. (35) found C3a, the other anaphylatoxin of the complement system, to be present in advanced human atherosclerosis, and to increase the expression of proinflammatory
cytokines in vitro. The CODAM study from the Netherlands, another population-based study, found that increased C3a was associated with increased intima-media thickness as well as decreased ankle-arm index (both signs of advanced atherosclerosis) (36). C3 showed similar results, but not when simultaneously considering other risk factors of cardiovascular disease.
However, Carter et al. found increased levels of C3 to be an independent risk factor for myocardial infarction (37).
Deposition of the membrane attack complex, C5b-9, has been detected in atherosclerotic lesions by several authors (26, 28, 35, 38-41). Helske et al. (35) noted that the deposition of C5b-9 was more prominent in advanced compared to early lesions of atherosclerosis, a finding also reported by others (26, 28, 38). C5b-9 deposits have been demonstrated to be most distinct in the deeper parts of the intimal layer (28, 39-41), and to co-localize with properdin (40), CRP (41), C1q, C3c, C4, IgG, and IgM (28). Another report, however, did not find IgM or C4 to localize with C5b-9 (40). The complement regulators factor H and C4b- binding protein have only been demonstrated present in the superficial intima (where they co- localized with C3 and C4), with little or no overlap with C5b-9 (39, 40). Hertle et al. (42) have studied soluble C5b-9 systemically and found that it was not associated with markers for atherosclerosis.
17 Graft vascular disease
Graft vascular disease is an important long-term complication of allografts, and is a driving factor in late solid organ graft loss and antibody-mediated rejection (22, 43). Although graft vascular disease lesions are greatly heterogeneous, they are typically characterized by intimal accumulation of smooth muscle cells and extracellular matrix, constricting the lumen. Like atherosclerosis, graft vascular disease affects arteries. Graft vascular disease differs from atherosclerosis in that only the intimal layer is affected, with relative sparing of the media and adventitia, whereas in atherosclerotic lesions, all three layers of the artery are involved (Fig.
3A, B). Furthermore, graft vascular disease lesions often affect the vessels diffusely instead of focally, which means that graft vascular disease affects larger parts of or even the entire vascular bed, but is strictly confined to vessels of the donated organs – host vessels are spared (22, 44). The lesion can in some instances develop rather rapidly – over the course of 12 months after transplantation, around 50% of cardiac allografts will have developed graft vascular disease, and within 10 years post-transplant, the rate of allografts with graft vascular disease rises to 90% (43). Although graft vascular disease has been reported to occur in several different solid organ transplants (22, 43), the majority of the research presented in this project thesis focuses on cardiac graft vascular disease due to the vast interest in cardiac transplant research.
In a study of local, graft-derived gene expression, Keslar et al. (45) found that cardiac grafts that histologically showed moderate antibody-mediated rejection expressed more factor B, properdin, C3, C3a receptor and C5a receptor genes than grafts with less severe rejection.
There was no difference in gene expression for C4, C5, or factor D.
Studies using immunohistochemistry or immunofluorescence have found significant deposition of C4d in grafts with graft vascular disease (46, 47), and that presence of circulating donor specific antibodies (host-derived antibodies to donor HLA, involved in antibody-mediated rejection (48)) significantly increases the rate of graft vascular disease as well as reduce the time from transplantation to graft vascular disease formation (46). Another study further found a relationship between antibodies against non-classical MHC molecules (which can also induce complement activation) and graft vascular disease as well as between
18
donor specific antibodies and graft vascular disease. Presence of either one type of antibody or both in combination increased the rate of graft vascular disease (49).
Jane-Wit et al. (50) found that the membrane attack complex deposited on graft endothelial cells in vitro and in vivo in presence of donor specific antibodies. They further saw that MAC did not induce cytolysis, but rather up-regulated inflammatory genes, which in turn
augmented formation of graft vascular disease. In a rat model where hearts were transplanted into either C6 sufficient or C6 deficient rats, researchers discovered that graft rejection and graft vascular disease formation happened earlier and more frequently in grafts of C6 sufficient rats compared to C6 deficient rats (51).
