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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. 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 with T 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). In addtion, acetylation of the histone substrates H3K9, H3K18 and H3K14 was increased in AAA compared to control aortae. 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.
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R E S E A R C H Open Access
Histone acetylation and histone
acetyltransferases show significant
alterations in human abdominal aortic
aneurysm
Yanshuo Han
1,2,3
, Fadwa Tanios
1
, Christian Reeps
1,4
, Jian Zhang
2
, Kristina Schwamborn
5
, Hans-Henning Eckstein
1,7
,
Alma Zernecke
6,1*
and Jaroslav Pelisek
1,7*
Abstract
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 (P0.019). Highest expression was observed for KAT2B, KAT3A, KAT3B, and
KAT6B (P0.007). 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: alma.zernecke@uni-wuerzburg.de;jaroslav.pelisek@tum.de
Equal contributors
6
Institute of Experimental Biomedicine, University Hospital, University of
Würzburg, Würzburg, Germany
1
Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar
der Technische Universität München, Ismaninger Str. 22, 81675 Munich,
Germany
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
DOI 10.1186/s13148-016-0169-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
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 [1]. 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 [4]. 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 [5]. The
histone acetylation process is regulated by the balanced
activities of two key enzyme families of transferases,
namely lysine [K] histone acetyltransferases (KATs) [6],
and histone deacetylases (HDACs) [7]. The function of
KATs is to add an acetyl group to the lysine residue,
resulting in chromatin opening and gene activation [8].
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 [11], 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 [12].
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 [13]. Krishna et al. have
hypothesized that epigenetic mechanisms may also play a
role in the pathogenesis of AAA [14]. 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
progression.
Results
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
acetyltransferases KAT2A,KAT2B,KAT3A,KAT3B,KAT5,
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
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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.
KAT8 0.648**
KAT4
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
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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 [17]. 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
+
leukocytes, CD68
+
macrophages, and CD3
+
T cells (Fig. 3a). In addition,
staining of intramural CD31
+/
CD34
+
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
AB
CD
EF
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
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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
source
In order to determine the acetylation of the main histone
substrates of KAT2B, KAT3B, and KAT6B (Additional
file 1: Table S2) [8], 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. 4ad). 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
+
AB
C
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
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leukocytes, CD68
+
macrophages, and CD3
+
Tcells
(Fig.5a).Furthermore,CD34
+
and CD31
+
neovessels
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
+
leukocytes, CD68
+
macrophages, and CD34
+
/CD31
+
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
+
leukocytes and
CD68
+
macrophages (Fig. 5c). In contrast, only some
CD34
+
/CD31
+
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
AB
CD
EF
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
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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 [18]. Furthermore, we also analyzed the expression of
vascular cell adhesion molecule (VCAM)-1, which plays
an important role in the development of AAA [17]. 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).
AB
C
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
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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).
Discussion
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
[21]. 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 [22]. 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 [23] 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 [26], 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
diseased aorta.
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
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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 [27]. 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 [30]. 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 [17]. 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 [33]. 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 [34]. 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 [35]. 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 [3]. However, Wilson
et al. [36] 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
the future.
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 [37]. 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 [38]. 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 [39]. Consequently, KAT6B may facilitate but
also inhibit processes leading to AAA formation. Thus, a
potential role of KAT6B in AAA has to be further
elucidated.
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 [4042]
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 [43] 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[44]. Based on our
Han et al. Clinical Epigenetics (2016) 8:3 Page 9 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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
over-expressionofKATsisfoundinCD45andCD3
positive cells is based on our results in AAA, with-
out any comparison with other inflammatory cells,
e.g., from peripheral blood.
Conclusions
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
disease.
Methods
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 patients.
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 manufacturers 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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
for all samples were independently repeated at least
two times, and in the case of heterogeneous results,
additional PCR was performed.
Immunohistochemistry (IHC)
Histological and immunohistochemical analyses were
performed on representative sections of aortic tissue
samples (23μm). 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),
13
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-
facturersinstructions.
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 manufacturers
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 12 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).
Statistical analysis
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 Pearsons correlation coefficient for nor-
mally distributed samples or Spearmans 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 files
Additional file 1: Tables S1S4. 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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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)
Abbreviations
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.
Competing interests
The authors declare that they have no competing interests.
