R E S E A R C H Open Access
Histone acetylation and histone
acetyltransferases show significant
alterations in human abdominal aortic
, Fadwa Tanios
, Christian Reeps
, Jian Zhang
, Kristina Schwamborn
, Hans-Henning Eckstein
and Jaroslav Pelisek
Background: Epigenetic modifications may play a relevant role in the pathogenesis of human abdominal aortic
aneurysm (AAA). The aim of the study was therefore to investigate histone acetylation and expression of
corresponding lysine [K] histone acetyltransferases (KATs) in AAA.
Results: A comparative study of AAA tissue samples (n= 37, open surgical intervention) and healthy aortae (n=12,
trauma surgery) was performed using quantitative PCR, immunohistochemistry (IHC), and Western blot. Expression of
the KAT families GNAT (KAT2A, KAT2B), p300/CBP (KAT3A, KAT3B), and MYST (KAT5, KAT6A, KAT6B, KAT7, KAT8) was
significantly higher in AAA than in controls (P≤0.019). Highest expression was observed for KAT2B, KAT3A, KAT3B, and
KAT6B (P≤0.007). Expression of KAT2B significantly correlated with KAT3A, KAT3B, and KAT6B (r= 0.705, 0.564, and
0.528, respectively, P< 0.001), and KAT6B with KAT3A, KAT3B, and KAT6A (r= 0.407, 0.500, and 0.531, respectively,
P< 0.05). Localization of highly expressed KAT2B, KAT3B, and KAT6B was further characterized by immunostaining.
Significant correlations were observed between KAT2B with endothelial cells (ECs) (r= 0.486, P< 0.01), KAT3B with
T cells and macrophages, (r= 0.421 and r= 0.351, respectively, P< 0.05), KAT6A with intramural ECs (r= 0.541,
P< 0.001) and with a contractile phenotype of smooth muscle cells (SMCs) (r= 0.425, P<0.01),and KAT6B withT
cells (r=0.553,P< 0.001). Furthermore, KAT2B was associated with AAA diameter (r=0.382,P< 0.05), and KAT3B,
KAT6A, and KAT6B correlated negatively with blood urea nitrogen (r=−0.403, −0.408, −0.478, P<0.05).Inaddtion,
acetylation of the histone substrates H3K9, H3K18 and H3K14 was increased in AAA compared to control aortae.
Conclusions: Our results demonstrate that aberrant epigenetic modifications such as changes in the expression of
KATs and acetylation of corresponding histones are present in AAA. These findings may provide new insight in the
pathomechanism of AAA.
Keywords: AAA, Epigenetics, Histone acetylation, Acetyltransferases, KAT/HAT
* Correspondence: email@example.com;firstname.lastname@example.org
Institute of Experimental Biomedicine, University Hospital, University of
Würzburg, Würzburg, Germany
Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar
der Technische Universität München, Ismaninger Str. 22, 81675 Munich,
Full list of author information is available at the end of the article
© 2016 Han et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Han et al. Clinical Epigenetics (2016) 8:3
In the past decades, abdominal aortic aneurysm (AAA)
has been increasingly recognized as a leading cause of
sudden death in men older than 65 years . Despite
considerable advances in surgical treatment, the only re-
liable diagnostic option so far is the measurement of the
diameter of AAA. Exceeding 5.5 cm, patients generally
undergo surgical or endovascular repair [2, 3]. Thus, a
better understanding of the pathophysiologic processes
leading to AAA wall destabilization until rupture remains
an important issue to identify patients at increased risk.
Pathophysiological changes in gene expression of vari-
ous factors within the vessel wall are the reason of many
cardiovascular diseases. Among others, epigenetics have
been recognized as a powerful tool to activate or silence
gene transcription by changes in the chromatin structure
without alterations of the DNA sequence . Many fac-
tors are involved in the establishment of epigenetic
traits, including DNA methylation and multitudinous
modifications of histones such as methylation, acetyl-
ation, or phosphorylation [4, 5]. In particular, targeted
histone alterations determine the epigenetic state of the
genome. One of the most important histone modifica-
tions is attachment or removal of an acetyl group, lead-
ing either to gene activation or repression . The
histone acetylation process is regulated by the balanced
activities of two key enzyme families of transferases,
namely lysine [K] histone acetyltransferases (KATs) ,
and histone deacetylases (HDACs) . The function of
KATs is to add an acetyl group to the lysine residue,
resulting in chromatin opening and gene activation .
In the context of histone acetylation, four families of
KATs have been described so far (GNAT, p300/CBP,
MYST, and TF-related family), comprising in total 11
acetyltransferases [9, 10].
Although the role of epigenetics as a potential mech-
anism to control gene activity has been proposed in
cardiovascular diseases , few and inconsistent studies
have investigated such epigenetic changes to date. For
example, genomic DNA isolated from human athero-
sclerotic lesions was found to be hypomethylated .
Recently, our group demonstrated significant differences
in histone and DNA methylation and the expression of
corresponding methyltransferases at different stages of
atherosclerosis in carotid arteries . Krishna et al. have
hypothesized that epigenetic mechanisms may also play a
role in the pathogenesis of AAA . However, epigenetics
in AAA have not been addressed experimentally.
