Print - Arteriosclerosis, Thrombosis, and Vascular Biology

Original Article
Proteomic Analysis of Intraluminal Thrombus
Highlights Complement Activation in Human Abdominal
Aortic Aneurysms
Roxana Martinez-Pinna, Julio Madrigal-Matute, Carlos Tarin, Elena Burillo, Margarita Esteban-Salan,
Carlos Pastor-Vargas, Jes S. Lindholt, Juan Antonio Lopez, Enrique Calvo, Melina Vega de Ceniga,
Olivier Meilhac, Jesus Egido, Luis M. Blanco-Colio, Jean-Baptiste Michel, Jose Luis Martin-Ventura
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Objective—To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological
mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA).
Approach and Results—Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry–based
strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity
software highlighted the presence of several circulating proteins, among them were proteins from the complement
system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA
diameter=3–5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA
patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large
and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA
thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in
AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally,
the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that
C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen
species production.
Conclusions—A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local
complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation,
playing a detrimental role in AAA progression. (Arterioscler Thromb Vasc Biol. 2013;33:00-00.)
Key Words: aortic aneurysm, abdominal ◼ complement system proteins ◼ inflammation ◼ neutrophils ◼ thrombosis
C
linical and pathophysiological evidence indicates that
intraluminal thrombus (ILT) plays a role in the evolution
of abdominal aortic aneurysms (AAA).1 The eccentric distribution of the ILT was associated with continuous expansion,2
and aortic ILT volume is associated with AAA growth.3–5 It
has been reported that large ILT areas were significantly associated with increased AAA expansion.3 Speelman et al4 demonstrated that larger ILT in AAA was not only associated with
a higher AAA growth rate, but also with a lower wall stress.
These data suggest that weakening of the AAA wall, under
the biological dynamics of ILT, might play a more imminent
role in the process of AAA growth than the stress acting on the
wall. A recent study has confirmed the association of ILT volume with AAA growth and also with cardiovascular events.5
Finally, radiological signs of ILT lysis could precede aortic
rupture (crescent sign).1
In parallel, accumulating data suggest that biological
activities associated with leukocyte, platelet, and red blood
cell accumulation in ILT play an important role in AAA
progression.6–11 Thus, the identification of novel proteins
related to ILT biological activities could help to find novel
pathogenic mechanisms, as well as therapeutic targets, of
AAA. In previous studies, following a strategy based on
the analysis of AAA tissue-conditioned media by gel- or
array-based proteomic techniques, we identified proteins
related to different pathological processes involved in AAA,
such as oxidation10 and proteolysis.12 To further explore the
pathophysiology of ILT in human AAA by increasing the
number of identified proteins, ILT and wall-conditioned media
were analyzed in this study using liquid chromatography and
tandem mass spectrometry (MS). Global pathway analysis of
identified peptides/proteins by Ingenuity software highlighted
Received on: November 29, 2012; final version accepted on: May 9, 2013.
From the Vascular Research Lab (R.M.-P., J.M.-M., C.T., E.B.) and Immunology Lab (C.P.-V.), IIS-Fundación Jiménez Diaz-Autonoma University,
Madrid, Spain; Hospital de Galdakao, Vizcaya, Spain (M.E.-S., M.V.d.C.); Departments of Cardiovascular and Thoracic Surgery, University Hospital of
Odense and Viborg, Denmark (J.S.L.); Unidad de Proteómica, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (J.A.L.); and Inserm,
U698, Univ Paris, Paris, France (O.M., J.-B.M.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.301191/-/DC1.
Correspondence to Jose Luis Martin-Ventura, PhD, Vascular Research Lab, IIS-Fundacion Jimenez Diaz, Autonoma University, Av Reyes Católicos 2,
28040 Madrid, Spain. E-mail [email protected]
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1
DOI: 10.1161/ATVBAHA.112.301191
2 Arterioscler Thromb Vasc Biol August 2013
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that complement system components were highly enriched in
AAA tissue-conditioned media.
The complement system plays a major role in innate immunity, participating in host defense responses against microorganisms via opsonization, chemoattraction of leukocytes, cell
activation, and bridging innate and adaptive immunity.13–16
However, disturbances in this defense machinery contribute
to the pathogenesis of various autoimmune diseases, such as
systemic lupus erythematosus. Systemic lupus erythematosus
is characterized by decreased circulating complement components associated with their deposition and activation in host
tissues. As complement proteins are mainly synthesized by
the liver, we hypothesized that the high levels of complement
peptides/proteins identified in AAA thrombus-conditioned
media could be related to its trapping from the blood and by
increased proteolytic activation. To test this hypothesis, we
first assessed C3 concentration and activity in blood of AAA
patients at different stages of the disease. Second, we analyzed the presence and activation of C3 in AAA tissue and
tissue-conditioned media. Finally, we studied the effect of
complement activation in human AAA thrombus on neutrophil chemotaxis and oxidation, key mechanisms involved in
AAA pathogenesis.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Results
Liquid Chromatography and Tandem MS Analysis
of Proteins From AAA Tissue-Conditioned Media
Proteins obtained from AAA thrombus- and wall-conditioned
media were trypsin digested, and the resulting peptides were
then fractionated by 2-dimensional liquid chromatography
using a strong cation exchange column followed by C18
reversed phase chromatography. Finally, the MS and MS/
MS spectra were used for protein identification. Table II in
the online-only Data Supplement lists all the proteins identified in AAA tissue supernatants, where 60% of them were
classically secreted. To organize identified proteins, Ingenuity
software was used to find the most enriched canonical pathways in our samples. A total of 257 proteins extracted from
the protein lists corresponding to thrombus and wall layer
supernatants were analyzed in the same data set. Among others, coagulation and complement systems have been found as
relatively enriched in the AAA tissue supernatants (compared
with the human genome database; Figure 1A). Interestingly,
several complement-related proteins (eg, C3, C9, clusterin,
factor H) were identified, which are represented in gray color
in Figure 1B.
Systemic C3 Concentrations and
Activity in AAA Patients
As C3 is the central molecule in the complement cascade,
we analyzed serum concentrations of C3 in a first cohort of
healthy controls (n=28) and AAA patients at follow-up (small
AAA, AAA diameter=3–5 cm [n=62]) or at surgery (large
AAA, AAA diameter >5 cm, [n=28]). Clinical characteristics
are shown in Tables 1 and 2. Increased C3 concentrations were
observed in small AAA patients compared with both controls and large AAA patients (controls=148±5 versus small
AAA=177±4 versus large AAA=124±8 mg/dL; P<0.01).
Logistic regression analysis showed that association between
increased C3 in small AAA patients and controls remained
significant when adjusted by age but was lost when adjusted
for risk factors (not shown), whereas the decreased C3 in large
versus small AAA patients persisted after adjustment for risk
factors (Table III in the online-only Data Supplement). Plasma
C3 concentrations correlated with lipid levels (r=0.4 for lowdensity lipoprotein and triglycerides and r=−0.4 for highdensity lipoprotein; P<0.001 for all) and aortic size (r=−0.4;
P<0.005). Linear regression analysis between C3 and aortic
size was also independent of risk factors (Table IV in the
online-only Data Supplement).
To confirm previous data, we further analyzed a second
cohort of patients (Tables 1 and 2), showing that large AAA
patients (n=39) have significantly decreased C3 plasma concentrations compared with small AAA patients (n=26; 122±4
versus 138±4 mg/dL; P<0.01; Figure 2A), which persisted
after adjustment for risk factors (Table III in the online-only
Data Supplement). A nonsignificant negative correlation was
shown for C3 and aortic size (r=−0.2; P=0.1; Figure 2C).
To test whether complement activity is modified in plasma
of AAA patients at different stages of evolution, we performed an alternative pathway (AP) 50 assay that measures
the ability of the patient’s plasma to lyse rabbit erythrocytes.