Authorscontributions
YH and FT performed the experiments. YH, JP, and KS participated in data analysis.
CR and BL collected tissue samples and patientsdata. 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.
Author details
1
Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar
der Technische Universität München, Ismaninger Str. 22, 81675 Munich,
Germany.
2
Department of Vascular and Surgery, The First Hospital of China
Medical University, Shenyang, China.
3
Department of General Surgery,
Shengjing Hospital of China Medical University, Shenyang, China.
4
Department for Visceral, Thoracic and Vascular Surgery at the University
Hospital, Technical University Dresden, Dresden, Germany.
5
Institute of
Pathology, Klinikum rechts der Isar der Technische Universität München,
Munich, Germany.
6
Institute of Experimental Biomedicine, University Hospital,
University of Würzburg, Würzburg, Germany.
7
DZHK (German Centre for
Cardiovascular Research), partner site Munich Heart Alliance, Munich,
Germany.
Received: 5 October 2015 Accepted: 4 January 2016
References
1. Thompson RW. Detection and management of small aortic aneurysms.
N Engl J Med. 2002;346:14846.
2. Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm. Lancet.
2005;365:157789.
3. Han YS, Zhang J, Xia Q, Liu ZM, Zhang XY, Wu XY, et al. A comparative
study on the medium-long term results of endovascular repair and open
surgical repair in the management of ruptured abdominal aortic aneurysms.
Chin Med J (Engl). 2013;126:47719.
4. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. An operational
definition of epigenetics. Genes Dev. 2009;23:7813.
5. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:107480.
6. Allis CD, Berger SL, Cote J, Dent S, Jenuwien T, Kouzarides T, et al.
New nomenclature for chromatin-modifying enzymes. Cell. 2007;131:6336.
7. McKinsey TA, Zhang CL, Olson EN. Control of muscle development by
dueling HATs and HDACs. Curr Opin Genet Dev. 2001;11:497504.
8. Narlikar GJ, Fan HY, Kingston RE. Cooperation between complexes that
regulate chromatin structure and transcription. Cell. 2002;108:47587.
9. Santos-Rosa H, Caldas C. Chromatin modifier enzymes, the histone code
and cancer. Eur J Cancer. 2005;41:2381402.
10. Furdas SD, Kannan S, Sippl W, Jung M. Small molecule inhibitors of histone
acetyltransferases as epigenetic tools and drug candidates. Arch Pharm
(Weinheim). 2012;345:721.
11. Sharma P, Sharma P, Kumar J, Garg G, Kumar A, Patowary A, et al. Detection
of altered global DNA methylation in coronary artery disease patients. DNA
Cell Biol. 2008;27:35765.
12. Turunen MP, Aavik E, Ylä-Herttuala S. Epigenetics and atherosclerosis.
Biochim Biophys Acta. 1790;2009:88691.
13. Greißel A, Culmes M, Napieralski R, Wagner R, Gebhard H, Schmitt M, et al.
Alternation of histone and DNA methylation in human atherosclerotic
carotid plaques. Thromb Haemost. 2015;114:390402.
14. Krishna SM, Dear AE, Norman PE, Golledge J. Genetic and epigenetic mechanisms
and their possible role in abdominal aortic aneurysm. Atherosclerosis. 2010;212:1629.
15. Sheikh BN. Crafting the brainrole of histone acetyltransferases in neural
development and disease. Cell Tissue Res. 2014;356:55373.
16. Wang Z, Zang C, Cui K, Schones DE, Barski A, Peng W, et al. Genome-wide
mapping of HATs and HDACs reveals distinct functions in active and
inactive genes. Cell. 2009;138:101931.
17. Reeps C, Pelisek J, Seidl S, Schuster T, Zimmermann A, Kuehnl A, et al.
Inflammatory infiltrates and neovessels are relevant sources of MMPs in
abdominal aortic aneurysm wall. Pathobiology. 2009;76:24352.
18. Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular
smooth muscles cell phenotypic diversity. Heth Heart J. 2007;15:1008.
19. Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C. Core histone
hyperacetylation co-maps with generalized DNase I sensitivity in the
chicken beta-globin chromosomal domain. EMBO J. 1994;13:182330.