The aim of the present study was therefore to analyze
the expression profiles of known KATs in AAA and
healthy aortic tissue. Furthermore, we examined their
main histone substrates in individual cell types within
AAA. Our results provide interesting data about a
possible role of specific KATs and histone acetylation
in the epigenetic regulation of AAA development and
KAT mRNA expression levels and their correlations in AAA
We first determined the expression of KATs in AAA at
the messenger RNA (mRNA) level using quantitative
real-time reverse transcriptase polymerase chain reaction
(RT-PCR) and compared our results with that of control
aortic tissue samples. The mRNA expression of histone
KAT6A,KAT6B,KAT7,andKAT8 belonging to the GNAT,
p300/CBP, and MYST family of KATs was significantly
higher in AAA than in healthy control tissue (Fig. 1). In
contrast, KAT8, a member of the TF-related family of
KATs, was detected neither in AAA nor in healthy
aortic tissue. Among the above analyzed acetyltransferases,
KAT2A,KAT3A,KAT5,KAT7,andKAT8 transcripts were
not detected in control aorta. In contrast, KAT4,belonging
to the family of TF-related KATs, was significantly de-
creased in AAA specimens compared to controls (Fig. 1).
For a better overview of the expression levels of the KATs
analyzed in our study, mRNA expression levels normalized
to GAPDH are depicted in Additional file 1: Table S3. The
highest expression in AAA tissue was observed for KAT2B
(2.5-fold higher in AAA compared to control aorta),
KAT3A (not expressed in healthy aorta), KAT3B (3.9-fold
higher in AAA), and KAT6B (2.8-fold higher in AAA).
KATs are frequently activated in clusters [15, 16]. We
therefore in addition analyzed the inter-relationships of
the expression of the individual KATs in human AAA
tissue samples (Table 1). KAT2B correlated significantly
with KAT3A,KAT3B,andKAT6B (r= 0.705, 0.564, and
0.528, P< 0.001 and <0.01, respectively), KAT3A correlated
with KAT6B (r=0.407, P<0.05), KAT3B correlated with
KAT6B and KAT8 (r= 0.500 and 0.342, P< 0.01 and <0.05,
respectively), KAT5 correlated with KAT7 and KAT8
(r= 0.357 and 0.443, P< 0.05 and <0.01, respectively),
KAT6A correlated with KAT6B (r= 0.532, P< 0.01), and
KAT4 correlated with KAT8 (r=0.648,P< 0.01), suggesting
corporate activity especially of KAT2B,KAT3A,KAT3B,
and KAT6B. Selected examples of the correlation analysis
with a statistically significant outcome are depicted as dot
blots in Additional file 2: Figure S1.
Protein expression and cellular localization of KATs in
inflammatory cells in AAA
In order to further evaluate the protein expression of se-
lected KATs that showed highest expression on mRNA
level, Western blot analyses were performed. Corrobor-
ating our results on the mRNA level, protein expression
of KAT2B, KAT3B, and KAT6B was significantly higher
in AAA tissue compared to the protein expression in
healthy control aortae (4.1-fold, 2.8-fold, and 2.2-fold,
Han et al. Clinical Epigenetics (2016) 8:3 Page 2 of 13
Fig. 1 Expression analysis of lysine [K] histone acetyltransferases (KATs) in AAA and healthy aorta at mRNA level. Quantification was performed by
SYBR green-based RT-PCR. Relative expression indicates expression of individual KATs related to the expression of GAPDH set as 1 (100 % expression).
AAA specimens of abdominal aortic aneurysm (n=37),ctrl control healthy aorta (n=12).*P<0.05,#P<0.001
Table 1 Inter-related correlation between KATs in AAA
rKAT2A KAT2B KAT3A KAT3B KAT5 KAT6A KAT6B KAT7 KAT8 KAT4
KAT2A –n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c.
KAT2B –0.705*** 0.564** n.c. n.c. 0.528** n.c. n.c. n.c.
KAT3A –n.c. n.c. n.c. 0.407* n.c. n.c. n.c.
KAT3B –n.c. n.c. 0.500** n.c. 0.342* n.c.
KAT5 –n.c. n.c. 0.357* 0.443** n.c.
KAT6A –0.531** n.c. n.c. n.c.
KAT6B –n.c. n.c. n.c.
KAT7 –n.c. n.c.
Significant differences between individual KATs: *P<0.05, **P<0.01, ***P<0.001; n.c. no correlation
Han et al. Clinical Epigenetics (2016) 8:3 Page 3 of 13
P< 0.001, <0.001, and 0.033, respectively; see Fig. 2).
As we could not detect KAT3A mRNA expression in
control tissue and no appropriate antibodies are com-
mercially available to detect KAT3A in formalin-fixed
paraffin-embedded (FFPE) tissue samples, we omitted
to analyze KAT3A at the protein level.