Regarding MBL and graft vascular disease, differing study conclusions exist. One report found that MBL deficiency was significantly more common in human cardiac grafts with graft vascular disease compared to grafts without, but found no deficiencies of C1q, factor B, C3 or C4 (52). Conversely, a later study did not find significant differences in MBL
concentrations in human cardiac grafts with and without graft vascular disease (53).
Pavlov et al. (54) studied the effects of the complement inhibitor decay-accelerating factor (DAF) in mice and found that in donor hearts deficient of DAF, rejection happened
significantly sooner than in donor hearts from wildtype mice. Significant graft vascular disease lesions were only observed in mice with DAF-deficiency. Furthermore, they observed a protective role of C3 deficiency, as C3 deficient grafts survived longer than wild-type grafts.
Another study of complement inhibition demonstrated that blocking antibodies to C5 significantly reduced formation of C5b-9 as well as graft vascular disease lesions (55).
Acute atherosis
During pregnancy, the spiral arteries supplying the placenta with blood become altered (remodeled) by a process involving maternal uterine immune cells and invading “foreign”
fetal cells (extravillous trophoblasts), as reviewed by my supervisor Staff and coauthor Redman (56). The fetal cells interact with maternal immune cells and induce the replacement of the muscular layer of the arteries with fibrinoid, a fibrin-like matter (57). These
physiological changes are most prominent in the basal decidua (the endometrial lining of
19
pregnancy) and the inner third of the myometrium underlying the placenta, collectively termed the placental bed (58). The failure of this remodeling process is considered to be of importance in the pathogenesis of preeclampsia (59), with which acute atherosis is associated.
Acute atherosis occurs in 20-40 % of preeclamptic pregnancies, compared to approximately 10 % of normotensive pregnancies (60-62). The lesion is found in the spiral arteries of the placental bed (Fig. 3C) and is classically characterized by accumulation of lipid-filled foam cells, fibrinoid necrosis, and leukocyte perivascular infiltrate (11). A revised and evidence- based definition for acute atherosis, including only the presence of CD68+ foam cells and fibrinoid has been proposed by the research group I am affiliated with (61). Acute atherosis is morphologically similar to early lesions of atherosclerosis with the presence of subendothelial foam cells and leukocyte infiltrate. As previously mentioned, the exact mechanism of lesion initiation and progression is unknown, but immune mechanisms have been proposed (60).
Several authors have investigated complement activation in the placental bed, both in relation to spiral arteries with acute atherosis, and in normal spiral arteries. Due to the link between preeclampsia and acute atherosis, certain interesting papers focusing on complement activation in preeclampsia are discussed here as well.
Labarrere and colleagues investigated decidual tissue from both normal pregnancies as well as from pregnancies with intrauterine growth restriction and acute atherosis using
immunohistochemistry. They found “massive deposits” of IgM, along with deposits of C3, C1q, IgG, and IgA in the vessel wall of spiral arteries with acute atherosis, but no deposition of immunoglobulins or complement factors in normal spiral arteries without acute atherosis (63). They later found similar results in smaller studies (64, 65). However, they did find deposits of IgG, IgM, IgA, and C3 also in normal spiral arteries without acute atherosis in one of their studies (66) These deposits were smaller than the deposits found in acute atherosis arteries, and were located on the endothelial surface of the arteries, in contrast to the intramural deposits observed in spiral arteries with acute atherosis.
Complementing investigations to those of Labarrere et al. have been performed by Weir (67) and Wells (68) who focused solely on spiral arteries from women with normal pregnancies.
