Down-Regulation of mir-424 Contributes to the
Abnormal Angiogenesis via MEK1 and Cyclin E1 in Senile
Hemangioma: Its Implications to Therapy
Taiji Nakashima, Masatoshi Jinnin*, Tomomi Etoh, Satoshi Fukushima, Shinichi Masuguchi, Keishi
Maruo, Yuji Inoue, Tsuyoshi Ishihara, Hironobu Ihn
Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
Background: Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically
seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear.
Methodology/Principal Findings: In this study, we found that senile hemangioma consisted of clusters of proliferated small
vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the
mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis
revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1
and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other
anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs)
using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were
not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while
the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1.
Conclusions/Significance: Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile
hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for
cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in
the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma.
Citation: Nakashima T, Jinnin M, Etoh T, Fukushima S, Masuguchi S, et al. (2010) Down-Regulation of mir-424 Contributes to the Abnormal Angiogenesis via
MEK1 and Cyclin E1 in Senile Hemangioma: Its Implications to Therapy. PLoS ONE 5(12): e14334. doi:10.1371/journal.pone.0014334
Editor: Christophe Egles, Tufts University, United States of America
Received June 25, 2010; Accepted October 8, 2010; Published December 14, 2010
Copyright: ? 2010 Nakashima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by grants from Rohto Award, from JSID’s Fellowship Shiseido Award, and from Mitsubishi Pharma Research
Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: This study was supported in part by grants from a commercial source (Rohto, Shiseido and Mitsubishi Pharma). The funders do not alter
the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Mature blood vessels are composed of two distinct cell types: a
continuous monolayer of endothelial cells (ECs) forming the inner
surface of the vessel wall and an outer layer of perivascular
supporting cells including pericytes and smooth muscle cells .
On the other hand, the term ‘vascular anomalies’ generically
indicates various conditions including developmental error or
dysregulated developmental processes of vascular morphogenesis.
According to a classification proposed by Mulliken and Glowacki
in 1982 and 1996, cutaneous vascular anomalies can be divided
into vascular tumor characterized by cellular hyperplasia (too
many normal cells), and vascular malformations characterized by
enlargement of dysplastic vessels . Vascular tumors include
infantile hemangioma, kaposiform hemangioendothelioma, and
tufted angioma. Vascular malformations are further classified into
capillary, venous, lymphatic, and arteriovenous malformations.
Malignant vascular tumors such as angiosarcoma or Kaposi’s
sarcoma were not included in this classification.
Senile hemangioma, so-called cherry angioma, is a smooth
reddish dome-shaped tumor, mainly found on the trunk of the
elderly person . A venous lake is also smooth dark bluish dome-
shaped papule/nodule that appears on the lower lip, face and ears
. They are known as the most common vascular anomalies
specifically seen in the aged skin. These tumors are usually
asymptomatic, but sometimes become problematic because of
bleeding and disfigurement. However, there have been few
therapeutic options, such as surgical resection or laser treatments,
in spite of recent advances in the development of anti-angiogenic
therapies against various vascular anomalies [5–7].
These tumors are not described in the above classification
system, and the pathogenesis of these tumors has been poorly
investigated. Venous lake is frequent in lower lip, indicating the
correlation with sunlight [8,9]. On the other hand, senile
hemangioma is not likely to be associated with UV exposure
because of their distribution on the trunk. Tuder et al. reported
that senile hemangiomas are overgrowths made up of ECs with
terminal differentiation, based on the low immunoreactivity of
PLoS ONE | www.plosone.org1December 2010 | Volume 5 | Issue 12 | e14334
tumor ECs with Ki-67 and activation-related antibody in vivo and
in vitro . Thus, the tumor is thought to have different etiology
from abnormal angiogenesis seen in intrinsic aged skin or
photoaged skin, which is characterized by an age-dependent
reduction of cutaneous microvasculature [11,12].