The most common cells in AAA are smooth muscle
cells (SMCs) and inflammatory cells, such as macro-
phages and lymphocytes . We analyzed the cellular
localization of KAT2B, KAT3B, and KAT6B within the
AAA wall by immunohistochemistry (IHC) in consecu-
tively stained sections. KAT2B expression was found to
predominantly colocalize to CD45
macrophages, and CD3
T cells (Fig. 3a). In addition,
staining of intramural CD31
endothelial cells in
neovessels was also found to colocalize with the expres-
sion of KAT2B. In contrast, an only marginal staining of
KAT2B was detected in smooth muscle cells (Fig. 3a).
Staining patterns of KAT3B similarly showed a strong
colocalization with leucocytes and T cells but not with
macrophages, medial SMCs, or neovessels (Fig. 3b).
Staining for KAT6B predominantly localized to leuko-
cytes, macrophages, and T cells, while luminal endothe-
lial cells (ECs) and neovessels as well as SMCs did not
show co-staining with this histone acetyltransferase
(Fig. 3c). In contrast to the AAA tissue samples, KAT2B,
KAT3B, and KAT6B could not be detected in healthy
aortic tissue (data not shown). Summarizing the IHC
Fig. 2 Expression analysis of KAT2B, KAT3B, and KAT6B in AAA and healthy aorta at protein level. a,c,eWestern blot analysis. b,d,fQuantification of
the band intensities relative to the expression of GAPDH. Ratio (% of Ctrl) indicates relative expression to Ctrl set as 100 %). C and ctrl, control healthy
aorta (n=8),A/AAA specimens of abdominal aortic aneurysm (n=24).*P<0.05,#P<0.001
Han et al. Clinical Epigenetics (2016) 8:3 Page 4 of 13
results, our data demonstrate that KAT2B, KAT3B, and
KAT6B expression is predominantly found in inflamma-
tory cells in AAA.
Expression of main histone substrates and their cellular
In order to determine the acetylation of the main histone
substrates of KAT2B, KAT3B, and KAT6B (Additional
file 1: Table S2) , expression of H3K9ac, H3K14ac,
and H3K18ac was determined in AAA tissue samples
compared with healthy aortic tissue specimens by
Western blotting. As no appropriate antibody against
H3K36ac was available, analysis of this histone substrate
had to be omitted. Acetylation of H3K9 (H3K9ac) and
H3K18 (H3K18ac) was 2.8-fold and 1.8-fold higher in
AAA than healthy aortic tissue (P= 0.004 and 0.019, re-
spectively, Fig. 4a–d). Expression levels of acetylated
H3K14 was 1.9-fold higher in AAA than in control aortae;
however, without reaching statistical significance due
to the heterogeneity of the individual values (P=0.338,
Fig. 4e, f).
We further assessed the cellular localization of H3K9ac,
H3K14ac, and H3K36ac by IHC in consecutive sections.
H3K9ac staining was found to colocalize with CD45
Fig. 3 Expression analysis of KAT2B (a), KAT3B (b), and KAT6B (c) in AAA using IHC for cellular localization. Overview image (left panel)ofthewhole
AAA tissue sample with areas selected for cellular localization of KAT expression (haemalum-eosin staining). The magnified images depict consecutive
staining of cells as revealed by staining for indicated markers within the AAA wall and indicated KATs. Scale bar, overview image 1000 μm
Han et al. Clinical Epigenetics (2016) 8:3 Page 5 of 13
macrophages, and CD3
were positive for acetylated H3K9 (H3K9ac). In con-
trast, only weak or negative staining of H3K9ac was
detectable in SMCs (Fig. 5a). Similar to H3K9ac, acety-
lated H3K14 (H3K14ac) was mainly colocalized to CD45
macrophages, and CD34
neovessels. Again, staining in SMCs was very weak and
not all cells were positive (Fig. 5b). Acetylation of H3K18
(H3K18ac) was most intensive in CD45
macrophages (Fig. 5c). In contrast, only some
neovessels and SMCs were weakly positive
for H3K18ac (Fig. 5c).
Correlation analysis of KATs with cell markers
Additional experiments were performed to further con-
firm the localization of KATs in the individual cells
within AAA observed by IHC. Because we were not able
to extract individual cells from AAA tissue samples, we
correlated mRNA expression of the selected KATs with
markers indicative of different cell types. CD45 was se-
lected for leukocytes, CD3 for T cells, and MSR1 for
Fig. 4 Analysis of acetylated H3K9 (H3K9ac), H3K14 (H3K14ac), and H3K18 (H3K18ac) in AAA and healthy aorta at protein level. a,c,eWestern
blot analysis. b,d,fQuantification of the band intensities relative to the expression of GAPDH. Ratio (% of Ctrl) indicates relative expression to Ctrl
set as 100 %). C and ctrl control healthy aorta (n= 8), A and AAA specimens of abdominal aortic aneurysm (n= 24). *P< 0.05, #P< 0.001
Han et al. Clinical Epigenetics (2016) 8:3 Page 6 of 13
macrophages. As different phenotypes of SMCs co-exist in
AAA, namely synthetic and contractile SMCs, we selected
markers for both cell types, with smoothelin (SMTN) and
SM myosin heavy chain (SM-MHC, MYH11) representing
the contractile phenotype and SMemb/non-muscle MHCB
(MYH10) and collagen I representing the synthetic pheno-
type . Furthermore, we also analyzed the expression of
vascular cell adhesion molecule (VCAM)-1, which plays
an important role in the development of AAA . A sig-
nificant positive correlation was found for KAT2B with
MSR1, indicative of macrophages, and VCAM-1 (Table 2,
Additional file 3: Figure S2A). In addition, significant
correlations were observed of KAT3B with CD45,
MSR1,andCD3, indicative of leukocytes, macro-
phages, and T cells, respectively (Table 2, Additional
file 3: Figure S2B). Positive correlations were also
observed for KAT6B with leukocyte and T cell
markers (Table 2, Additional file 3: Figure S2C),
further corroborating the association of KATs with
inflammatory cell infiltrates. Among the expression
of other KATs, KAT3B and KAT5 correlated with
CD45 and KAT6A with CD3,MYH10,andVCAM-1,
and a negative correlation was found for KAT7 with
MYH11 (Table 2).