Weir (67) observed spiral arteries free of acute atherosis, but with C3 deposition in the vessel
20
wall in nearly half of the collected samples, similar to Labarrere (66). Unlike Labarrere’s findings, Weir observed no deposition of C1q, IgA, IgM, or IgG neither intramurally nor interstitially. Wells et al. (68) studied the placental bed from hysterectomy samples with varying gestational ages (4-40 weeks), and found deposition of C1q, C3d, C4, C6 and C9 in the spiral artery wall. They reported that C9 and C3d were the most intense in reactivity.
Lichtig et al. (69) sampled the placental bed in patients undergoing abortion in first trimester.
They observed lesions similar to acute atherosis in some of the biopsies, primarily in biopsies from primipara women. At the same time, C3 deposition in the spiral artery wall was
observed, also here primarily in primipara. The authors did not mention whether complement deposition was observed more frequently in arteries with presence of vascular lesions or not.
However, they did state that the combination of most severe vascular lesions and heaviest C3 deposition only occurred in primipara.
A study from Hustin et al. (70) found that acute atherosis was present in 50% of decidua samples of women with preeclampsia. Immunohistochemistry was performed on a minority of the samples collected, where deposits of IgG in the arterial walls were found in more than half of these preeclamptic samples. C3 was also observed in some samples, always co-localizing with IgG. Conversely, on samples from healthy pregnant women, C3 deposition was only found in one sample.
In decidual tissue from rats with preeclampsia, C3 expression in uteroplacental vessels has also been found increased as compared with healthy pregnant rats. Similarly, C3 expression in human spiral arteries with acute atherosis has been shown to be increased compared to spiral arteries without acute atherosis (71), a study that used decidua basalis samples from our biobank (Fig. 4).
21
Although the literature search yielded no results for studies of systemic complement activation in acute atherosis, it has been investigated in patients with preeclampsia.
Lynch et al. (72, 73) found that elevated plasma levels of factor Bb and C3a in early pregnancy (before gestational week 20) were associated with increased risk of
developing preeclampsia and any hypertensive disease, respectively, later in pregnancy. Derzsy et al. (74) found that circulating levels of CRP, C4d, C3a, and soluble C5b-9 (MAC) were significantly higher, and that C3 was significantly lower, in preeclamptic patients compared with healthy pregnant women. Locally, deposition of C4d has been shown to be increased in placentas of preeclamptic women compared to placentas of normotensive pregnant women (75), however this study did not report significant MAC deposition, and rarely co-localization with MAC and C4d. Burwick et al. (76) found elevated C5a and C5b-9 levels in both urine and plasma in women with severe preeclampsia.
Regarding C3a, they only found elevated levels in urine samples, but not in plasma.
Figure 4. Decidual tissue from our group’s pregnancy biobank, showing increased C3 expression in preeclamptic decidua with acute atherosis compared to control (healthy) decidua without acute atherosis. Reproduced with permission from (71), DOI:(10.1161/HYPERTENSIONAHA.107.102905).
22 Discussion
The project thesis search results demonstrate the presence of an activated complement system in each of the three arterial lesions studied, and that various parts of the system are involved.
Fig. 5 shows the similarities and differences in complement activation in atherosclerosis, graft vascular disease and acute atherosis, respectively.
The articles presented in this project thesis show that the classical, lectin, alternative and terminal pathways are activated in
atherosclerosis, and that the activation is both systemic and local. Complement activation is shown to be important during all stages of atherogenesis, with LDL and CRP as the agents to set off the complement cascade at the earliest stages, and C5b-9 amounts increasing with lesion progression. The results further demonstrate the importance of balance between
complement activation and inhibition, as demonstrated by the fact that both too much and too little MBL is associated with cardiovascular disease. Some reports moreover suggest a
clinical applicability of the complement system as serological markers of severe disease, such
Figure 5. Activation pathways of the complement system and their role in atherosclerosis (black dot), graft vascular disease (red dot), and acute atherosis (green dot). CP = classical pathway. LP = lectin pathway. AP = alternative pathway. MAC = membrane attack complex.