Accordingly, large AAA patients have decreased complement
activity compared with small AAA patients (42±5 versus
75±4% lysis; P<0.01; Figure 2B). Logistic regression analysis showed that the significant association between AP50
in large AAA compared with small AAA patients persisted
after adjustment for risk factors (Table III in the online-only
Data Supplement). AP50 correlated with aortic size (r=−0.4;
P<0.005; Figure 2D), which persisted after adjustment for risk
factors (Table IV in the online-only Data Supplement).
Genetic Association Study
No association of single-nucleotide polymorphisms (SNPs)
in the complement cascade in AAA patient and control
studies has been recently described.17,18 To get further insight
into a potential genetic association between complement
and AAA evolution, we analyzed whether the decrease in
C3 concentrations and activity in large versus small AAA
patients could be related to a particular combination of
SNPs in complement genes (complotypes), as described for
other disorders, such as age-related macular degeneration.19
However, no association was found between these
complotypes and AAA in patients at different stages of the
disease (large versus small AAA). In plasma samples available
from these patients (n=138), we further confirmed that C3
concentrations were decreased in large AAA patients (n=66)
compared with small AAA patients (n=72; 194±5 versus
210±5 mg/dL; P<0.05). Logistic regression analysis showed
that the significant association between decreased plasma C3
in large AAA compared with small AAA patients persisted
after adjustment for risk factors (Table III in the online-only
Data Supplement). A nonsignificant negative correlation was
shown for C3 and aortic size (r=−0.2; P=0.07).
Martinez-Pinna et al Complement Activation in Human AAA 3
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Figure 1. Ingenuity pathway analysis of identified proteins by liquid chromatography and tandem mass spectrometry (MS). A, The bar
graphic shows the canonical pathway distribution represented by gene enrichment. Ratios show the number of genes associated with
each pathway found in our experiment with respect to the human genome database. Fisher exact test provides P<0.05 for the 7 most
abundant canonical pathways. B, Detailed inspection of the complement system cascade, where proteins identified by MS in abdominal
aortic aneurysm tissue-conditioned media are represented in gray color. Protein groups or complex are yellow encircled, and receptors of
different complement components are represented at the bottom of the figure.
Local Complement Retention
and Activation in AAA
We analyzed the presence of C3 in AAA tissue by immunohistochemistry, showing an intense extracellular staining in the
ILT and, to a lesser extent, in the wall, whereas healthy wall
shows weak staining (Figure 3A). Similarly, high C9 staining was observed in AAA thrombus compared with wall and
healthy wall (Figure I in the online-only Data Supplement).
Interestingly, C3 and C9 deposition was observed in similar areas of the thrombus, suggesting complement activation
(Figure 3B). Whereas C3 was mainly present in acellular
areas of the thrombus, C3 in the wall was also associated with
α-actin–positive cells in the media (Figure II in the onlineonly Data Supplement) and, to a lesser extent, with CD15/
Table 1. Clinical Characteristics of AAA Patients in First
Cohort
Table 2. Clinical Characteristics of AAA Patients in Second
Cohort
Small AAA (n=62)
Sex (men/women)
Age, y±SD
Large AAA (n=28)
62/0
28/0
69.9±6.7
73.4±6.6
Small AAA (n=26)
Large AAA (n=39)
Sex (men/women)
24/2
37/2
Age, y±SD
79±5
70±10
Dyslipidemia, %
56.5
37
Dyslipidemia, %
46
53
Current smoking, %
37.1
42.9
Current smoking, %
15
30
Diabetes mellitus, %
19.4
3.6
Diabetes mellitus, %
30
7
Hypertension, %
64.5
64.3
Hypertension, %
73
46
Heart disease, %
32.3
21.4
Heart disease, %
23
23
AAA indicates abdominal aortic aneurysm.
AAA indicates abdominal aortic aneurysm.
4 Arterioscler Thromb Vasc Biol August 2013
Figure 2. Systemic C3 concentration and activity in abdominal
aortic aneurysm (AAA) patients.
C3 concentration (A) and activity (alternative pathway [AP]
50, B) in plasma of small AAA
patients (n=26) and large AAA
patients (n=39). Correlation of
C3 concentration (C) and activity (D) with aortic size.
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CD68-positive cells mainly in adventitia (Figure III in the
online-only Data Supplement), suggesting the possible synthesis by resident or infiltrating cells in the wall. To test this
hypothesis, we performed both real-time polymerase chain
reaction and Western blot of tissue homogenates. No detectable C3 mRNA was obtained from 3 of 6 thrombi analyzed,
and very low levels were observed in the rest (0.01±0.02 a.u.).
No differences were observed in C3 mRNA levels between
healthy and AAA wall (0.22±0.1 versus 0.28±0.05 a.u., not
shown). In contrast, C3 protein levels in tissue homogenates
were higher in ILT compared with pathological wall and
healthy wall, confirming the results observed by immunohistochemistry (Figure 4A).
We further tested C3 concentration and activation in the
conditioned media of human ILT and wall of AAA, as well
as in healthy media. C3 levels were increased in the AAA
thrombus compared with the pathological wall and healthy
wall (5.6±0.5 versus 2.4±0.2 versus 0.9±0.2 µg/mL; P<0.001
for all comparisons). Furthermore, C3 proteolytic fragments
of 35 to 40 kDa (corresponding to the molecular weight of
C3c/C3dg; Figure 4A–4C) appear mainly in tissue and tissueconditioned media of ILT and, to a lesser extent, in the wall
(media and adventitia) of AAA, whereas almost no proteolytic
fragments were observed in healthy wall. Because complement activation could involve proteolytic degradation of C3
by proteases, such as plasmin or elastase,13 abundantly present
in ILT,6,7 we assessed whether proteolysis of C3 in the thrombus of AAA could take place ex vivo. In this respect, when
native C3 was incubated with the luminal part of the thrombus, the 35 to 40 kDa fragments observed in ILT-conditioned
media were increased (Figure 4D).
Role of Complement Activation in AAA ThrombusInduced PMN Chemotaxis and Activation
As we did not observe the first fragment of C3 activation, C3a,
by Western blot probably as a result of its low molecular weight,
we analyzed the presence of C3a in tissue-conditioned media
by ELISA. We have observed that C3a levels are increased
in ILT compared with the wall of AAA and also the healthy
wall (P<0.001 for all comparisons; Figure 5A). Furthermore,
because C3a is involved in PMN chemotaxis and reactive
oxygen species production, we address the functional role
of complement activation by proteolysis in ILT. Neutrophils
were allowed to migrate through a filter into a lower chamber containing thrombus-conditioned media, and the effect
of C3 blocking was assessed. Neutrophils were attracted by
luminal thrombus, and C3 blockade by anti-C3 antibody was
able to decrease such effect (P<0.001; Figure 5B). Similar
effect was observed when native C3 was used as a positive
control (not shown). Furthermore, incubation of thrombusconditioned media with fresh neutrophils increased reactive
oxygen species levels, which was prevented by anti-C3 antibody (Figure 5C).
Discussion
In the present article, the combination of nano-liquid chromatography and LTQ-Orbitrap MS allowed us to identify
larger lists of proteins from AAA tissue-conditioned media
compared with array or gel-based approaches.10,12 Several proteins previously associated with different AAA pathological
mechanisms have been identified (eg, immune–inflammatory
response [clusterin], thrombosis [fibrinogen]). Interestingly,
a recent proteomic study has also shown increased levels of
clusterin, a complement lysis inhibitor able to block the terminal complement cascade in AAA thrombus-conditioned
media.20 In contrast, clusterin concentrations were decreased
in AAA plasma, and the authors suggested that ILT could
sequester systemic proteins.20 In agreement, the functional
distribution of the identified proteins in our study has shown
an enrichment in circulating proteins (eg, coagulation and
complement systems). As complement proteins are mainly
synthesized by the liver, we hypothesized that the high levels
Martinez-Pinna et al Complement Activation in Human AAA 5
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Figure 3. Local complement retention in abdominal aortic aneurysm (AAA) tissue. Immunohistochemistry of C3 in thrombus (A), wall
(C), and healthy wall (D). Negative control (nonspecific IgG; B). C3 (E) and C9 (F) immunostaining is performed in serial sections of AAA
thrombus. Magnification, ×20 (inset, ×120, showing areas with polymorphonuclears).
of complement peptides/proteins identified in AAA thrombus-conditioned media could be related to its trapping from
the blood and by increased proteolytic activation. We first
analyzed C3 concentrations in AAA patients and controls.