Fig. 5 Analysis of acetylated H3K9 (a), H3K14 (b), and H3K18 (c) in AAA using IHC for cellular localization. Overview image (left panel) of the
whole AAA tissue sample with areas selected for cellular localization of KAT expression (hemalum-eosin staining). The magnified images depict
consecutive staining of individual cell types, as revealed by staining for indicated markers, within the AAA wall and indicated corresponding
substrates. Scale bar, overview image 1000 μm, detailed images 100 μm
Han et al. Clinical Epigenetics (2016) 8:3 Page 7 of 13
Correlation analysis of KATs with blood parameter
We finally evaluated a possible correlation of the expres-
sion of KATs with blood parameters available for the AAA
patients of our study. A summary of these correlations is
provided in Additional file 1: Table S4. Interestingly, AAA
diameter positively correlated with the expression of
KAT2B (r=0.353, P< 0.05). Furthermore, three different
KATs, namely KAT3A, KAT6A, and KAT6B were nega-
tively associated with the concentration of blood urea ni-
trogen (r=−0.403, −0.408, and −0.478, P<0.05, P<0.05,
and P< 0.01, respectively; Additional file 4: Figure S3).
In the current study, we analyzed histone acetylation
and the expression of corresponding histone acetyltrans-
ferases in human AAA compared to healthy aortic tissue.
Our results show that members of all three families of
KATs, GNAT, p300/CBP, and MYST were significantly
overexpressed in the AAA wall compared to healthy aorta,
particularly KAT2B, KAT3A, KAT3B, and KAT6B. These
acetyltransferases were predominantly found in colocaliza-
tion with macrophages and Tcells, and their main histone
substrates were H3K9, H3K14, and H3K18. Interestingly,
some histone acetyltransferases such as KAT2B correlated
also with AAA diameter and KAT3B, KAT6A, and
KAT6B were associated with blood urea nitrogen. These
results demonstrate for the first time that aberrant histone
acetylation occurs in AAA.
Expression analyses showed that mRNA levels of
members of the GNAT, CBP, and MYST family of KATs
were significantly increased in the vessel wall of AAA
patients compared with healthy aortae. In general, an in-
creased expression of KATs entails an enhanced histone
acetylation, and a plethora of previous studies in other
tissues have provided evidence that hyperacetylated his-
tone lysine residues are related to transcriptionally active
chromatin, facilitating accessibility of the DNA template
to the transcriptional machinery [19, 20]. For instance,
KAT2B has been described to activate the MMP-9 pro-
moter either independently or in a synergistic manner
. MMP-9 has been established as one of the key me-
diators of the degradation of extracellular matrix pro-
teins in the arterial wall in AAA. Another example is
KAT3A, which acetylates not only histones but also
transcription factors such as p53 and thereby facilitates
their binding to DNA . p53 is furthermore closely
associated to SMC apoptosis. Accelerated apoptosis and
necrosis of SMCs, which are the main producers of
extracellular matrix proteins, lead to a weakening of the
aortic wall stability  and can entail the expression of
inflammatory cytokines and proteolytic enzymes in the
vessel wall, contributing to the accumulation of inflam-
matory cells within the AAA wall and consequently to
the progression of AAA.
In contrast to KATs of the GNAT, CBP, and MYST
family, the expression of members of the TF-related
family was either significantly lower in AAA (KAT4) or
were detected in neither AAA nor control tissue (KAT12).
KAT4 is a component of the TFIID complex, which is a
general transcription factor allowing RNA Pol II to bind
to the promoters of protein-coding genes in living cells to
initiate mRNA synthesis [24, 25]. Furthermore, the tran-
scription factor TFIIIC has a barrier function mediated by
the RNA Pol III and genome organization , leading to
cell growth arrest and impairment of proper cellular
function. The expression of KAT4 may thus be important
for the maintenance of normal cellular functions in
the healthy aortic tissue, which is impaired in the
Interestingly, a significant inter-relationship between
individual KATs in AAA determined by correlation ana-
lyses was observed in many cases, suggesting that the
expression of some of these KATs is regulated in clus-
ters. For instance, KAT6A correlated with KAT6B. This
may be explained by the fact that these two KATs are
highly homologous and share the same lysine substrate
Table 2 Correlation between KAT expression and expression of markers of cells in AAA
rCD45 CD3 MSR1 SMTN MYH11 MYH10 Coll I VCAM-1
KAT2A n.c. n.c. n.c. n.c. n.c. n.c. –0.528*** n.c.