20. Vettese-Dadey M, Grant PA, Hebbes TR, Crane-Robinson C, Allis CD, Workman
JL. Acetylation of histone H4 plays a primary role in enhancing transcription
factor binding to nucleosomal DNA in vitro. EMBO J. 1996;15:250818.
21. Zhao X, Benveniste EN. Transcriptional activation of human matrix
metalloproteinase-9 gene expression by multiple co-activators. J Mol Biol. 2008;
383:94556.
22. Gu W, Shi XL, Roeder RG. Synergistic activation of transcription by CBP and
p53. Nature. 1997;387:81923.
23. López-Candales A, Holmes DR, Liao S, Scott MJ, Wickline SA, Thompson RW.
Decreased vascular smooth muscle cell density in medial degeneration of
human abdominal aortic aneurysms. Am J Pathol. 1997;150:9931007.
24. Thomas MC, Chiang CM. The general transcription machinery and general
cofactors. Crit Rev Biochem Mol Biol. 2006;41:10578.
25. Kirkland JG, Raab JR, Kamakaka RT. TFIIIC bound DNA elements in nuclear
organization and insulation. Biochim Biophys Acta. 1829;2013:41824.
26. Varon R, Gooding R, Steglich C, Marns L, Tang H, Angelicheva D, et al. Partial
deficiency of the C-terminal-domain phosphatase of RNA polymerase II is
associated with congenital cataracts facial dysmorphism neuropathy syndrome.
Nat Genet. 2003;35:1859.
27. Pelletier N, Champagne N, Stifani S, Yang XJ. MOZ and MORF histone
acetyltransferases interact with the Runt-domain transcription factor Runx2.
Oncogene. 2002;21:272940.
28. Doyon Y, Côté J. The highly conserved and multifunctional NuA4 HAT
complex. Curr Opin Genet Dev. 2004;14:14754.
29. Utley RT, Lacoste N, Jobin-Robitaille O, Allard S, Côté J. Regulation of NuA4
histone acetyltransferase activity in transcription and DNA repair by
phosphorylation of histone H4. Mol Cell Biol. 2005;25:817990.
30. Bastiaansen AJ, Ewing MM, de Boer HC, van der Pouw Kraan TC, de Vries MR,
Peters EA, et al. Lysine acetyltransferase PCAF is a key regulator of
arteriogenesis. Arterioscler Thromb Vasc Biol. 2013;33:190210.
31. Hupp TR, Meek DW, Midgley CA, Lane DP. Regulation of the specific DNA
binding function of p53. Cell. 1992;71:87586.
32. Grossman SR. p300/CBP/p53 interaction and regulation of the p53 response.
Eur J Biochem. 2001;268:27738.
33. Martinez-Balbas MA, Bauer UM, Nielsen SJ,BrehmA,KouzaridesT.Regulationof
E2F1 activity by acetylation. EMBO J. 2000;19:66271.
34. Pillai S, Kovacs M, Chellappan S. Regulation of vascular endothelial growth factor
receptors by Rb and E2F1: role of acetylation. Cancer Res. 2010;70:493140.
35. Gu W, Roeder RG. Activation of p53 sequence specific DNA binding by
acetylation of the p53 C-terminal domain. Cell. 1997;90:595606.
36. Wilson WR, Wills J, Furness PN, Loftus IM, Thompson MM. Abdominal aortic
aneurysm rupture is not associated with an up-regulation of inflammation
within the aneurysm wall. Eur J Vasc Endovasc Surg. 2010;40(2):1915.
Han et al. Clinical Epigenetics (2016) 8:3 Page 12 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
37. Kraus WL, Kadonaga JT. p300 and estrogen receptor cooperatively activate
transcription via differential enhancement of initiation and reinitiation.
Genes Dev. 1998;12:33142.
38. Ying AK, Hassanain HH, Roos CM, Smiraglia DJ, Issa JJ, Michler RE, et al.
Methylation of the estrogen receptoralpha gene promoter is selectively
increased in proliferating human aortic smooth muscle cells. Cardiovasc Res.
2000;46:1729.
39. Champagne N, Bertos NR, Pelletier N, Wang AH, Vezmar M, Yang Y, et al.
Identification of a human histone acetyltransferase related to monocytic
leukemia zinc finger protein. J Biol Chem. 1999;274:2852836.
40. Pearce WH, Koch AE. Cellular components and features of immune response
in abdominal aortic aneurysms. Ann N Y Acad Sci. 1996;800:17585.