Circulating C3 levels were increased in small AAA patients
compared with controls, probably suggesting an initial hepatic
response to vascular injury. However, when we performed a
multivariate analysis, including risk factors, no significant differences were observed between small AAA patients and controls in agreement with previous data,21 discarding its potential
use as a diagnostic biomarker. Regarding risk factors, we have
observed a positive correlation between C3 and lipid levels.
Lipids have been previously suggested as a potential mechanism leading to complement activation in experimental and
human hypercholesterolemia.22 Furthermore, it has been proposed that IgG is an initial mechanism leading to C3 activation in an experimental model of AAA,23 and increased IgG
concentrations have been recently observed in small AAA
patients versus controls. However, IgG concentrations decline
in large AAA patients.24 In agreement, we have shown that
C3 concentrations are decreased in large compared with small
AAA patients, which was independent of different risk factors. In addition, when we performed an AP50 assay to test
whether complement activity is modified during AAA evolution, we showed a negative association of complement activity
with later stages of disease and with aortic size, independent of
risk factors. On the whole, our data suggest that systemic C3
increases in the initial phases of AAA, probably as a response
to injury of the wall to increased lipid and IgG concentrations,
whereas a decrease in systemic complement concentration
and activation takes place in the later stages of AAA.
Complement deficiencies (inherited or acquired) could
be linked to the development of autoimmunity, as shown in
systemic lupus erythematosus where decreased complement
6 Arterioscler Thromb Vasc Biol August 2013
A
C
T
W
H
B
T
M
D
M
T
H C3dg
C3c C3b iC3b
C3
ADV
T
HM
T+C3
HADV
C3
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Figure 4. Local complement activation in abdominal aortic aneurysm (AAA) tissue and tissue-conditioned media. A, Representative Western blot of C3 in tissue homogenates of human AAA thrombus (T), pathological wall (W=media+adventitia), and healthy wall
(H=media+adventitia). B, Representative Western blot of C3 in tissue-conditioned media of human AAA thrombus (T), pathological wall
(media, M, and adventitia, ADV), and healthy wall (media, HM, and adventitia, HADV). C, Representative Western blot of C3 in tissue-conditioned media of human AAA pathological wall (media, M), thrombus (T), and healthy wall (H). Fragments of C3 (C3dg, C3c, C3b, iC3b) or
native C3 purified as described.20 D, Representative Western blot of C3 in thrombus-conditioned media incubated with native C3. Arrows
indicate proteolytic fragments of C3.
components are observed. To check whether genetic anomalies in complement genes could take place in AAA evolution,
we performed a genetic study analyzing a particular combination of SNPs in complement genes (complotypes). This
approach has been also described for other disorders, such as
age-related macular degeneration.19 No association on any of
the combination of SNPs analyzed was found between small
and large AAA patients, similar to previous studies where
those individual SNPs were assessed in AAA patients and
controls.17,18 It has been suggested that the acquired diminution in circulating complement proteins in autoimmune diseases could be associated with deposition of complement
components in host tissue.13 In this respect, complement proteins have been previously detected in human AAA wall.17,25,26
We observed that C3 protein levels were increased in tissue
and tissue-conditioned media of AAA wall compared with
healthy aortic wall, whereas C3 mRNA is similarly present in
pathological and healthy wall in agreement with Hinterseher
et al.17 But, Hinterseher et al17 did not show C3 staining in
the thrombus. In contrast, we have shown that human AAA
thrombus displays an intense extracellular staining, along with
increased protein levels in tissue homogenates and tissueconditioned media of ILT compared with wall. Differences
between both immunohistological studies could be related to
the different antibodies used. Furthermore, C3 in thrombus
from acute myocardial infarction has also been observed.27
Because the liver is the major source of complement proteins in humans and low/undetectable C3 mRNA levels were
observed in thrombus homogenates, the high extracellular levels of C3 observed in AAA thrombus should be associated
with its retention from serum and subsequent activation. In
this regard, high C9 immunostaining was also shown in acellular areas of the thrombus and wall compared with healthy
wall. In agreement, Pagano et al26 showed C5B9 in the luminal
side of the wall, whereas no staining was observed in healthy
wall. Similarly, Tulamo et al28 also observed complement activation and C5B9 formation in the less cellular part of intracranial artery aneurysm wall. Finally, we showed intense C9
immunostaining associated with C3 deposition, suggesting
complement activation in AAA thrombus.
Complement activation involves the classical pathway,
the lectin pathway, the AP, and the extrinsic pathway.13 The
classical pathway can be activated by antibodies or by other
stimulus, such as CRP. The AP is part of the innate (nonantigen-specific) immune system and is important in antibodyindependent defense against bacterial infection. The extrinsic
pathway involves proteolytic degradation of C3 by proteases,
such as elastase, and phagocytes. Furthermore, other components of the coagulation system, such as plasmin, could also
participate in complement activation.16 In this respect, human
AAA thrombus could be a privileged site for complement
activation because proteases and PMNs are abundant in the
ILT of AAA.6,7 When we analyzed C3 levels in AAA tissue
and tissue-conditioned media by Western blot, we observed
proteolytic fragments of C3 in AAA thrombus and thrombus-conditioned media. Furthermore, these products of C3
Martinez-Pinna et al Complement Activation in Human AAA 7
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Figure 5. Role of complement activation in abdominal aortic aneurysm (AAA) thrombus-induced chemotaxis. A, ELISA of C3a in tissueconditioned media of human AAA thrombus (T, n=10), pathological wall (media [M, n=10] and adventitia [ADV, n=10]), and healthy wall
(media [HM, n=10] and adventitia [HADV, n=10]). * and † P<0.001 for T vs M and ADV and M and ADV vs HM and HADV, respectively. B,
Chemotaxis assay showing polymorphonuclear (PMN) migration toward the thrombus (T), thrombus preincubated with anti-C3 (T+antiC3 at 1:100 or 1:10 dilution), and thrombus preincubated with anti-IgG (T+IgG, for nonspecific chemotaxis). *P<0.001 for anti-C3 vs T. C,
NADPH-dependent reactive oxygen species (ROS) production in PMNs stimulated during 2 minutes with thrombus-conditioned media,
thrombus preincubated with anti-C3 (T+anti-C3 at 1:100 or 1:10 dilution), or thrombus preincubated with anti-IgG (T+IgG). *P<0.05 for
anti-C3 vs T.
proteolysis and activation were increased when native C3
protein was coincubated with the luminal part of AAA, supporting that in vivo proteolysis of C3 protein could take place
within the thrombus of AAA. In addition, C3c/C3dg levels
were increased in AAA wall compared with healthy wall.
These proteolytic products could participate in the shift from
innate to adaptive immunity, characteristic of the adventitial
response in AAA.29 Finally, given that C3a is released in the
initial step of the proteolytic processing of C3, C3a levels were
assessed in AAA tissue and healthy wall. In agreement with
the results obtained for C3, C3a was increased in conditioned
media of ILT and wall compared with healthy aorta wall, further supporting proteolysis of C3 in AAA tissue. C3aR mRNA
is upregulated in AAA tissue,17 favoring the potential interaction with C3a in AAA and its functional consequences. In
this respect, it is already described that the complement acts
as a critical mediator of neutrophil recruitment in AAA mice
lesions.26 Interestingly, we have observed that PMN chemotaxis induced by the ILT could be modulated by incubation
with an anti-C3 antibody. Furthermore, C3a has been involved
in PMN respiratory burst.30 We observed that NADPHdependent reactive oxygen species production is increased
when fresh PMNs are incubated with ILT-conditioned media,
and this effect was prevented by C3 blockade. In any case, we
should take into account that these functional activities of the
thrombus could be also related to the presence of other factors,
such as tissue factor, clotting factors (eg, Xa), or thrombin,
among others. Thrombin is involved in the activation of the
complement system, further confirming a coordinated action
of the coagulation and complement systems.16 Interestingly,
both systems have been associated with innate immunity
into what Engelmann and Massberg31 recently described as
immunothrombus. Thrombin also modulates fibrinolysis by
activating the plasma carboxypeptidase, thrombin-activatable
procarboxypeptidase B. In this respect, enhanced AAA formation in pCPB−/− mice was observed to be associated with
plasmin generation.32 On the whole, all these data suggest that
complement activation by proteases present in the thrombus
could contribute to AAA pathogenesis.