Abs = antibodies. C4bp = C4-binding protein. DAF = decay accelerating factor.
23
as in myocardial infarction. Studies of the role of complement activation in atherosclerosis are however somewhat diverging, especially regarding the value of C3 as a marker for severe cardiovascular disease. Large population studies are warranted to conclude on C3’s applicability as a marker for atherosclerosis and adverse outcomes such as myocardial infarction or stroke. Conversely, C5a as a biomarker for severe consequences of advanced atherosclerosis seems to be better established.
The classical, alternative, and terminal pathways are all activated in graft vascular disease, while data regarding the lectin pathway are controversial. The importance of complement in graft vascular disease is established on both a local and systemic basis. Further, the
complement system is also of importance in antibody-mediated rejection, with which graft vascular disease is associated. There is some evidence that terminal activation of complement can cause graft vascular disease, not by cytolysis, but by up-regulating inflammatory genes. In addition, complement inhibition may present therapeutic options in the future, exemplified by the protective roles of C6 deficiency and blocking antibodies to C5, and the deleterious effects of defective endogenous complement inhibition. Further studies to determine with certainty the role of the lectin pathway in graft vascular disease are needed.
The results regarding acute atherosis indicate local activation of the classical complement pathway in uteroplacental spiral arteries with acute atherosis, and systemic activation of the alternative, classical and terminal pathways in patients with preeclampsia. The presence of complement components both in normal spiral arteries as well as systemically in
normotensive pregnant women indicate a role for complement also in normal pregnancy.
One limitation of this project thesis is the relatively limited scope of the thesis, due to the time constraints set for the project thesis. Only one database (PubMed) was used, which could affect the results both qualitatively and quantitatively. Reaching the latest, up-to-date research is also a challenge, due to the rapid and ever-changing nature of biological research.
24
Possible future approaches for complement related research on uteroplacental acute atherosis
As of today, there is little information available regarding acute atherosis and systemic complement activation. A possible approach to elucidate this topic could be to sample blood and decidua basalis tissue from pregnant women at delivery, evaluate presence of acute atherosis in tissue samples (by histology and immunohistochemistry) and complement components in the blood (e.g. by ELISA). It would be useful to investigate which of the complement pathways are activated, by selection of proper markers for each pathway. My proposal would be mannose-binding lectin for the lectin pathway, CRP, C1q or antibodies for the classical pathway, and properdin or factor B for the alternative pathway. The extent of complement activation is also of importance, and thus detection of C3, C5 and the membrane attack complex could provide useful information.
There is also a need for further analysis of complement activation on a local level. For this, immunohistochemistry or immunofluorescence studies would be appropriate methods. The lectin and alternative pathways in particular need further investigation as their role in acute atherosis remains unclear. Mannose-binding lectin (MBL), properdin, factor B could serve as useful markers. Similar to the proposal of systemic complement activation, determination of the extent of complement activation would also be of importance on a local level.
Studies of expressed complement genes (mRNA expression level) on both a local (decidua basalis or myometrium tissue) and systemic (blood samples) could similarly be useful in describing complement activation in acute atherosis.
Both normotensive pregnant women and preeclamptic patients should be included in future research, because complement activation already has been shown in preeclampsia, and preeclampsia and acute atherosis are closely related. It would be useful to determine the contribution of preeclampsia and acute atherosis, respectively, to complement activation in one patient. Therefore, comparison of preeclamptic women with and without acute atherosis, as well as normotensive pregnant women with and without acute atherosis, would be suitable.
Also, it remains to be elucidated what is the initiating step of the complement activation in
25
preeclampsia and acute atherosis, or if it could result from several of the multiple steps that lead to the syndromic clinical picture of preeclampsia, and the likely multifactorial pathway to uteroplacental acute atherosis.