41. Zhang J, Böckler D, Ryschich E, Klemm K, Schumacher H, Schmidt J, et al.
Impaired Fas-induced apoptosis of T lymphocytesin patients with
abdominal aortic aneurysms. J Vasc Surg. 2007;45:103946.
42. Yin M, Zhang J, Wang Y, Wang S, Böckler D, Duan Z, et al. Deficient CD4
+CD25+ T regulatory cell function in patients with abdominal aortic
aneurysms. Arterioscler Thromb Vasc Biol. 2010;30:182531.
43. Sheppard KA, Rose DW, Haque ZK, Kurokawa R, McInerney E, Westin S, et al.
Transcriptional activation by NF-kappaB requires multiple coactivators.
Mol Cell Biol. 1999;19:636778.
44. Kiernan R, Brès V, Ng RW, Coudart MP, El Messaoudi S, Sardet C, et al.
Post-activation turn-off of NF-kappa B-dependent transcription is regulated
by acetylation of p65. J Biol Chem. 2003;278:275866.
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... Epigenetic alterations, such as DNA methylation [13], histone modification [14] or RNA modification [15], has been found to exert vital parts in AAA due to their strong impacts on regulating gene levels. N6methyladenosine (m6A) has been well investigated and most frequently observed in mRNA, which is found to be related to pre-mRNA translation, processing, mRNA decay and miRNA biogenesis. ...
... The typical 2-3-µm aortic tissue sections were utilized to carry out histological and IHC analyses. In brief, we used hematoxylin-eosin (HE) to stain paraffin-embedded sections for assessing the inflammatory cell composition, morphology and infiltration extent in each AAA sample according to previous description [14]. ...
... The symptoms of the AAA patients are summarized in Table 2. Altogether 22% AAA cases had aneurysmal rupture, whereas 5 out of the 32 cases had iliac aneurysms. Among AAA cases, each AAA sample was semi-quantitatively and histologically characterized for evaluating each histopathological characteristic degree within AAA wall as previously [14]. In Table 3, IHC was used to differentiate between the four main cell types in AAAs, i.e., endothelial cells, lymphocytes, macrophages, and smooth muscle cells to assess the extension of the individual histopathological features in AAA wall. ...
Article
Background: It remains largely unclear about the function of 5-methylcytosine (m5C) RNA modification in the context of abdominal aortic aneurysm (AAA). In this regard, the present work focused on investigating m5C RNA methylation and related modulator expression levels in AAA. Materials and methods: To this end, we quantified the m5C methylation levels in AAA tissues (n = 32) and normal aortic tissues (n = 12) to examine the mRNA m5C status and m5C modulator expression at mRNA and protein levels. Meanwhile, modulator localization within AAA tissue samples was detected by immunohistochemistry (IHC). Moreover, RNA immunoprecipitation-sequencing (RIP-seq) was also used to analyze the lncRNAs and mRNA binding to Aly/REF, as an m5C reader. Results: m5C expression markedly elevated in AAA in comparison with normal aortic samples in the AAA cases. The major 5-methylcytosine modulators, including NSUN2, NSUN5, and Aly/REF, which represented the major parameters related to the abnormal m5C modification level, were observed up-regulating in AAA tissues at both protein and mRNA levels. In addition, NSUN2 mRNA level remarkably related to Aly/REF expression, and they were co-expressed in the same cells in AAA group. Regarding the cellular location, Aly/REF was associated with inflammatory (CD45+, CD3+) infiltrates. Simultaneously, after screening for reads in AAA tissue compare with anti-Aly/REF group relative to IgG as control, we obtained totally 477 differentially expressed Aly/REF-binding lncRNAs and 369 differentially expressed Aly/REF-binding mRNAs in AAA tissue. The functions of Aly/REF-interacting lncRNA were involved in immune system process and macrophages infiltration. Through regulatory network (lncRNA-mRNA) analysis, our findings predicted the potential mechanism of Aly/REF-induced lncBCL2L1 and Aly/REF-lncFHL1 axis in AAA and inspire the understanding of m5C and lncRNA in AAA. Conclusions: This study is the first to examine m5A modification within human AAA samples. Our results indicate that m5C modulators, namely, Aly/REF and NUSN2, play vital parts in the human AAA pathogenic mechanism, which shed new lights on the function of m5C modification within AAA. Taken together, findings in this work offer a possible RNA methylation modification mechanism within clinical AAA.