Several studies have recently demonstrated that genetic
modification of different mediators of the complement pathway could reduce experimental AAA formation.23,26 However,
complement activation is modulated by several complement
inhibitors (eg, CD59, vitronectin). In this respect, Tulamo et
al28 showed a differential distribution of complement inhibitors
in different areas of the wall of intracranial artery aneurysms.
These data suggest that a disturbed complement regulation
is associated with an increased susceptibility to complement
activation and inflammation and may be also cell loss.28 In our
previous study, vitronectin was shown to be downregulated in
tissue of ruptured AAA compared with nonruptured AAA.33
Finally, deficiency of CD59 accelerated, whereas transgenic
overexpression of human CD59 attenuated, the progression
of experimental AAA.34 At the therapeutic level, anti-C5
8 Arterioscler Thromb Vasc Biol August 2013
blockade was able to reverse atherosclerosis associated with a
decrease in complement deposition.35 Because atherosclerosis
and AAA share some pathological mechanisms, it could be
interesting to address whether the therapeutic modulation of
complement activators or inhibitors may have a protective role
in AAA progression.
On the whole, the decrease of systemic C3 concentration
and activity in the later stages of AAA associated with local
complement retention, consumption, and proteolysis in the
thrombus could induce PMN activation, playing a detrimental
role in AAA. Future studies targeting complement activation
could be an attractive therapeutic strategy to prevent AAA
progression.
Acknowledgments
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We thank Patricia Llamas Granada for technical assistance and
Santiago Rodriguez de Cordoba for the reagents provided and for his
helpful comments, suggestions, and critical revision of the content
of the article.
Sources of Funding
The article has been supported by the EC, FAD project (FP-7,
HEALTH F2-2008–200647), the Spanish MICIN (SAF2010/21852),
Fundacion Ramon Areces, Ministerio de Sanidad y Consumo,
Instituto de Salud Carlos III, Redes RECAVA (RD12/0042/00038),
and biobancos (RD09/0076/00101).
Disclosures
None.
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Significance
Downloaded from http://atvb.ahajournals.org/ by guest on November 20, 2016
Clinical and pathophysiological evidence indicates that intraluminal thrombus plays a role in evolution of abdominal aortic aneurysms (AAA).
Intraluminal thrombus and wall-conditioned media were analyzed using a proteomic approach. Global pathway analysis of identified peptides/proteins highlighted that complement system components were highly enriched in AAA tissue-conditioned media. This could be related
to its trapping from the blood (because decreased complement C3 concentration was associated with later stages of AAA in 3 different
cohorts) and by increased proteolytic activation (as C3 fragments were observed in intraluminal thrombus). The functional consequences of
complement retention and activation in AAA thrombus are related to increased polymorphonuclear chemotaxis and reactive oxygen species
production, main mechanisms involved in AAA progression. Our data support an important role of complement activation not only in the initial
phases of AAA formation (as demonstrated in experimental models of AAA) but also in human AAA progression associated with polymorphonuclear recruitment and activation in intraluminal thrombus.
Downloaded from http://atvb.ahajournals.org/ by guest on November 20, 2016
Proteomic Analysis of Intraluminal Thrombus Highlights Complement Activation in
Human Abdominal Aortic Aneurysms
Roxana Martinez-Pinna, Julio Madrigal-Matute, Carlos Tarin, Elena Burillo, Margarita
Esteban-Salan, Carlos Pastor-Vargas, Jes S. Lindholt, Juan Antonio Lopez, Enrique Calvo,
Melina Vega de Ceniga, Olivier Meilhac, Jesus Egido, Luis M. Blanco-Colio, Jean-Baptiste
Michel and Jose Luis Martin-Ventura
Arterioscler Thromb Vasc Biol. published online May 23, 2013;
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/early/2013/05/23/ATVBAHA.112.301191
Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2013/05/23/ATVBAHA.112.301191.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
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Supplemental material online
Supplemental tables
Table I online.- Clinical characteristics of patients from the VIVA trial 1st degree relative with AAA
Current smokers
Diabetes
Hypertension
Use of ACE-inhibitors
Use of beta-blocker
Statin
Low dose aspirin
Peripheral arterial disease (PAD)
Intermittent claudication
Previous AMI
Current angina pectoris:
Age (years)
Body mass index (BMI)
Systolic blood pressure (mmHg)
Diastolic bloodpressure (mmHg)
Lowest ABI
Maximal aortic diameter (mm)
Aneurysmal growth rate
(mm/year)
ABI: Ankle brachial bloodpressure index
AMI: Acute myocardial infarction
N=
186
15
76
15
95
45
61
93
84
40
35
44
25
Mean
69.66
27.60
154.30
88.40
0.96
48.75
3.10
All
AAA Small AAA
(%)
(%)
8.4
8.5
42.5
47.4
8.4
4.2
53.4
50.5
26.0
24.7
35.5
31.9
52.8
51.6
48.3
44.6
22.3
22.1
20.1
17.9
25.4
25.3
14.4
14.7
SD Mean (SD)
2.91
69.4 (2.9)
3.54
27.6(3.6)
21.67
156 (22.7)
12.00
88.3(12.3)
0.17
0.95(0.18)
15.42
37.0(5.18)
2.95
2.98(2.77)
Large AAA
(%)
8.0
36.4
12.5
52.4
29.8
40.0
57.5
54.7
21.6
21.7
27.7
16.9
Mean (SD)
70.0(2.8)
27.6 (3.5)
152(20.5)
88.5(11.8)
0.96(0.15)
70.0(2.84)
5.97(2.84)
Table II online.- Proteins identified by LTQ-Orbitrap mass spectrometry
Uniprot
Code
A4D0S4
A5D8V7
A6NJS3
A8K8V0
B2RMS9
D6RAK8
E9PQD6
F6THE9
G3V0G4
I3L499
O00400
O00574
O14672
O14744
O14994
O15264
O15466
O43749
O60508
O60673
O60885
O75179
O75385
O75920
O75970
O94776
O95218
O95477
O95831
P00450
P00734
P00738
P00747
P00846
P01011
P01023
P01024
P01042
P01583
Protein Name
Laminin subunit beta-4
Coiled-coil domain-containing protein 151
Putative V-set and immunoglobulin domain
Zinc finger protein 785
Inter-alpha (Globulin) inhibitor H4 (Plasma Kallikreinsensitive glycoprotein)