In this study, we aimed to clarify the pathogenesis of these
tumors. First, we tried to characterize these tumors based on the
above classification system, and presented that senile hemangioma
is vascular tumor and venous lake is vascular malformation. We
then investigated the mechanism(s) underlying the abnormally
increased endothelial proliferation in senile hemangioma, focusing
on microRNA (miRNA). miRNAs, short ribonucleic acid molecules
on average only 22 nucleotides long, are post-transcriptional
regulators that bind to complementary sequences in the three
prime untranslated regions (39 UTRs) of mRNAs, leading to gene
silencing. There are thought to be more than 1000 miRNAs in the
human genome, which may target about 60% of mammalian genes
. Recent vigorous efforts of research in this field indicated that
miRNAs play a role in angiogenesis as well as immune response or
carcinogenesis in vivo [14–18]. Our study demonstrated a
regulatory mechanism of angiogenesis in the aged skin by miRNAs.
Materials and Methods
Patient material and Ethics Statement
This research was approved by the Ethics Review Committee in
Kumamoto University (No. 177). Skin specimens were obtained
from 7 senile hemangioma, 3 venous malformation, 4 angiosar-
coma, 4 venous lake, and 3 infantile hemangioma (Table 1). Seven
control skin samples are obtained from routinely discarded skin of
healthy human subjects undergoing skin graft. Control and patient
samples were collected and processed immediately after resection
in parallel. Written informed consent was obtained according to
the Declaration of Helsinki.
RNA isolation and quantitative real-time polymerase
chain reaction (PCR)
Skin specimens were obtained from 7 senile hemangioma, 3
venous malformation, 4 angiosarcoma, 4 venous lake, 3 infantile
hemangioma and 7 healthy controls. They were fixed in 10%
neutral-buffered formalin, embedded in paraffin, and sliced. Total
RNA isolation from paraffin-embedded section of normal skin and
tumor tissues were performed with RNeasy FFPE kit (Qiagen,
Valencia, CA) following the manufacturer’s instructions. cDNA
was synthesized from total RNA with PrimeScript RT rea(Takara
Bio Inc, Shiga, Japan). Quantitative real-time PCR with a Takara
Thermal Cycler Dice (TP800)H used primers and templates mixed
with the SYBR Premix Ex gent Kit TaqII (Takara Bio Inc).
Primer sets for vascular endothelial growth factor receptor
(VEGFR) 1, VEGFR2 and GAPDH were purchased from SA
Biosciences (Frederick, MD). Primers for HIF-1a, MEK1 and
cyclin E1 were from Takara. These primer sets were prevalidated
to generate single amplicons. MEK1 was amplified for 50 cycles of
denaturation for 5s at 95uC, annealing for 30s at 70uC, whereas
annealing temperature was set at 60uC for the other primers. Data
generated from each PCR reaction were analyzed using Thermal
Cycler Dice Real Time System ver2.10B (Takara Bio Inc).
Specificity of reactions was determined by melting curve analysis.
Transcript levels were normalized to GAPDH.
miRNA extraction and PCR array analysis of miRNA
Small RNAs were extracted using a miRNeasy FFPE kit
(Qiagen). Then, RNAs were reverse-transcribed into first strand
cDNA using an RT2miRNA First Strand Kit (SABiosciences,
Frederick, MD). For RT2Profiler PCR Array (SABioscience), the
cDNA was mixed with RT2SYBR Green/ROX qPCR Master
Mix and the mixture was added into a 96-well RT2miRNA PCR
Array (SABiosciences) that included primer pairs for 88 human
miRNAs. PCR was performed on a Takara Thermal Cycler Dice
(TP800H) following the manufacture’s protocol. Threshold cycle
(Ct) for each miRNA was extracted using Thermal Cycler Dice
Real Time System ver2.10B. The raw Ctwas normalized using the
values of small RNA housekeeping genes.
For quantitative real-time PCR, primers for mir-424, mir-1 or
U6 (SABioscience) and templates were mixed with the SYBR
Premix Ex TaqII (Takara Bio Inc). DNA was amplified for 50
cycles of denaturation for 5s at 95uC, annealing for 30s at 60uC.