... This modification makes chromatin to be more compact such that gene transcription could be inhibited. While HAT has the opposite function which could transfer the acetyl group of acetyl-CoA to the specific lysine residue at the amino terminus of histones, it would make the chromatin sparse and promote gene transcription (Han et al., 2016). HDAC is mechanosensitive and could inhibit osteogenic differentiation and decrease the level of BMSC mineralization through the Wnt signaling pathway. ...
... HDACs can reverse the acetylation of the N-tail lysine residue of the core histone, resulting in chromatin condensation and inhibition of gene transcription. On the contrary, HAT transfers the acetyl group of acetyl-CoA to the specific lysine residue at the amino terminus of histones, loosening the chromatin structure and promoting transcription of the gene (Han et al., 2016). Previous literature has reported that HAT played an important role in bone formation and bone loss (Szuhanek et al., 2020;Wu et al., 2020). ...
Article
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Bone marrow mesenchymal stem cells (BMSCs) play a critical role in bone formation and are extremely sensitive to external mechanical stimuli. Mechanical signals can regulate the biological behavior of cells on the surface of titanium-related prostheses and inducing osteogenic differentiation of BMSCs, which provides the integration of host bone and prosthesis benefits. But the mechanism is still unclear. In this study, BMSCs planted on the surface of TiO 2 nanotubes were subjected to cyclic mechanical stress, and the related mechanisms were explored. The results of alkaline phosphatase staining, real-time PCR, and Western blot showed that cyclic mechanical stress can regulate the expression level of osteogenic differentiation markers in BMSCs on the surface of TiO 2 nanotubes through Wnt/β-catenin. As an important member of the histone acetyltransferase family, GCN5 exerted regulatory effects on receiving mechanical signals. The results of the ChIP assay indicated that GCN5 could activate the Wnt promoter region. Hence, we concluded that the osteogenic differentiation ability of BMSCs on the surface of TiO 2 nanotubes was enhanced under the stimulation of cyclic mechanical stress, and GCN5 mediated this process through Wnt/β-catenin.
... Additionally, recent studies have shown that epigenetics is strongly associated with various vascular diseases. At chromatin level, study has demonstrated that histone acetylation and histone acetyltransferases play significant roles in human AAA (12). At mRNA level, increased N6-methyladenosine (m6A) modification level, 5methylcytosine (m5C) methylation level and the up-regulating expression of their regulatory genes were verified in human AAA (13,14). ...
Article
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Objectives This study aimed to identify key AAA-related m1A RNA methylation regulators and their association with immune infiltration in AAA. Furthermore, we aimed to explore the mechanism that m1A regulators modulate the functions of certain immune cells as well as the downstream target genes, participating in the progression of AAA. Methods Based on the gene expression profiles of the GSE47472 and GSE98278 datasets, differential expression analysis focusing on m1A regulators was performed on the combined dataset to identify differentially expressed m1A regulatory genes (DEMRGs). Additionally, CIBERSORT tool was utilized in the analysis of the immune infiltration landscape and its correlation with DEMRGs. Moreover, we validated the expression levels of DEMRGs in human AAA tissues by real-time quantitative PCR (RT-qPCR). Immunofluorescence (IF) staining was also applied in the validation of cellular localization of YTHDF3 in AAA tissues. Furthermore, we established LPS/IFN-γ induced M1 macrophages and ythdf3 knockdown macrophages in vitro , to explore the relationship between YTHDF3 and macrophage polarization. At last, RNA immunoprecipitation-sequencing (RIP-Seq) combined with PPI network analysis was used to predict the target genes of YTHDF3 in AAA progression. Results Eight DEMRGs were identified in our study, including YTHDC1, YTHDF1-3, RRP8, TRMT61A as up-regulated genes and FTO, ALKBH1 as down-regulated genes. The immune infiltration analysis showed these DEMRGs were positively correlated with activated mast cells, plasma cells and M1 macrophages in AAA. RT-qPCR analysis also verified the up-regulated expression levels of YTHDC1, YTHDF1 , and YTHDF3 in human AAA tissues. Besides, IF staining result in AAA adventitia indicated the localization of YTHDF3 in macrophages. Moreover, our in-vitro experiments found that the knockdown of ythdf3 in M0 macrophages inhibits macrophage M1 polarization but promotes macrophage M2 polarization. Eventually, 30 key AAA-related target genes of YTHDF3 were predicted, including CD44, mTOR, ITGB1, STAT3 , etc. Conclusion Our study reveals that m1A regulation is significantly associated with the pathogenesis of human AAA. The m1A “reader,” YTHDF3 , may participate in the modulating of macrophage polarization that promotes aortic inflammation, and influence AAA progression by regulating the expression of its target genes.