Vitamin D-binding protein
Serum amyloid A protein
Butyrophilin-like protein 2
2-aminoadipic 6-semialdehyde dehydrogenase, isoform
CRA_c
Patched domain-containing protein 3
Acetyl-coenzyme A transporter 1
C-X-C chemokine receptor type 6
Disintegrin and metalloproteinase domain-containing
protein 10
Protein arginine N-methyltransferase 5
Synapsin-3
Mitogen-activated protein kinase 13
Alpha-2,8-sialyltransferase 8E
Olfactory receptor 1F1
Pre-mRNA-processing factor 17
DNA polymerase zeta catalytic subunit
Bromodomain-containing protein 4
Ankyrin repeat domain-containing protein 17
Serine/threonine-protein kinase ULK1
Small EDRK-rich factor 1
Multiple PDZ domain protein
Metastasis-associated protein MTA2
Zinc finger Ran-binding domain-containing protein 2
ATP-binding cassette sub-family A member 1
Apoptosis-inducing factor 1, mitochondrial
Ceruloplasmin
Prothrombin
Haptoglobin
Plasminogen
ATP synthase subunit a
Alpha-1-antichymotrypsin
Alpha-2-macroglobulin
Complement C3
Kininogen-1
Interleukin-1 alpha
Secretory
Predictions *
S
S
S
S
S
S
S
NC
S
NC
NC
NC
S
NC
S
NC
S
S
S
S
NC
S
NC
NC
S
NC
NC
S
S
S
S
S
S
NC
S
S
S
S
S
P02042
P02647
P02649
P02652
P02656
P02671
P02675
P02679
P02743
P02748
P02749
P02751
P02760
P02763
P02768
P02786
P02787
P02790
P02792
P02794
P03891
P03999
P04040
P04114
P04196
P04217
P05155
P06396
P06703
P07305
P07951
P07996
P08294
P08670
P0C0L4
P0C0L5
P10636
P10828
P10909
P11487
P12259
P13688
P16499
P16860
Hemoglobin subunit delta
Apolipoprotein A-I
Apolipoprotein E
Apolipoprotein A-II
Apolipoprotein C-III
Fibrinogen alpha chain
Fibrinogen beta chain
Fibrinogen gamma chain
Serum amyloid P-component
Complement component C9
Beta-2-glycoprotein 1
Fibronectin
Protein AMBP
Alpha-1-acid glycoprotein 1
Serum albumin
Transferrin receptor protein 1
Serotransferrin
Hemopexin
Ferritin light chain
Ferritin heavy chain
NADH-ubiquinone oxidoreductase chain 2
Short-wave-sensitive opsin 1
Catalase
Apolipoprotein B-100
Histidine-rich glycoprotein
Alpha-1B-glycoprotein
Plasma protease C1 inhibitor
Gelsolin
Protein S100-A6
Histone H1.0
Tropomyosin beta chain
Thrombospondin-1
Extracellular superoxide dismutase [Cu-Zn]
Vimentin
Complement C4-A
Complement C4-B
Microtubule-associated protein tau
Thyroid hormone receptor beta
Clusterin
Fibroblast growth factor 3
Coagulation factor V
Carcinoembryonic antigen-related cell adhesion molecule 1
Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit
alpha
Natriuretic peptides B
NC
S
S
S
S
NC
NC
S
S
S
S
S
S
S
S
S
S
S
NC
S
NC
NC
NC
S
NC
S
S
S
S
NC
NC
NC
S
S
S
S
NC
S
S
S
S
S
NC
S
P17980
P20273
P20339
P22557
P22670
P22792
P23142
P24530
P26006
P27482
P29144
P29218
P31327
P32119
P35221
P35228
P35626
P36980
P37231
P40429
P41134
P43652
P46100
P46977
P47224
P47989
P48637
P49321
P49418
P49815
P49916
P50416
P51580
P51826
P51884
P52737
P54132
P59923
P60709
P63010
P68873
P69891
P69905
26S protease regulatory subunit 6A
B-cell receptor CD22
Ras-related protein Rab-5A
5-aminolevulinate
synthase,
erythroid-specific,
mitochondrial
MHC class II regulatory factor RFX1
Carboxypeptidase N subunit 2
Fibulin-1
Endothelin B receptor
Integrin alpha-3
Calmodulin-like protein 3
Tripeptidyl-peptidase 2
Inositol monophosphatase 1
Carbamoyl-phosphate synthase [ammonia], mitochondrial
Peroxiredoxin-2
Catenin alpha-1
Nitric oxide synthase, inducible
Beta-adrenergic receptor kinase 2
Complement factor H-related protein 2
Peroxisome proliferator-activated receptor gamma
60S ribosomal protein L13a
DNA-binding protein inhibitor ID-1
Afamin
Transcriptional regulator ATRX
Dolichyl-diphosphooligosaccharide
protein
glycosyltransferase subunit STT3A
Guanine nucleotide exchange factor MSS4
Xanthine dehydrogenase/oxidase
Glutathione synthetase
Nuclear autoantigenic sperm protein
Amphiphysin
Tuberin
DNA ligase 3
Carnitine O-palmitoyltransferase 1, liver isoform
Thiopurine S-methyltransferase
AF4/FMR2 family member 3
Lumican
Zinc finger protein 136
Bloom syndrome protein
Zinc finger protein 445
Actin, cytoplasmic 1
AP-2 complex subunit beta
Hemoglobin subunit beta
Hemoglobin subunit gamma-1
Hemoglobin subunit alpha
NC
S
S
S
S
S
S
NC
S
S
NC
S
S
NC
NC
S
NC
S
S
S
S
NC
S
NC
NC
NC
S
NC
NC
NC
S
S
NC
S
NC
S
NC
S
NC
NC
NC
NC
NC
P80192
P82673
P98160
Q01524
Q04446
Q04725
Q06710
Q07020
Q07954
Q08379
Q08ER8
Q12788
Q12797
Q13017
Q13108
Q13114
Q13402
Q13683
Q14004
Q14767
Q14789
Q15063
Q15067
Q15431
Q15528
Q16666
Q4J6C6
Q4KMG0
Q53FM8
Q53G59
Q53WY6
Q59ER9
Q59FE5
Q59H18
Q5FWF5
Q5KSL6
Q5T5U3
Q5T686
Q5VT06
Q5VTL8
Q5VV42
Q5ZF01
Mitogen-activated protein kinase kinase kinase 9
28S ribosomal protein S35, mitochondrial
Basement membrane-specific heparan sulfate proteoglycan
core protein
Defensin-6
1,4-alpha-glucan-branching enzyme
Transducin-like enhancer protein 2
Paired box protein Pax-8
60S ribosomal protein L18
Prolow-density lipoprotein receptor-related protein 1
Golgin subfamily A member 2
Zinc finger protein 543
Transducin beta-like protein 3
Aspartyl/asparaginyl beta-hydroxylase
Rho GTPase-activating protein 5
Melanoma ubiquitous mutated protein
TNF receptor-associated factor 3
Unconventional myosin-VIIa
Integrin alpha-7
Cyclin-dependent kinase 13
Latent-transforming growth factor beta-binding protein 2
Golgin subfamily B member 1
Periostin
Peroxisomal acyl-coenzyme A oxidase 1
Synaptonemal complex protein 1
Mediator of RNA polymerase II transcription subunit 22
Gamma-interferon-inducible protein 16
Prolyl endopeptidase-like
Cell
adhesion
molecule-related/down-regulated
by
oncogenes
Fucose-1-phosphate guanyltransferase variant
Kelch-like protein 12
Transthyretin
B aggressive lymphoma gene variant
A disintegrin and metalloproteinase with thrombospondin
motifs 10
Serine/threonine-protein kinase TNNI3K
N-acetyltransferase ESCO1
Diacylglycerol kinase kappa
Rho GTPase-activating protein 21
Arginine vasopressin-induced protein 1
Centrosome-associated protein 350
Pre-mRNA-splicing factor 38B
Threonylcarbamoyladenosine tRNA methylthiotransferase
Activating signal cointegrator 1 complex subunit 3-like 1
S