KAT2B n.c. n.c. 0.388* n.c. n.c. n.c. n.c. 0.486**
KAT3A 0.396* n.c. n.c. n.c. n.c. n.c. n.c. n.c.
KAT3B 0.421* 0.361* 0.351* n.c. n.c. n.c. n.c. n.c.
KAT5 0.378* n.c. n.c. n.c. n.c. n.c. n.c. n.c.
KAT6A n.c. 0.389* n.c. n.c. n.c. 0.425** n.c. 0.541***
KAT6B 0.609*** 0.553*** n.c. n.c. n.c. n.c. n.c. n.c.
KAT7 n.c. n.c. n.c. n.c. –0.377* n.c. n.c. n.c.
KAT8 n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c.
KAT4 n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c.
*P<0.05, **P<0.01, ***P<0.001. n.c. no correlation detected
Han et al. Clinical Epigenetics (2016) 8:3 Page 8 of 13
H3K14. In addition, KAT6A and KAT6B are promiscu-
ous transcriptional co-activators involved in the tran-
scriptional activation mediated by Runx1 and Runx2
that interact with these KATs . Furthermore, a sig-
nificant positive correlation was found between mRNA
levels of KAT5 and KAT7 and of KAT5 and KAT8 in
AAAs. These three KATs belong to the same family of
histone acetyltransferases called MYST, show high hom-
ology, and share the same substrate histone H4 [9, 10].
However, little is known about their role in chromatin
modification. The yeast NuA4 histone acetyltransferase
complex, a homolog of human KAT5, is known to be
involved in transcription, cell cycle control, and DNA re-
pair [28, 29]. In this regard, the MYST family of KATs
might also be involved in these processes.
A significant over-expression of KAT2B was found in
the AAA wall. Recently, Bastiaansen et al. demonstrated
that KAT2B acts as master switch in inflammatory pro-
cesses required for effective arteriogenesis . In our
work, we found a significant colocalization of KAT2B
with macrophages and endothelial cells of neovessels
within the intima in AAA, which may suggest its associ-
ation with inflammation and neovascularization in AAA
development . Furthermore, two important cell cycle
regulators, E2F1 and p53, can interact with KAT2B
[31, 32]. Transcription factor E2F1 induces S-phase-
specific gene expression and is involved in promoting
S-phase entry. In contrast, p53 inhibits cell cycle pro-
gression and entry in the S-phase by posttranslational
protein modifications. Acetylation by KAT2B has three
functional consequences on E2F1 activity: an increased
DNA-binding ability and gene activation and an increase
in the half-life of the protein . In endothelial cells,
acetylation is associated with the VEGF signaling pathway
and appears to be predominantly mediated by KAT2B,
and inhibition of KAT2B expression is sufficient to hinder
angiogenesis . These data may indicate that KAT2B
can promote inflammation and neovascularization in
AAA. In contrast, acetylation of the p53 region is ob-
served after DNA damage, leading to cell cycle arrest or
apoptosis . So, the overexpression of KAT2B may en-
tail growth arrest and/or apoptosis of SMCs. Interestingly,
SMCs within the aortic wall were mostly negative for this
acetyltransferase, suggesting protection from apoptosis
and cell death in AAA.
Finally, results of our present work demonstrate that
the expression of KAT2B is significantly associated with
the diameter of AAA. This is an important finding, as
KAT2B is a master switch in inflammation, which again
is a driving force in AAA progression. In consequence,
KAT2B may also be a potential biological marker of
patients at increased risk of AAA rupture. Such an
assertion needs to be confirmed in further studies. On
the other hand, it is to mention that the expression of
KAT2B negatively correlated with the amount of blood
leukocytes (WBC), which was surprising, because in-
flammatory cells play a crucial role during develop-
ment and progression of AAA . However, Wilson
et al.  demonstrated e.g., no elevation of inflam-
matory cells in ruptured aneurysms. The authors
also suggested other mechanisms leading to the rup-
ture of AAA. Our results showed a significant cor-
relation between the expression of KAT2B and the
macrophage marker MSR-1. These data are some-
what inconsistent and require additonal studies in
Our data regarding KAT3B (p300) were contradictory.
On the one hand, inflammatory cells were strongly posi-
tive for this acetyltransferase within the AAA wall, par-
ticularly in T cells. On the other hand, we found only a
weak correlation of KAT3B with markers of inflamma-
tory cells. No evidence is available of a possible role of
KAT3B in cardiovascular disease. However, KAT3B and
estrogen receptor (ER) function cooperate to increase
the efficiency of transcription initiation . Further-
more, estrogen receptor-α(ER-α) promoter methylation is
increased in atherosclerotic lesions and a similar promoter
methylation was found also in SMCs obtained from athero-
mata . This may imply that KAT3B can also contribute
to AAA formation.