... These acetylated histone marks at both promoter and intragenic regions mediate cell-restricted gene expression. Altered HBO1 expression has been reported in human abdominal aortic aneurysm (36), a vascular disease closely related to endothelial dysfunction (37). Hbo1-knockout mice are embryonic lethal (35). ...
Article
Full-text available
The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 and histone acetylation were downregulated in NGBR deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of both HBO1 and acetylated H4K5/K12 on the promotor of CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs. .
... Current research mainly focuses on the potential mechanism between epigenetic modification and AD (10). A variety of epigenetic modifications have been detected, including the most commonly seen DNA methylation, histone methylation and acetylation, and chromosomal remodeling (11,12). Of course, there are also modification models at the RNA level (13). ...
Article
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Objective To identify the feature of N6-methyladenosine (m6A) methylation modification genes in acute aortic dissection (AAD) and explore their relationships with immune infiltration. Methods The GSE52093 dataset including gene expression data from patients with AAD and healthy controls was downloaded from Gene Expression Omnibus (GEO) database in order to obtain the differentially expressed genes (DEGs). The differentially methylated m6A genes were obtained from the GSE147027 dataset. The differentially expressed m6A-related genes were obtained based on the intersection results. Meanwhile, the protein-protein interaction (PPI) network of differentially expressed m6A-related genes was constructed, and hub genes with close relationships in the network were selected. Later, hub genes were verified by using the GSE153434 dataset. Thereafter, the relationships between these genes and immune cells infiltration were analyzed. Results A total of 279 differentially expressed m6A-related genes were identified in the GSE52093 and GSE147027 datasets. Among them, 94 genes were up-regulated in aortic dissection (AD), while the remaining 185 were down-regulated. As indicated by Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, these genes were mainly associated with extracellular matrix (ECM) and smooth muscle cells (SMCs). The seven hub genes, namely, DDX17, CTGF, FLNA, SPP1, MYH11, ITGA5 and CACNA1C, were all confirmed as the potential biomarkers for AD. According to immune infiltration analysis, it was found that hub genes were related to some immune cells. For instance, DDX17, FLNA and MYH11 were correlated with Macrophages M2. Conclusion Our study identifies hub genes of AD that may serve as the potential biomarkers, illustrates of the molecular mechanism of AD, and provides support for subsequent research and treatment development.
... These acetylated histone marks at both promoter and intragenic regions mediate cell-restricted gene expression. Altered HBO1 expression has been reported in human abdominal aortic aneurysm (36), a vascular disease closely related to endothelial dysfunction (37). Hbo1-knockout mice are embryonic lethal (35). ...
Article
Cerebral cavernous malformations (CCMs) are enlarged leaking vascular lesions in the brain caused by loss-of-function mutations in CCM1/2/3 genes or loss of expression. Although we previously showed that Nogo-B receptor (NgBR) knockout in endothelial cells (ECs) results in CCMs-like cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NgBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of NgBR in ECs at both the postnatal and adult stages results in CCM1/2 expression deficiency and consequently CCMs-like lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and intracerebral hemorrhage. These cerebrovascular defects in the brain of NgBR endothelial-specific knockout (ecKO) mice can be rescued by adeno-associated virus (AAV)-mediated overexpression of CCM1 and CCM2 genes. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 was downregulated in NgBR deficient ECs. The mechanistic study elucidated that NgBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NgBR impairs the binding of both the HBO1 and acetylated H4K5/K12 at the promoter of CCM1 and CCM2 genes. Similarly, AAV-mediated overexpression of HBO1 restores the acetylation of H4K5/K12 and rescues the CCMs-like cerebrovascular defects in the brain of NgBR ecKO mice. Our findings on epigenetic regulation of CCM1 and CCM2 provide a perspective that NgBR and HBO1-mediated histone H4 acetylation may be targeted for preventing the onset of CCMs-like cerebrovascular disease.