S
S
S
S
S
NC
NC
S
NC
S
S
NC
S
S
NC
S
S
NC
S
NC
S
NC
NC
NC
NC
NC
S
NC
S
S
NC
S
NC
NC
S
NC
S
NC
NC
S
S
Q68CZ2
Q6IBW4
Q6P9A3
Q6U7Q0
Q6ZT12
Q70Z53
Q75T13
Q76I76
Q7Z4N2
Q7Z7C8
Q7Z7G8
Q861H1
Q86VE9
Q86Z14
Q8IVE3
Q8IWQ3
Q8IXT1
Q8IY21
Q8IYA6
Q8IYF1
Q8IYI6
Q8IYM0
Q8N119
Q8NBP0
Q8NE79
Q8NEL9
Q8NEN0
Q8NF91
Q8NI22
Q8TBF4
Q8TD19
Q8TEK3
Q8WZ42
Q92794
Q92876
Q92994
Q93100
Q969Y0
Q96DZ1
Q96EB6
Q96EC4
Q96I76
Q96J94
Tensin-3
Condensin-2 complex subunit H2
Zinc finger protein 549
Zinc finger protein 322
E3 ubiquitin-protein ligase UBR3
Protein FRA10AC1
GPI inositol-deacylase
Protein phosphatase Slingshot homolog 2
Transient receptor potential cation channel subfamily M
member 1
Transcription initiation factor TFIID subunit 8
Vacuolar protein sorting-associated protein 13B
MHC class I antigen
Serine incorporator 5
Beta-klotho
Pleckstrin homology domain-containing family H member 2
Serine/threonine-protein kinase BRSK2
Nitric oxide-inducible gene protein
Probable ATP-dependent RNA helicase DDX60
Cytoskeleton-associated protein 2-like
RNA polymerase II transcription factor SIII subunit A2
Exocyst complex component 8
Protein FAM186B
Matrix metalloproteinase-21
Tetratricopeptide repeat protein 13
Blood vessel epicardial substance
Phospholipase DDHD1
Armadillo repeat-containing protein 2
Nesprin-1
Multiple coagulation factor deficiency protein 2
Zinc finger CCHC-type and RNA-binding motif-containing
protein 1
Serine/threonine-protein kinase Nek9
Histone-lysine N-methyltransferase, H3 lysine-79 specific
Titin
Histone acetyltransferase KAT6A
Kallikrein-6
Transcription factor IIIB 90 kDa subunit
Phosphorylase b kinase regulatory subunit beta
NXPE family member 3
Endoplasmic reticulum lectin 1
NAD-dependent protein deacetylase sirtuin-1
AHNAK nucleoprotein
G patch domain-containing protein 3
Piwi-like protein 1
NC
NC
S
S
S
NC
S
S
NC
NC
NC
S
NC
S
S
NC
S
S
NC
S
NC
NC
S
S
S
NC
NC
S
S
S
NC
NC
S
NC
S
S
NC
S
S
S
S
NC
S
Q96JS3
Q96NW4
Q96P70
Q96Q83
Q96RD9
Q96RW7
Q96S42
Q96SU4
Q96T58
Q99457
Q9BVI0
Q9BWV1
Q9BYK8
Q9BZ29
Q9BZF1
Q9BZH6
Q9GZS0
Q9H2D6
Q9H2X9
Q9H3L0
Q9H7D0
Q9H7X0
Q9H9P8
Q9HAH1
Q9NPC7
Q9NPI1
Q9NQR4
Q9NQW7
Q9NS00
Q9NU22
Q9NX55
Q9NYF8
Q9NYP7
Q9P0K1
Q9P212
Q9P2K8
Q9P2N7
Q9P2R3
Q9UHC7
Q9UKT4
Q9ULV0
PiggyBac transposable element-derived protein 1
Ankyrin repeat domain-containing protein 27
Importin-9
Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3
Fc receptor-like protein 5
Hemicentin-1
Nodal homolog
Oxysterol-binding protein-related protein 9
Msx2-interacting protein
Nucleosome assembly protein 1-like 3
PHD finger protein 20
Brother of CDO
Peroxisomal proliferator-activated receptor A
Dedicator of cytokinesis protein 9
Oxysterol-binding protein-related protein 8
WD repeat-containing protein 11
Dynein intermediate chain 2, axonemal
TRIO and F-actin-binding protein
Solute carrier family 12 member 5
Methylmalonic aciduria and homocystinuria type D protein,
mitochondrial
Dedicator of cytokinesis protein 5
N-alpha-acetyltransferase 60
L-2-hydroxyglutarate dehydrogenase, mitochondrial
Zinc finger protein 556
Myoneurin
Bromodomain-containing protein 7
Omega-amidase NIT2
Xaa-Pro aminopeptidase 1
Glycoprotein-N-acetylgalactosamine
3-betagalactosyltransferase 1
Midasin
Huntingtin-interacting protein K
Bcl-2-associated transcription factor 1
Elongation of very long chain fatty acids protein 5
Disintegrin and metalloproteinase domain-containing
protein 22
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase
epsilon-1
Eukaryotic translation initiation factor 2-alpha kinase 4
Kelch-like protein 13
Ankyrin repeat and FYVE domain-containing protein 1
E3 ubiquitin-protein ligase makorin-1
F-box only protein 5
Unconventional myosin-Vb
S
S
S
S
S
NC
S
NC
NC
NC
S
S
NC
S
NC
NC
NC
S
NC
S
NC
NC
S
NC
S
NC
S
S
S
NC
S
NC
NC
S
NC
S
S
NC
S
S
S
Q9Y2G5
Q9Y4A5
Q9Y4D8
Q9Y6D5
Q9Y6J0
GDP-fucose protein O-fucosyltransferase 2
Transformation/transcription domain-associated protein
Probable E3 ubiquitin-protein ligase C12orf51
Brefeldin A-inhibited guanine nucleotide-exchange protein
2
Calcineurin-binding protein cabin-1
S
S
NC
S
S
* Secretory Predictions data provided by SecretomeP.1 Software (S: Classically
secreted proteins; NC: Non-classically secreted proteins. Classically secreted: 155
proteins, 60% of total; Non-classically secreted: 102 proteins, 40% of total)
Table III online. Logistic regression analysis with AAA stage as dependent variable
a) First cohort
B
SE
OR
P-value
(Constant)
-,931
3,842
,394
,808
Age
,089
,050
1,093
,075
Dyslipidemia
,-,823
,733
,439
,261
Current smoking
1,094
,747
2,986
,143
Diabetes
-1,828
1,277
,161
,152
Hypertension
,533
,710
1,703
,453
Heart disease
-,862
,765
,422
,260
C3
-,042
,011
,959
,000
B
SE
OR
P-value
(Constant)
26,759
7,499
4,2E11
,000
Sex
-,262
1,190
,769
,826
Age
-,242
,079
,785
,002
Dyslipidemia
,877
,959
2,404
,360
Current smoking
-1,567
1,176
,209
,183
Diabetes
-4,096
1,594
,017
,010
Hypertension
-,875
,979
,417
,372
Heart disease
1,247
1,099
3,479
,257
C3
-0,052
,019
,949
,007
AP50
-0,102
,036
,903
,005
b) Second cohort
c) Third cohort (VIVA trial)
C3
Body mass index (kg(m2)
Current smoking
Diabetes mellitus
Diastolic blood pressure
(mmHg)
Peripheral arterial disease
(ABI<0.9)
Constant
B
-,013
,017
-1,141
,974
S.E.
,006
,065
,449
,977
OR
,987
1,018
,320
2,647
Pvalue
,020
,787
,011
,319
,016
,017
1,016
,370
-,174
,525
,840
,740
4,619 479,459
,181
6,173
Table IV online. Linear regression analysis with AAA diameter as dependent variable
a) First cohort
Coefficients(a)
Unstandardized
Coefficients
Standardized
Coefficients
Model
T
Sig.
2,402
,019
B
Std.
Error
(Constant)
33,971
14,141
Sex
4,482
5,693
,083
,787
,434
Age
,226
,165
,147
1,374
,173
Dyslipidemia
-3,303
2,280
-,165
-1,449
,152
Current smoking
5,251
2,242
,257
2,343
,022
Diabetes
-2,526
3,044
-,091
-,830
,409
Hypertension
,873
2,249
,042
,388
,699
Heart disease
-,138
2,402
-,006
-,057
,954
C3
-8,02E02
,025
-,330
-3,247
,002
Beta
b) Second cohort
Coefficients(a)
Unstandardized
Coefficients
Standardized
Coefficients
Model
T
Sig.
6,204
,000
B
Std.