Data generated from each PCR reaction were analyzed using
Thermal Cycler Dice Real Time System ver2.10B (Takara Bio
Inc). Transcript levels were normalized to U6.
Paraffin sections were deparaffinized in xylen and rehydrated in
a graded ethanol series. Antigens were retrieved by incubation
with citrate buffer pH 6 for 5 min with microwave oven.
Endogenous peroxidase activity was inhibited, after which sections
were incubated with 5% milk for 30min and then reacted with the
antibodies for a-smooth muscle actin or type IV collagen (Abcam,
Cambridge, MA, 1:300) overnight at 4uC. After excess antibody
was washed out with PBS, samples were incubated with HRP-
labeled goat anti-mouse antibody (Nichirei, Tokyo, Japan) for
60min. The reaction was visualized by the diaminobenzidine
Table 1. Clinical data: ages of patients at the time of
resection and location of the lesions.
senile hemangioma1M 79trunk
3M 45upper extremity
5M 57upper extremity
7F 69 face
venous malformaiton1F 66 upper extremity
3M 50upper extremity
venous lake1M 55 lip
infantile hemangioma1F0 lower extremity
2M2 upper extremity
Sample No.5 and No.6 of senile hemangioma were obtained from same patient.
mir-424 in Senile Hemangioma
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substrate system (Dojin, Kumamoto, Japan). Slides were counter-
stained with Mayer’s haematoxylin, and examined under a light
microscope (OLYMPUS BX50, Tokyo, Japan).
In situ hybridization
In situ hybridization was performed with 59-locked digoxigenin-
labeled nucleic acid (LNA) probes complementary to human
mature mir-424 and scrambled negative control (Exiqon, Vedbaek,
Denmark) [19,20]. Briefly, human tissues were deparaffinized and
deproteinized with protease K for 5 min. Slides were then washed
in 0.2% Glycine in PBS and fixed with 4% paraformaldehyde.
Hybridization was performed at 48uC overnight followed by
blocking with 2% fetal bovine serum and 2% bovine serum
albumininPBSand 0.1% Tween 20(PBST) for1 hour. The probe-
target complex was detected immunologically by a digoxigenin
antibody conjugated to alkaline phosphatase acting on the
chromogen nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate (Roche Applied Science, Mannheim, Germany). Slides
were counterstained with nuclear fast red, and examined under a
light microscope (OLYMPUS BX50; Tokyo, Japan).
Paraffin sections were deparaffinized in xylen and rehydrated in
a graded ethanol series. Antigens were retrieved by incubation
with 0.1% trypsin at 37uC for 5 min. The slides were
permeabilized in 0.5% Triton-PBS for 5min and blocked in 5%
nonfat dry milk for 30min at room temperature. As the primary
antibodies, rabbit anti-MEK1 (1:50, Santa Cruz Biotechnology) or
rabbit anti-cyclin E1 (1:50, Santa Cruz Biotechnology) with mouse
anti-CD34 (1:25, DakoCytomation, Carpinteria, CA) diluted in
5% milk in PBS, were applied to the sections. The sections were
incubated overnight at 4uC, followed by PBS-0.05% Triton X-100
washes. Matching isotype IgG was used as a negative control.
Then, Alexa Fluor 488 anti-rabbit (1:200, Invitrogen, Carlsbad,
CA) and Rhodamine-conjugated anti-mouse secondary antibodies
(1:100, Jackson ImmunoResearch, Suffolk, UK) were applied to
the sections. After 1 hour at room temperature, sections were
washed and mounted with VECTASHIELD mounting medium
with DAPI (Vector, Burlingame, CA). Zeiss Axioskop 2 micro-
scope (Carl Zeiss, Oberkochen, Germany) was used for fluores-
Figure 1. Clinical presentation and histological findings of vascular anomalies. Panel (a) shows clinical pictures of senile hemangioma (SH),
vascular malformation (VM), angiosarcoma (AS), venous lake (VL) and infantile hemangioma (IH). Panel (b) (magnification,610) and (c) (magnification,
6100) show hematoxylin-eosin staining of each anomalies. SH showed clusters of proliferated small vascular channels in upper dermis and dilated
vessels in the deep dermis. AS consisted of diffuse proliferation of atypical ECs accompanied with irregular vessel-like spaces. Diffuse proliferation of
tumor cells and dilated vessels were seen in IH. VM and VL were characterized by thin-walled, dilated vessels accompanied with thrombosis
throughout the dermis.