... Regarding histone modifications, a recent study comparing AAA and healthy aortic samples reported a wide variety of histone acetylation transferases that were significantly higher in disease [59]. KAT2B, KAT3A, KAT3B, and KAT6B were among the highest expressed in AAA tissue. ...
Article
Full-text available
Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high morbidity and mortality in the setting of acute rupture. Recently, advances in surgical and endovascular repair of AAA have been achieved; however, pharmaceutical therapies to prevent AAA expansion and rupture remain lacking. This highlights an ongoing need to improve the understanding the pathological mechanisms that initiate formation, maintain growth, and promote rupture of AAA. Over the past decade, epigenetic modifications, such as DNA methylation, posttranslational histone modifications, and non-coding RNA, have emerged as important regulators of cellular function. Accumulating studies reveal the importance of epigenetic enzymes in the dynamic regulation of key signaling pathways that alter cellular phenotypes and have emerged as major intracellular players in a wide range of biological processes. In this review, we discuss the roles and implications of epigenetic modifications in AAA animal models and their relevance to human AAA pathology.
... Changes in VSMCs pattern, vascular inflammation, and degradation of the extracellular matrix (ECM) are precursors of progressive thinning of the aortic wall [97]. Emerging evidence documented epigenetic events involving phenotypic alteration of VSMCs, i.e., proliferation and dedifferentiation, mainly related to ECM remodeling of the vascular wall and increased inflammation in various in vitro and in vivo models [98][99][100]. Of interest, a novel role of histone 3 lysine 9 demethylation (H3K9me2) in VSMCs has been uncovered for atherosclerosis and vascular injury since protein expression of H3K9me2 is significantly reduced in murine atherosclerotic and restenotic lesions. ...
Article
Vascular inflammation is one of the main activating stimuli of cardiovascular disease and its uncontrolled development may worsen the progression and prognosis of these pathologies. Therefore, the search for new therapeutic options to treat this condition is undoubtedly needed. In this regard, it may be better to repurpose endogenous anti-inflammatory compounds already known, in addition to synthesizing new compounds for therapeutic purposes. It is well known that vitamin D, anandamide, and melatonin are promising endogenous substances with powerful and widespread anti-inflammatory properties. Currently, the epigenetic mechanisms underlying these effects are often unknown. This review summarizes the potential epigenetic mechanisms by which vitamin D, anandamide, and melatonin attenuate vascular inflammation. This information could contribute to the improvement in the therapeutic management of multiple pathologies associated with blood vessel inflammation, through the pharmacological manipulation of new target sites that until now have not been addressed.
... Heterogeneous nuclear ribonucleoprotein C-like 1 (DLQAIK, LQAIKQ, QAKQE), alterations of which are found in sporadic and suspected Lynch syndrome endometrial cancer [36]. • Histone acetyltransferases KAT2A and KAT2B (DGRVIG), when altered, are associated with cardiovascular pathology [37]. • Islet cell autoantigen 1 (MKDLQA) Islet autoantibodies are typically associated with type 1 diabetes, but have been found in patients diagnosed with type 2 diabetes in whom they are associated with lower adiposity [38]. ...
Article
Full-text available
Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number of human proteins, thus potentially instituting the basis for cross-reactions. We found a potential for cross-reactivity between the virus and the human proteome and described an epitope platform to be used in order to avoid it, thus obtaining effective, specific, and safe immunization. Potential advantages for mRNA and peptide-based vaccine formulations are discussed.