Error
(Constant)
99,069
15,968
Sex
-2,562
7,550
-,045
-,339
,736
Age
-,408
,202
-,301
-2,024
,049
Dyslipidemia
-5,107
3,499
-,195
-1,459
,151
Current smoking
-2,918
4,295
-,098
-,679
,500
Diabetes
-5,968
4,717
-,177
-1,265
,212
Hypertension
1,253
3,775
,047
,332
,742
Heart disease
1,730
4,345
,057
,398
,692
AP50
-,133
,060
-,327
-2,230
,031
Beta
AAA thrombus
A
Negative
B
Healthy wall
AAA wall
C
D
Supplemental figure I. Immunohistochemistry of C9 in thrombus (A), wall (C) and Healthy wall (D). NegaBve control (non-­‐specific IgG) (B). MagnificaBon 20x. C3
A
SMC
B
Supplemental figure II. Immunohistochemistry of C3 (A) and immunofluorescence of alpha-­‐acBn (B) in AAA wall. MagnificaBon 40x. C3
A
CD15
B
CD68
C
Supplemental figure III. Immunohistochemistry of C3 (A), CD15 (B) and CD68 (C) in serial secBons of AAA wall. MagnificaBon 10x. Material and methods
AAA patients
Spanish patients
In a first cohort, serum from 62 male patients with an asymptomatic infrarenal AAA
was collected during clinical examination (aortic size = 3-5 cm, small AAA).
Additionally, serum from 28 male patients with an asymptomatic infrarenal AAA was
collected before surgical repair (aortic size > 5 cm, large AAA). Twenty-eight healthy
male controls with non-dilated infrarenal aortas (aortic size < 3 cm, confirmed with
abdominal ultrasound) and no risk factors were obtained from a screening program
undertaken in our area of care. All these samples were obtained from Galdakao
Ursansolo Hospital (Bilbao, Spain). In a second cohort, plasma samples were
obtained from the biobank of IIS-FJD (Madrid, Spain) including 26 small AAA
patients and 39 large AAA patients. Hypertension was defined as systolic blood
pressure (sBP) >140 mmHg and/or diastolic pressure (dBP) ≥90 mmHg measured
during the examination, after the participant had been sitting for at least 30 minutes,
or the participant was already taking hypotensive medication. A patient was
considered diabetic if he was under treatment (supervised diet, hypoglycaemic oral
medication, insulin) or we found basal glycaemia >120 mg/dL and/or HbA1c
>=6.5%. Hypercholesterolemia was defined as total basal cholesterol levels ≥200
mg/dl, LDL levels ≥100 mg/dl or the patients were receiving specific medication or a
supervised diet. Cardiac disease included coronary heart disease, valvular disease,
cardiomyopathy and arrhythmia. Clinical characteristics are summarized in Table 1.
The studies were approved by Spanish center’s Research and Ethics Committees, and
informed consent from the patients and the controls for their inclusion in the study
was obtained.
Danish patients
Blood cells were obtained from 186 patients from the randomised population based
Viborg Vascular (VIVA) screening trial screening 65-74 year old men for AAA,
peripheral arterial disease and unrecognised hipertension (1). Informed consent was
obtained from all subjects before participation, and the study was approved by the
Local Ethics Committee of the Viborg Hospital, Denmark, and performed in
accordance with the Helsinki Declaration. Cases were selected according to initial
size and growth rate. Clinical characteristics of the patients are included in table 1
online.
AAA tissue and tissue-conditioned media
Sixteen AAA thrombus and wall samples were collected from patients enrolled in the
RESAA protocol (2) undergoing surgery (three for MS analysis, ten for ELISA,
western-blot and immunohistochemistry and six for homogenization). One part was
included in paraffin for immunohistochemistry and the rest was dissected into
thrombus and wall (media and adventitia) for incubation in a RPMI protein-free
medium. All patients gave their informed written consent and the protocol was
approved by a French ethics committee (CPB, Cochin Hospital). Twelve control
aortas (six for immunohistochemistry and six for homogenization) were sampled from
dead organ donors with the authorization of the French Biomedicine Agency (PFS 09007). These control aortic samples were macroscopically normal, devoid of early
atheromatous lesions. Different layers of AAA thrombus and wall, as well as healthy
walls, were cut into small pieces (5 mm2) and separately incubated in RPMI 1640
medium containing antibiotics and an antimycotic (Gibco) for 24 hours at 37°C (6
ml/g of wet tissue). The conditioned medium (supernatant containing proteins
released by the tissue sample) was obtained after centrifugation as 3,000 g for 10
minutes at 20°C. In some cases, native C3 [purified as previously described in
Alcorlo M et al, (3)] was incubated for 90 min at 37ºC with 1 µl of thrombusconditioned media and then subjected to western-blot. In addition, tissues were snapfrozen in N2 liquid and homogenates (0.2 g) were divided and resuspended for mRNA
and protein analysis.
Bidimensional nanoLC-MS/MS analysis
Proteins from AAA–tissue conditioned media obtained from 3 patients were
precipitated using 2D clean-up kit (GE Healthcare) and resuspended in
Triethylammonium bicarbonate (TEAB) buffer for protein concentration measure by
Bradford assay (Biorad). A total of 50 µg of protein was reduced with 5 mM Tris (2carboxiethyl) phosphine (TCEP) for 1 hour at 60ºC and alkylated using 10 mM smethylmethenethiosulponate (MMTS) at room temperature during 10 minutes. Later,
proteins were digested with trypsin, at 37oC for 5h, at a ratio of 1:50 trypsin to
protein. Digested samples were subjected to nano-liquid chromatography coupled to
MS for protein identification. Peptides were injected onto a strong cation exchange
(SCX) microprecolumn (500 µm I.D. and 15mm BioX-SCX TM, LC Packings,
Amsterdam, The Netherlands) with a flow rate of 30µL/min as a first dimension
separation. Peptides were eluted from the column as fractions by injecting three salt
steps of increasing concentration of ammonium acetate (10, 100 and 2000 mM). Each
of the three fractions together with the nonretained fraction was on line injected onto
a C-18 reversed phase (RP) nano-column (100 mm I.D. and 12 cm, Mediterranea sea,
Teknokroma) and analyzed in a continuous acetonitrile gradient consisting of 0-50%
B in 90 min, 50-90% B in 1 min (B=95% acetonitrile, 0.5% acetic acid). A flow rate
of 300 nL/min was used to elute peptides from the RP nano-column to an emitter
nanospray needle for real time ionization and peptide fragmentation on an LTQOrbitrap XL mass spectrometer (Thermo Fisher). An enhanced FT-resolution
spectrum (resolution = 30000) followed by the MS/MS spectra from most intense
three parent ions (dissociated using CID activation) was analyzed along the
chromatographic run (130 min). Dynamic exclusion was set at 1 min.
Database Searching
Tandem mass spectra were extracted by Proteome Discoverer v1.0 software (Thermo
Fisher). Charge state deconvolution and deisotoping were not performed. For protein
identification, fragmentation spectra were searched against a curated subset of a
human database (human_ref.fasta; 2003, April; 39414 entries) using Sequest (Thermo
Fisher Scientific version 1.0.43.2) and X-Tandem (The GPM, thegpm.org; version
2007.01.01.1) engines. Sequest and X-Tandem were searched allowing two missed
trypsin cleavages, and a tolerance of 15 ppm or 0.8 Da was set for full MS or MS/MS
spectra searches, respectively. Methane thiosulfate alkylation of cysteine residues and
oxidation of methionine were allowed as variable modifications. Finally, Scaffold
v.3.00.02 software (Proteome Software Inc) was used to validate MS/MS based
peptide and protein identifications.
Bioinformatics
Pathway analysis were created using Ingenuity System software (Ingenuity System
Software, Inc.). Enriched canonical pathways were calculated as the ratio between the
number of genes for one pathway found in the experiment and the total number of
genes destinated to that pathway. All the ratios shown are associated to p-values
provided by Fisher’s exact test. These analyses were derived from all protein
identifications obtained from the MS analyses. Predictions of protein secretions were
made using a software package, publically available, hosted at the Technical
University of Denmark, as described (4).