26 Literature
1. Zipfel PF, Skerka C. Complement regulators and inhibitory proteins. Nat Rev Immunol. 2009;9(10):729-40.
2. Hovland A, Jonasson L, Garred P, Yndestad A, Aukrust P, Lappegård KT, et al. The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis. Atherosclerosis. 2015;241(2):480-94.
3. Mollnes TE, Lachmann PJ. Regulation of complement. Scand J Immunol.
1988;27(2):127-42.
4. Bogen B, Munthe LA. Komplementsystemet. Immunologi. 2. utg. ed. Oslo:
Universitetsforl.; 2007. p. 136-42.
5. Walport MJ. Complement. First of two parts. N Engl J Med. 2001;344(14):1058-66.
6. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature.
1993;362(6423):801-9.
7. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005;352(16):1685-95.
8. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006;6(7):508-19.
9. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999;340(2):115- 26.
10. Wehner J, Morrell CN, Reynolds T, Rodriguez ER, Baldwin WM, III. Antibody and Complement in Transplant Vasculopathy. Circulation research. 2007;100(2):191-203.
11. Labarrere C. Acute atherosis. A histopathological hallmark of immune aggression?
Placenta. 1988;9(1):95-108.
12. Staff AC, Johnsen GM, Dechend R, Redman CW. Preeclampsia and uteroplacental acute atherosis: immune and inflammatory factors. J Reprod Immunol. 2014;101-102:120-6.
13. Hertle E, Stehouwer CD, van Greevenbroek MM. The complement system in human cardiometabolic disease. Mol Immunol. 2014;61(2):135-48.
14. Colvin RB, Smith RN. Antibody-mediated organ-allograft rejection. Nat Rev Immunol. 2005;5(10):807-17.
15. Chaplin H, Jr. Review: the burgeoning history of the complement system 1888-2005.
Immunohematology. 2005;21(3):85-93.
16. Nesargikar PN, Spiller B, Chavez R. The complement system: history, pathways, cascade and inhibitors. Eur J Microbiol Immunol (Bp). 2012;2(2):103-11.
17. Kaufmann SH. Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat Immunol. 2008;9(7):705-12.
18. Section A - Cardiovascular diseases (CVDs) due to atherosclerosis. Global atlas on cardiovascular disease prevention and control. Genevea2011. p. 1-56.
19. Lusis AJ. Atherosclerosis. Nature. 2000;407(6801):233-41.
20. Libby P. Changing concepts of atherogenesis. J Intern Med. 2000;247(3):349-58.
21. Volger OL, Fledderus JO, Kisters N, Fontijn RD, Moerland PD, Kuiper J, et al.
Distinctive expression of chemokines and transforming growth factor-beta signaling in human arterial endothelium during atherosclerosis. Am J Pathol. 2007;171(1):326-37.
22. Tan CD, Baldwin WM, 3rd, Rodriguez ER. Update on cardiac transplantation pathology. Arch Pathol Lab Med. 2007;131(8):1169-91.
23. Vlaicu SI, Tatomir A, Rus V, Mekala AP, Mircea PA, Niculescu F, et al. The role of complement activation in atherogenesis: the first 40 years. Immunol Res. 2016;64(1):1-13.
27
24. Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, Torzewski J, et al.
Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion.
Arterioscler Thromb Vasc Biol. 1998;18(3):369-78.
25. Bhakdi S, Torzewski M, Klouche M, Hemmes M. Complement and atherogenesis:
binding of CRP to degraded, nonoxidized LDL enhances complement activation. Arterioscler Thromb Vasc Biol. 1999;19(10):2348-54.
26. Bhakdi S, Torzewski M, Paprotka K, Schmitt S, Barsoom H, Suriyaphol P, et al.
Possible protective role for C-reactive protein in atherogenesis: complement activation by modified lipoproteins halts before detrimental terminal sequence. Circulation.
2004;109(15):1870-6.
27. Fraser DA, Tenner AJ. Innate immune proteins C1q and mannan-binding lectin enhance clearance of atherogenic lipoproteins by human monocytes and macrophages. J Immunol. 2010;185(7):3932-9.