Article
Aims Vascular stiffness increases with age and independently predicts cardiovascular disease risk. Epigenetic changes, including histone modifications, accumulate with age but the global pattern has not been elucidated nor are the regulators known. Smooth muscle cell-mineralocorticoid receptor (SMC-MR) contributes to vascular stiffness in aging mice. Thus, we investigated the regulatory role of SMC-MR in vascular epigenetics and stiffness. Methods and Results Mass spectrometry-based proteomic profiling of all histone modifications completely distinguished 3 from 12-month-old mouse aortas. Histone-H3 lysine-27(H3K27) methylation(me) significantly decreased in aging vessels and this was attenuated in SMC-MR-KO littermates. Immunoblotting revealed less H3K27-specific methyltransferase EZH2 with age in MR-intact but not SMC-MR-KO vessels. These aging changes were examined in primary human aortic (HA)SMC from adult versus aged donors. MR, H3K27 acetylation(ac), and stiffness gene (CTGF, Integrin-α5) expression significantly increased, while H3K27me and EZH2 decreased, with age. MR inhibition reversed these aging changes in HASMC and the decline in stiffness genes was prevented by EZH2 blockade. Atomic force microscopy revealed that MR antagonism decreased intrinsic stiffness and the probability of fibronectin adhesion of aged HASMC. Conversely, aging induction in young HASMC with H2O2; increased MR, decreased EZH2, enriched H3K27ac and MR at stiffness gene promoters by ChIP, and increased stiffness gene expression. In 12-month-old mice, MR antagonism increased aortic EZH2 and H3K27 methylation, increased EZH2 recruitment and decreased H3K27ac at stiffness genes promoters, and prevented aging-induced vascular stiffness and fibrosis. Finally, in human aortic tissue, age positively correlated with MR and stiffness gene expression and negatively correlated with H3K27me3 while MR and EZH2 are negatively correlated. Conclusion These data support a novel vascular aging model with rising MR in human SMC suppressing EZH2 expression thereby decreasing H3K27me, promoting MR recruitment and H3K27ac at stiffness gene promoters to induce vascular stiffness and suggests new targets for ameliorating aging-associated vascular disease. Translational perspective These findings provide a new epigenetic mechanism whereby rising MR in aging human SMC promotes vascular stiffness. Vascular stiffness contributes to common disorders of aging including hypertension, heart and kidney failure, and stroke, yet no therapies successfully target vascular stiffness. Drugs that inhibit MR are already approved and used in the elderly. In addition, drugs targeting histone-modifying enzymes, including EZH2, are being developed to treat cancer. Thus, these results provide preclinical support for drugs that could be immediately tested to treat aging-associated vascular stiffness and raise the potential for some cancer therapies to promote vascular stiffness.
Article
Atherosclerotic risk factors can be divided into two main categories-genetical and environmental issues. The latter ones include habitual factors since human habits manifest as environmental factors at the cellular level. Environmental issues can govern human health via epigenetic modification of chromatin structure. This review discusses the recent findings linking general epigenetic mechanisms of chromatin modifications to atherosclerosis development. © 2012 Springer Science+Business Media New York. All rights are reserved.
Article
Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.
Article
Objective: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-α gene (ERα) promoter blocks the expression of ERα, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERα is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. Methods: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERα in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). Results: We provide evidence that methylation of the ERα in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERα promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERα and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. Conclusions: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERα, could contribute to the switch in phenotype observed in these cells.
Article
抄録 腹部大動脈瘤破裂126例を対象に治療戦略変更の妥当性を検討した。2008年から閉腹により腹腔内圧が上昇すると予想される症例では,abdominal compartment syndromeの予防目的で積極的にopen abdominal managementを施行した。治療方針変更後,手術死亡は31%から8%へ,腸管虚血・壊死の合併率も24%から4%と著明に改善した。
Article
腹部大動脈瘤 (AAA) 240例を破裂群 (31例) と非破裂群 (209例) に分け, 非破裂群をASO合併群 (48例) とASO非合併群 (161例) に分けて検討した. 追跡期間は最長15年10か月, 平均4年2か月, 遠隔期追跡率は97%であった. 手術死亡率は破裂群41.9%, 非破裂群2.9%, ASO合併群6.3%, ASO非合併群1.9%であった. 遠隔期死亡原因は心疾患32%, 悪性腫瘍22%, 脳血管障害10%, 腎疾患10%などであったが, 手術時リスクファクターと関連したのは腎不全のみであった. 術後相対生存率は破裂群5年79%, 10年0%, 非破裂群5年90%, 10年70%で, 同年代一般人より低く, ASO非合併群は5年95%, 10年78%, ASO合併群は5年74%, 10年52%であり, ASO合併群ではさらに低値であった. ASO合併群は手術時, 虚血性心疾患, 糖尿病の合併が多く, 全体の遠隔期死亡原因は心疾患, 腎不全が多かった. これらを念頭においた遠隔期フォローアップが重要である.