Quantification of C3 and C3a
Soluble concentrations of C3 in human plasma samples were automatically measured
using VITROS chemistry products C3 reagents in the VITROS 5,1 FS and VITROS
5600 Integrated System analyzers, following the manufacturer´s instructions (OrthoClinical Diagnostics, Johnson & Johnson). Soluble concentrations of C3 in serum
samples from first cohort or in plasma samples from third cohort were assayed
automatically by timed nephelometry using a BNII Nephelometer (Siemens9). Both
methods were standardized against the international reference preparation CRM 470
(RPPHS). C3 and C3a in conditioned media was measured with commercial kits
(EC2101 Assaypro and 550499 BD, respectively) following the manufacturer’s
instructions.
AP50 assay
To test the hemolitic capacity of the complement system, red blood cells (RBCs) from
healthy rabbits were used together with human sera as described (5). Briefly, washed
RBCs were resuspended to 1% (v/v) in AP-CFTD buffer (5 mM sodium barbitone pH
7.4, 150 mM NaCl, 7 mM MgCl2, 10 mM EGTA). 40 ul of serum from control or
AAA patients, 50 ul of EDTA 0.2 M and 100 ul of VBS (NaCl 0.14 M, Sodium 5,5diethylbarbiturate 1.45 mM, acid 5,5-diethylbarbituric 2.5 mM) were added to 200 ul
of rabbit RBCs 1% (v/v) and incubated at 37°C for 30 minutes. To calculate lysis, 1.8
ml of VBS-EDTA (VBS= and 0.2 M EDTA) were added, cells were pelleted by
centrifugation at 2.500 rpm for 10 min and hemoglobin release was measured by
absorbance at at 412 nm. Control incubations included 0% lysis (buffer only) and
100% lysis (1.8 ml of H2O instead of VBS= 0.2 M EDTA). Percentage lysis
100*(A412 test sample-A412 0% control)/(A412 100% control-A412 0% control).
DNA isolation and genetic study
Genomic DNA was extracted from peripheral blood using EZ1 DNA Blood 350 µl
Kit in an EZ1 Advance Robot (Qiagen) following standard procedures. DNA samples
were genotyped for six single nucleotide polymorphisms (SNPs) (CFH Ile62Val, CFH
c.1696+2019G>A, CFHR1 Glu175Gln, CFB Leu9His, CFB Arg32Gln/Trp) (6). The
genotyping was performed using multiplex PCR and minisequencing methodology
(ABI Snapshot; Applied Biosystems). Minisequecing reations were run in an
automated sequencer (model 3730; ABI), and the fragments were analyzed with the
appropriate software (GeneMapper Software 4.0; ABI).
Immunohistochemistry
AAA and control aorta samples were fixed in 3.7% paraformaldehyde and embedded
in paraffin. Immunohistochemistry was performed using antiC3 (purified as described
in 7) and anti-C9 (mAb B7, a generous gift of Prof. Paul Morgan, Cardiff University)
as primary antibodies. Negative controls using the corresponding IgG were included
for checking non-specific staining. The secondary antibody and ABComplex/HRP
were added and sections were stained with 3,30-diaminobenzidine and mounted in
DPX. For colocalization of C3 with C9, CD15 (clone Carb-3, DAKO) and CD68
(clone PG-M1, DAKO), immunohistochemistry in serial sections was performed. For
colocalization of C3 with vascular smooth muscle cells (alpha-actin, clone 1A4
DAKO), immunohistochemistry followed by immunofluorescence was performed.
Western blot
Equal amounts of proteins from tissue or conditioned medium (30 µg or 5 µL
previously normalized to tissue weight: 1 g/6 mL, respectively) were loaded onto
12.5% polyacrylamide gels, electrophoresed and transferred to nitrocellulose
membranes. Then they were blocked with 7% milk powder in TBS-T for 1 hour and
incubated overnight at 4ºC with antiC3 (7). Then the membranes were washed with
TBS-T and incubated with anti-rabbit antibody (1:5000) for 1 hour at RT. After 4
washes, the signal was detected using the ECL chemiluminiscence kit (GE
Healthcare).
Real time PCR
Total RNA was isolated from cells using TRIzol reagent (Invitrogen). One microgram
of RNA was used to perform the reverse transcription with the high capacity cDNA
archive kit (Applied Biosystems). Real-time PCR reactions were performed on an
ABI Prism 7500 sequence detection PCR system (Applied Biosystems) according to
the manufacturer’s protocol, using the DDCt method. Human mRNA levels for C3
and 18S were done by amplification of cDNA using SYBRw Premix Ex TaqTM
(Takara Biotechnology). The primer sequences are: Forward C3 primer:
AAGCGCATTCCGATTGAGGA,
Reverse
C3
primer:
AAGACTTCCCCACCAGGTCT. The mRNA levels of C3 were normalized to the
18S mRNA content.
Cell isolation, chemotaxis assay and measurement of NADPH-dependent ROS
production
Neutrophils were isolated from venous blood of healthy volunteers (with informed
consent), sampled on EDTA. Red blood cells were aggregated by addition of 2%
dextran for 20 minutes at 20°C and the upper phase containing leukocytes was
centrifuged on Ficoll (20 minutes at 600 g, 20°C) (PAA Laboratories GmbH). The
pellet containing neutrophils was submitted to a hypo-osmotic shock to eliminate
residual erythrocytes.
Transwell migration assays were performed using 96-well disposable chemotaxis
chambers with a 8 µm polycarbonate filter (ChemoTX, Neuroprobe). Briefly, 29 ul of
3 different luminal thrombus conditioned media were added to the lower
compartment of each well. Luminal thrombus were preincubated for 30 min at 37ºC
in a humidified atmosphere (5% CO2), in the presence or in the absence of antiC3 or
IgG (0.4 ug/ul) in 30 µL of RPMI. Polymorphonuclear cells (200.000) were added to
the upper compartment. The chamber was then incubated at 37 °C in a humidified
atmosphere (5% CO2) for 2 h. A standard curve, consisting of a 1:2 dilution cascade
of polymorphonuclear cells (top standard, 200.000 cells in 29 µL), was constructed.
After incubation, the framed filter was carefully removed and the number of cells that
had migrated was determined reading fluorescence at 485ex/530em by comparison
with the standard curve. Each experiment was performed in triplicate.
Lucigenin-enhanced chemiluminescence assay was used to determine the NADPHdependent ROS production in fresh neutrophils as described (8). Briefly, 5 µL of 3
different luminal thrombus conditioned media were incubated for 2 min with 500,000
polymorphonuclear cells. Luminal thrombus were preincubated for 30 min at 37ºC in
a humidified atmosphere (5% CO2), in the presence or in the absence of antiC3 or
IgG (0,4 ug/ul) in 30 µL of RPMI. The reaction mixture comprised 50 mM phosphate
buffer containing 1mM EGTA, pH 7.0, 5 µM lucigenin and 0.1 mM NADPH. The
chemiluminescence, which was measured for 5 minutes after the addition of NADPH,
was recorded in a luminometer Sirius (Berthold Detection System). No activity could
be measured in the absence of NADPH. The ROS production was determined from
the ratio of relative light units.
Statistics
Normality of data was checked by probability plots. Normally distributed C3
concentrations and activity are expressed as mean±SEM. P < 0.05 was considered to
be statistically significant. Difference among the groups in the first cohort (control,
small aaa and large aaa) was analyzed by one-way ANOVA test followed by post hoc
Tukey Kramer test for multiple comparisons. Differences among the groups of the
second cohort and third cohort (small AAA vs large AAA) were analyzed by t-test.
Pearson correlation was used to determine correlations between two variables.
Logistic or linear regression analysis adjusted by risk factors was conducted with
AAA stage (small/large AAA) and aortic size as dependent variables, respectively.
The Wilcoxon paired test was used to analyze differences in C3 and C3a levels
between thrombus and wall supernatants of the same samples, while non-paired tests
were used for pathological wall vs healthy wall supernatants comparisons. All the
statistical analyses were performed by using SPSS 11.0 statistical package.
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