KAT6B was identified as another highly expressed his-
tone acetyltransferase in AAA. Here, a strong positive
correlation was observed between its expression and
markers of inflammatory cells, namely CD45 and CD3.
KAT6B contains multiple functional domains and may
be involved in both positive and negative regulation of
transcription. At its C-terminus, KAT6B possesses a po-
tent transcriptional activation domain, whereas a strong
transcriptional repression domain is located at its N-
terminus . Consequently, KAT6B may facilitate but
also inhibit processes leading to AAA formation. Thus, a
potential role of KAT6B in AAA has to be further
Interestingly, high expression of KAT2B, KAT3B, and
KAT6B was found in inflammatory cells in the diseased
aorta. AAA is characterized by chronic inflammation
throughout the media and adventitia, which leads to the
upregulation and release of multiple cytokines [40–42]
and the activation of a plethora of proteolytic enzymes
[2, 17], ultimately leading to a rapid expansion of AAA
and rupture. In this regard, it is of great interest that
several studies have already demonstrated that KAT2B
and KAT3B are involved in the modulation of NF-κBac-
tivity  and are required to co-activate p65-dependent
transcription to activate several NF-κB-regulated inflam-
matory genes, known to be involved in cardiovascular dis-
ease, such as eotaxin, GM-CSF (granulocyte-macrophage
colony-stimulating factor), and TNFα. Based on our
Han et al. Clinical Epigenetics (2016) 8:3 Page 9 of 13
results, inflammatory cells, particularly T-lymphocytes
and macrophages, seem to be experiencing the greatest
epigenetic changes in AAA. Further studies are necessary,
e.g., isolating the individual cells and analyzing them sep-
arately for histone acetylation and the expression of corre-
sponding KATs to elucidate the exact role of epigenetics
in these cells relating to AAA progression and potential
risk of rupture.
Some limitations of our current work should be con-
sidered. Our study comprises a relatively small sample
size. Furthermore, a large variation among the values
from the individual tissue specimens was observed. For
this reason, we attempted to adjust our data for the total
amount of cells within the AAA wall, extent of inflam-
mation, age, diameter, hyperlipidemia, smoking, and
rupture. Nevertheless, no significant correlation between
the factors used for adjustment and expression of KATs
was found and no improvement of our results was
achieved. Furthermore, as most of our samples were
formalin fixed, the cellular localization of KATs was
evaluated in consecutively stained sections and in-
directly by correlation analyses with specific cells
markers. In addition, the analysis of epigenetic
changes in inflammatory cells in AAA could not be
directly compared with controlhealthyaortictissue
samples because these specimens do not have many
inflammatory cells. Thus, the conclusion that an
positive cells is based on our results in AAA, with-
out any comparison with other inflammatory cells,
e.g., from peripheral blood.
Research on epigenetics is increasingly recognized to
play an important role during various pathophysiological
processes and diseases. Our current data provide evi-
dence that epigenetics and chromatin modification may
play an important role in AAA. As enzymatic epigenetic
regulators can be altered by natural or designed com-
pounds, their targeting may emerge as a potential novel
diagnostic and therapeutic strategy in cardiovascular
Patients and tissue collection
Samples of 37 patients (30 males, 7 females) with AAA
were obtained during elective open surgical repair. All
tissue samples were collected in a standardized manner
from the anterior sac of the infrarenal abdominal aorta.
Furthermore, all clinical data available were recorded for
each patient, including age, sex, AAA diameter, hyperten-
sion, hyperlipidemia, hypercholesterolemia, chronic kid-
ney disease, diabetes, and smoking within the preceding
6 months. Patients with Ehlers-Danlos syndrome, Marfan
syndrome, and other known vascular or connective tissue
disorders were excluded from the study. Aortic tissue
from 12 organ donors was used as a control (7 males, 5 fe-
males), obtained from the Department of Trauma Surgery.
Exclusion criteria for the control group included cancer,
infection, and any other immune-related disease. Baseline
characteristics of donors are summarized in Additional
file 1: Table S1. The study was performed according to the
Guidelines of the World Medical Association Declaration
of Helsinki. The Ethics Committee of Klinikum rechts
der Isar, Technische Universitaet Muenchen approved
the study, and written informed consent was given by
All tissue samples were divided into two parts. The
first part was fixed overnight in formalin embedded in
paraffin (FFPE) and used for histological and immuno-
histochemical analyses or quantitative real-time reverse
transcriptase-PCR (RT-PCR). The other part was imme-
diately frozen in liquid nitrogen and used for protein
extraction and quantitative Western blot analysis. The
nomenclature of the investigated KATs, their alternative
names, corresponding histone substrates, and proposed
functions are summarized in Additional file 1: Table S2.
RNA extraction and quantitative RT-PCR analyses (qPCR)
Total cellular RNA was isolated from FFPE sections
(20 μm thickness) adjacent to the sections used for
histological characterizations using the High Pure RNA
Paraffin Kit according to the manufacturer’s instructions
(Roche, Mannheim, Germany). The amount and the
purity of RNA was determined by spectrophotometry.