28. Vlaicu R, Niculescu F, Rus HG, Cristea A. Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous plaque. Atherosclerosis.
1985;57(2-3):163-77.
29. Cao W, Bobryshev YV, Lord RS, Oakley RE, Lee SH, Lu J. Dendritic cells in the arterial wall express C1q: potential significance in atherogenesis. Cardiovasc Res.
2003;60(1):175-86.
30. Troelsen LN, Garred P, Christiansen B, Torp-Pedersen C, Christensen IJ, Narvestad E, et al. Double role of mannose-binding lectin in relation to carotid intima-media thickness in patients with rheumatoid arthritis. Mol Immunol. 2010;47(4):713-8.
31. Vengen IT, Madsen HO, Garred P, Platou C, Vatten L, Videm V. Mannose-binding lectin deficiency is associated with myocardial infarction: the HUNT2 study in Norway. PLoS One. 2012;7(7):e42113.
32. Speidl WS, Kastl SP, Hutter R, Katsaros KM, Kaun C, Bauriedel G, et al. The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP-1 and MMP-9 in human macrophages in vitro. FASEB J. 2011;25(1):35- 44.
33. Speidl WS, Exner M, Amighi J, Kastl SP, Zorn G, Maurer G, et al. Complement component C5a predicts future cardiovascular events in patients with advanced
atherosclerosis. Eur Heart J. 2005;26(21):2294-9.
34. de Hoog VC, Timmers L, Schoneveld AH, Wang JW, van de Weg SM, Sze SK, et al.
Serum extracellular vesicle protein levels are associated with acute coronary syndrome. Eur Heart J Acute Cardiovasc Care. 2013;2(1):53-60.
35. Helske S, Oksjoki R, Lindstedt KA, Lommi J, Turto H, Werkkala K, et al.
Complement system is activated in stenotic aortic valves. Atherosclerosis. 2008;196(1):190- 200.
36. Hertle E, van Greevenbroek MM, Arts IC, van der Kallen CJ, Geijselaers SL, Feskens EJ, et al. Distinct associations of complement C3a and its precursor C3 with atherosclerosis and cardiovascular disease. The CODAM study. Thromb Haemost. 2014;111(6):1102-11.
37. Carter AM, Prasad UK, Grant PJ. Complement C3 and C-reactive protein in male survivors of myocardial infarction. Atherosclerosis. 2009;203(2):538-43.
38. Niculescu F, Rus HG, Vlaicu R. Immunohistochemical localization of C5b-9, S- protein, C3d and apolipoprotein B in human arterial tissues with atherosclerosis.
Atherosclerosis. 1987;65(1-2):1-11.
28
39. Oksjoki R, Jarva H, Kovanen PT, Laine P, Meri S, Pentikainen MO. Association between complement factor H and proteoglycans in early human coronary atherosclerotic lesions: implications for local regulation of complement activation. Arterioscler Thromb Vasc Biol. 2003;23(4):630-6.
40. Oksjoki R, Kovanen PT, Mayranpaa MI, Laine P, Blom AM, Meri S, et al.
Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway. Atherosclerosis.
2007;192(1):40-8.
41. Torzewski J, Torzewski M, Bowyer DE, Frohlich M, Koenig W, Waltenberger J, et al.
C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries. Arterioscler Thromb Vasc Biol.
1998;18(9):1386-92.
42. Hertle E, van Greevenbroek MM, Arts IC, van der Kallen CJ, Feskens EJ, Schalkwijk CG, et al. Complement activation products C5a and sC5b-9 are associated with low-grade inflammation and endothelial dysfunction, but not with atherosclerosis in a cross-sectional analysis: the CODAM study. Int J Cardiol. 2014;174(2):400-3.
43. Mitchell RN. Graft vascular disease: immune response meets the vessel wall. Annu Rev Pathol. 2009;4:19-47.
44. Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circulation research. 2006;99(8):801-15.
45. Keslar K, Rodriguez ER, Tan CD, Starling RC, Heeger PS. Complement gene expression in human cardiac allograft biopsies as a correlate of histologic grade of injury.
Transplantation. 2008;86(9):1319-21.
46. Frank R, Molina MR, Goldberg LR, Wald JW, Kamoun M, Lal P. Circulating donor- specific anti-human leukocyte antigen antibodies and complement C4d deposition are associated with the development of cardiac allograft vasculopathy. Am J Clin Pathol.
2014;142(6):809-15.
47. Poelzl G, Ullrich R, Huber A, Ulmer H, Antretter H, Hoefer D, et al. Capillary deposition of the complement fragment C4d in cardiac allograft biopsies is associated with allograft vasculopathy. Transpl Int. 2005;18(3):313-7.
48. Ma J, Patel A, Tinckam K. Donor-Specific Antibody Monitoring: Where Is the Beef?
Adv Chronic Kidney Dis. 2016;23(5):317-25.
49. Nath DS, Angaswamy N, Basha HI, Phelan D, Moazami N, Ewald GA, et al. Donor- specific antibodies to human leukocyte antigens are associated with and precede antibodies to major histocompatibility complex class I-related chain A in antibody-mediated rejection and cardiac allograft vasculopathy after human cardiac transplantation. Hum Immunol.
2010;71(12):1191-6.
50. Jane-Wit D, Manes TD, Yi T, Qin L, Clark P, Kirkiles-Smith NC, et al. Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through
noncanonical nuclear factor-kappaB signaling in endothelial cells. Circulation.
2013;128(23):2504-16.
51. Qian Z, Hu W, Liu J, Sanfilippo F, Hruban RH, Baldwin WM, 3rd. Accelerated graft arteriosclerosis in cardiac transplants: complement activation promotes progression of lesions from medium to large arteries. Transplantation. 2001;72(5):900-6.
52. Fiane AE, Ueland T, Simonsen S, Scott H, Endresen K, Gullestad L, et al. Low mannose-binding lectin and increased complement activation correlate to allograft
29
vasculopathy, ischaemia, and rejection after human heart transplantation. Eur Heart J.
2005;26(16):1660-5.
53. Fildes JE, Shaw SM, Walker AH, McAlindon M, Williams SG, Keevil BG, et al.
Mannose-binding lectin deficiency offers protection from acute graft rejection after heart transplantation. J Heart Lung Transplant. 2008;27(12):1353-6.
54. Pavlov V, Raedler H, Yuan S, Leisman S, Kwan WH, Lalli PN, et al. Donor
deficiency of decay-accelerating factor accelerates murine T cell-mediated cardiac allograft rejection. J Immunol. 2008;181(7):4580-9.
55. Qin L, Li G, Kirkiles-Smith N, Clark P, Fang C, Wang Y, et al. Complement C5 Inhibition Reduces T Cell-Mediated Allograft Vasculopathy Caused by Both Alloantibody and Ischemia Reperfusion Injury in Humanized Mice. Am J Transplant. 2016;16(10):2865- 76.
56. Staff AC, Redman CW. IFPA Award in Placentology Lecture: preeclampsia, the decidual battleground and future maternal cardiovascular disease. Placenta. 2014;35 Suppl:S26-31.
57. Pijnenborg R, Ball E, Bulmer JN, Hanssens M, Robson SC, Vercruysse L. In vivo analysis of trophoblast cell invasion in the human. Methods Mol Med. 2006;122:11-44.
58. Brosens I. Unraveling the anatomy. In: Pijnenborg R, Brosens I, Romero R, editors.
Placental Bed Disorders: Basic Science and its Translation to Obstetrics. Cambridge:
Cambridge University Press; 2010. p. 5-10.
59. Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis of preeclampsia. Obstet Gynecol Annu. 1972;1:177-91.