High-quality RNA samples used in the study had an
A260/A280 ratio >1.8. For PCR analysis, RNA was
reverse-transcribed into complementary DNA (cDNA)
with random hexamer primers and cDNA Synthesis Kit
RevertAid (Fermentas, St. Leon-Rot, Germany). Quanti-
tative real-time RT-PCR was performed using SYBR
green fluorescence dye (PeqLab, Erlangen, Germany) and
StepOnePlus real-time PCR-System (Applied Biosystems/
Life Technologies, Darmstadt, Germany). A modified amp-
lification protocol was applied to eliminate bias by primer
dimer using following PCR conditions: initialization step
5 min at 95 °C, denaturation 10 s at 95 °C, annealing
30 s at 60 °C, extension 10 s at 72 °C, and primer
dimer elimination 15 s at 77 °C, 45 cycles.
Amplification of a housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH) was used for nor-
malizing of the gene expression. All primers used in the
study were purchased from Qiagen as designed Quanti-
Tect Primer Assays: GAPDH (GAPDH_1), KAT2A, KA2B,
KAT3B (EP300), KAT4 (TAF1), KAT5, KAT6A, KAT6B,
KAT7, KAT8, KAT12; MSR-1, CD45 (PTPRC_5), CD3
(CD3D_1), SMTN, MYH10, MYH11, VCAM-1, and
Collagen I (COL1A_1). The quantitative PCR analyses
Han et al. Clinical Epigenetics (2016) 8:3 Page 10 of 13
for all samples were independently repeated at least
two times, and in the case of heterogeneous results,
additional PCR was performed.
Histological and immunohistochemical analyses were
performed on representative sections of aortic tissue
samples (2–3μm). Paraffin sections were routinely
stained with hematoxylin-eosin (HE) and Elastica van
Gieson (EvG) to assess tissue morphology, cellular com-
position, degree of infiltration with inflammatory cells,
and the content of elastin and collagen fibers in all AAA
samples. For immunohistochemistry, dewaxed and hy-
drated tissue sections were boiled to retrieve antigen epi-
topes, washed and treated with appropriate antibodies
(Abs) accordingly. For analysis of cells localized within
the AAA wall, smooth muscle cells were detected by
primary Abs targeting smooth muscle myosin heavy
chain 1 and 2 (SM-MHCII, rabbit monoclonal, dilution
1:1,000; Abcam, Cambridge, UK),
endothelial cells by
anti-CD31 (mouse monoclonal, dilution 1:40; Dako), and
anti-CD34 (mouse monoclonal; dilution 1:400; Dako).
Macrophage/monocytes were detected with anti-CD68
(mouse monoclonal, dilution 1:2000; Dako), leukocytes
with anti-CD45 (mouse monoclonal, dilution 1:200;
Dako), and T-lymphocytes with anti-CD3 (mouse mono-
clonal, dilution 1: 400; Dako). For detection of KATs, the
following Abs were applied: KAT2B (rabbit polyclonal,
dilution 1:200; Abcam), KAT3B (rabbit polyclonal, dilution
1:100; Abcam), and KAT6B (rabbit polyclonal, dilution
1:400; Abcam). Histone main substrates were detected with
the following Abs for acetylated forms of these locations:
H3K9ac (rabbit polyclonal, dilution 1:1500; Abcam),
H3K14ac (rabbit monoclonal, dilution 1:1000; Abcam),
and acH3K18 (rabbit polyclonal, dilution 1:1500; Abcam).
All primary Abs were detected and visualized by LSAB
ChemMate Detection Kit (Dako) according to the manu-
To detect the expression of KATs by immunohisto-
chemistry and also by PCR in the specific cell types,
corresponding consecutive slides were used in all cases.
Protein extraction and Western blot analyses
Fresh frozen samples corresponding to FFPE specimen
were homogenized in liquid nitrogen, suspended in lysis
buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 %
NP-40, 0.1 % sodium dodecyl sulfate, 0.5 % sodium
deoxycholate, 0.02 % sodium azide), and collected by
centrifugation, and the supernatants containing the
cellular proteins were used for analyses. Histone ex-
traction was carried out using EpiSeeker Histone Ex-
traction kit (Abcam) according to the manufacturer’s
instructions. Protein or histone concentrations of each
specimen were determined using BCA Protein Assay
Kit (Thermo Scientific, Bonn, Germany). Protein lysates
and histone extracts (30 μg of each sample) were subjected
to SDS-PAGE using either 7.5 or 15 % gel depending on
the protein size and transferred onto polyvinylidene-
difluoride (PVDF) membrane. The membranes were
blocked (5 % BSA in PBS with 0.05 % Tween 20, pH 7.4)
for 2 h, followed by incubation with primary antibody at a
dilution of 1:500 for anti-KAT3B, 1:1000 for anti-KAT2B,
anti-KAT6B, and GAPDH, overnight at 4 °C. The blots
were then incubated with appropriate horseradish perox-
idase conjugated secondary antibodies at a dilution of
1:1000 for 1–2 h at room temperature. Immunoreactive
bands were developed using a chemiluminescence detec-
tion system (SuperSignal West Pico Chemiluminescent
Substrate, Thermo Scientific, Bonn, Germany) and de-
tected with LAS1000 (Fuji Film, Tokyo, Japan). The densi-
tometry was performed with Image J software 1.44 (W.