60. Staff AC, Dechend R, Redman CW. Review: Preeclampsia, acute atherosis of the spiral arteries and future cardiovascular disease: two new hypotheses. Placenta. 2013;34 Suppl:S73-8.
61. Alnaes-Katjavivi P, Lyall F, Roald B, Redman CW, Staff AC. Acute atherosis in vacuum suction biopsies of decidua basalis: An evidence based research definition. Placenta.
2016;37:26-33.
62. Harsem NK, Roald B, Braekke K, Staff AC. Acute atherosis in decidual tissue: not associated with systemic oxidative stress in preeclampsia. Placenta. 2007;28(8-9):958-64.
63. Labarrere C, Alonso J, Manni J, Domenichini E, Althabe O. Immunohistochemical findings in acute atherosis associated with intrauterine growth retardation. Am J Reprod Immunol Microbiol. 1985;7(4):149-55.
64. Labarrere C, Althabe O, Caletti E, Muscolo D. Deficiency of blocking factors in intrauterine growth retardation and its relationship with chronic villitis. Am J Reprod Immunol Microbiol. 1986;10(1):14-9.
65. Labarrere CA, Catoggio LJ, Mullen EG, Althabe OH. Placental lesions in maternal autoimmune diseases. Am J Reprod Immunol Microbiol. 1986;12(3):78-86.
66. Labarrere C, Manni J, Salas P, Althabe O. Intrauterine growth retardation of unknown etiology. I. Serum complement and circulating immune complexes in mothers and infants.
Am J Reprod Immunol Microbiol. 1985;8(3):87-93.
67. Weir PE. Immunofluorescent studies of the uteroplacental arteries in normal pregnancy. Br J Obstet Gynaecol. 1981;88(3):301-7.
68. Wells M, Bennett J, Bulmer JN, Jackson P, Holgate CS. Complement component deposition in uteroplacental (spiral) arteries in normal human pregnancy. J Reprod Immunol.
1987;12(2):125-35.
69. Lichtig C, Deutsch M, Brandes J. Immunofluorescent studies of the endometrial arteries in the first trimester of pregnancy. Am J Clin Pathol. 1985;83(5):633-6.
30
70. Hustin J, Foidart JM, Lambotte R. Maternal vascular lesions in pre-eclampsia and intrauterine growth retardation: light microscopy and immunofluorescence. Placenta. 1983;4 Spec No:489-98.
71. Hering L, Herse F, Verlohren S, Park JK, Wellner M, Qadri F, et al. Trophoblasts reduce the vascular smooth muscle cell proatherogenic response. Hypertension.
2008;51(2):554-9.
72. Lynch AM, Gibbs RS, Murphy JR, Giclas PC, Salmon JE, Holers VM. Early elevations of the complement activation fragment C3a and adverse pregnancy outcomes.
Obstet Gynecol. 2011;117(1):75-83.
73. Lynch AM, Murphy JR, Byers T, Gibbs RS, Neville MC, Giclas PC, et al. Alternative complement pathway activation fragment Bb in early pregnancy as a predictor of
preeclampsia. Am J Obstet Gynecol. 2008;198(4):385 e1-9.
74. Derzsy Z, Prohaszka Z, Rigo J, Jr., Fust G, Molvarec A. Activation of the complement system in normal pregnancy and preeclampsia. Mol Immunol. 2010;47(7-8):1500-6.
75. Buurma A, Cohen D, Veraar K, Schonkeren D, Claas FH, Bruijn JA, et al.
Preeclampsia is characterized by placental complement dysregulation. Hypertension.
2012;60(5):1332-7.
76. Burwick RM, Fichorova RN, Dawood HY, Yamamoto HS, Feinberg BB. Urinary excretion of C5b-9 in severe preeclampsia: tipping the balance of complement activation in pregnancy. Hypertension. 2013;62(6):1040-5.