Rasband, Research Services Branch, NIMH, National
Institutes of Health, Bethesda, MD) and normalized
to the signal intensity of GAPDH for equal protein
loading control of each sample in each experiment
(Additional file 5: Figure S4).
All data were analyzed using SPSS for Windows version
20.0 (SPSS Inc, Chicago, IL, USA). First, data distribu-
tion was evaluated by one-sample Kolmogorov-Smirnov
test. Accordingly, continuous variables were compared
by either the parametric ttest for unpaired samples or
the non-parametric Mann-Whitney U test. The data
were monitored using either standard bar graphs or a
box plot diagram showing median and 25th/75th per-
centiles. Correlations between continuous variables were
quantified using Pearson’s correlation coefficient for nor-
mally distributed samples or Spearman’s rank correlation
coefficient for non-parametric values. All statistical com-
parisons were two-sided in the sense of an exploratory
data analysis using P< 0.05 as the level of significance.
Availability of supporting data
The data sets supporting the results of this article are
included within the article and its additional files
Additional file 1: Tables S1–S4. Table S1. Characteristics of the study
subjects. Table S2. Nomenclature of histone acetyltransferases used in
the study. Table S3. Summary of expression levels of KATs analyzed in
this study normalized to the expression of GAPDH. Table S4. Correlation
between KAT expression and clinical findings of AAA patients. (DOC 84 kb)
Additional file 2: Figure S1. Selected examples of scatter plot graphs
from correlation analysis of inter-relationships between KAT2B and KAT3B
(A), KAT2B and KAT6B (B), KAT3b and KAT6B (C) in AAA at mRNA level.
Quantification was performed by SYBR green-based RT-PCR using KATs
expression intensity normalized to GAPDH. AAA (n= 37). (PPTX 74 kb)
Han et al. Clinical Epigenetics (2016) 8:3 Page 11 of 13
Additional file 3: Figure S2. Selected examples of scatter plot graphs
from correlation analysis between expression of KATs and specific
markers of cells in AAA at mRNA level. KAT2B (A), KAT3B) (B), KAT6B (C).
Quantification was performed by SYBR green-based RT-PCR. The expression
of all factors was normalized to GAPDH. AAA (n=37).(PPTX152kb)
Additional file 4: Figure S3. Selected examples of scatter plot graphs
from correlation analysis between expression of KATs and clinical parameters.
KAT2B and AAA diameter (A), KAT3A, KAT6A, KAT6B against blood urea
nitrogen (B). Quantification was performed by SYBR green-based RT-PCR.
The KAT expression was normalized to GAPDH. AAA (n= 37). (PPTX 80 kb)
Additional file 5: Figure S4. Loading controls. Expression of GAPDH at
the protein level in all AAA tissue samples and all healthy aorta tissue
samples (Ctrl) used in western blot analyses. (PPTX 140 kb)
AAA: aortic abdominal aneurysm; Ab: antibody; DNA: deoxyribonucleic acid;
ECs: endothelial cells; EvG: Elastica van Gieson staining; FFPE: formalin-fixed
paraffin-embedded; GAPDH: glyceraldehyde 3-phosphate dehydrogenase;
HAT: histone acetyltransferase; HDACs: histone deacetylases; HE: hematoxylin-
eosin staining; IHC: immunohistochemistry; KAT: lysine [K] histone
acetyltransferase; MHC: myosin heavy chain; mRNA: messenger RNA;
PVDF: polyvinylidene-difluoride; RT-PCR: real-time reverse transcriptase
polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate
polyacrylamide gel electrophoresis; SMCs: smooth muscle cells.
The authors declare that they have no competing interests.
YH and FT performed the experiments. YH, JP, and KS participated in data analysis.
CR and BL collected tissue samples and patients’data. YH, JP and AZ wrote the
manuscript. JP and AZ conceived the research, and AZ, HHE, and JZ critically
reviewed the manuscript and interpreted the data. All authors read and approved
the final manuscript.
Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar
der Technische Universität München, Ismaninger Str. 22, 81675 Munich,
Department of Vascular and Surgery, The First Hospital of China
Medical University, Shenyang, China.
Department of General Surgery,
Shengjing Hospital of China Medical University, Shenyang, China.
Department for Visceral, Thoracic and Vascular Surgery at the University
Hospital, Technical University Dresden, Dresden, Germany.
Pathology, Klinikum rechts der Isar der Technische Universität München,
Institute of Experimental Biomedicine, University Hospital,
University of Würzburg, Würzburg, Germany.
DZHK (German Centre for
Cardiovascular Research), partner site Munich Heart Alliance, Munich,
Received: 5 October 2015 Accepted: 4 January 2016
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