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Figure 2. The expression of angiogenic factors or the structure of vessels. (a) Mean relative transcript levels of HIF-1a, VEGFR1 or VEGFR2
(normalized to GAPDH) in tissues from 7 normal skin (normal) or vascular anomalies such as 7 senile hemangioma (SH), 3 vascular malformation (VM),
4 angiosarcoma (AS), 4 venous lake (VL) and 3 infantile hemangioma (IH) by real-time quantitative PCR. The transcript levels in samples of normal skin
were set at 1. Error bars represent SD of +1. (b) The expression of a-smooth muscle actin (SMA) or type IV collagen (COL4) in the affected vessels of
senile hemangioma (SH), vascular malformation (VM) and venous lake (VL). Paraffin sections were subjected to immunohistochemical analysis with
antibodies against a-SMA or COL4 as described in ‘Materials and Methods’. Results are representative of several cases.
mir-424 in Senile Hemangioma
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Table 2. The expression profiles of miRNAs as measured with
the PCR array.
let-7d2.59 1.73 6.79
miR-9 5.765.01 6.66
miR-18b 3.132.23 7.01
miR-24 0.24 0.16 1.03
miR-141 5.37 5.286.42
miR-194 5.69 6.026.90
miR-196a 2.42 1.874.80
miR-2231.28 0.15 0.87
miR-518b 2.291.30 20.78
A mixture of equal amounts of miRNAs from 3 senile hemangioma, 3 venous
malformation or 3 angiosarcoma were prepared, and miRNA expression profile
in each tumor in vivo was evaluated using RT2Profiler PCR Array. The raw
threshold cycle (Ct) was normalized using the values of small RNA
housekeeping genes. DDCt (the raw Ct of each miRNA – Ct of small RNA
housekeeping genes) were shown.
Table 2. Cont.
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Human dermal microvascular ECs (HDMECs, CC-2543) were
obtained from Lonza (Walkersville, MD). HDMECs were grown
on 0.2% gelatin-coated dishes in EGM-2 (Clonetics, San Diego,
CA) with 20% fetal bovine serum (Hyclone, Logan, UT) and
Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA) in 5% CO2at
37uC [21,22]. Cells were passaged 1:3 every 4 to 6 days and used
between passages 3 to 9.
The transient transfection
siRNAs against MEK1 or cyclin E1 was purchased from Santa
Cruz Biotechnology. miRNA inhibitors were from Qiagen.
Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) was used
as transfection reagent. For reverse transfection, siRNA (6pmol) or
miRNA inhibitor (60pmol) mixed with transfection reagent were
added when cells were plated, followed by incubation for 48–
72 hours at 37uC in 5% CO2. Control experiments showed
transcript levels for target genes or miRNAs to be reduced by
.80% (data not shown).
Cell lysis and immunoblotting
HDMEC were washed with cold PBS twice and lysed in lysis
buffer (Denaturin Cell Extraction Buffer, BIOSOURCE, Camar-
illo, CA). Aliquots of cell lysates (normalized for protein
concentrations as measured by the Bio-Rad reagent) were
separated on sodium dodecyl sulfate-polyacrylamide gels and
transferred to PVDF membranes. The membranes were blocked
for 1 hour and incubated overnight at 4uC with antibody for
MEK1 (Santa Cruz Biotechnology, Santa Cruz, CA) or cyclin E1
(Santa Cruz Biotechnology). The membranes were washed in Tris-
buffered saline (TBS) and 0.1% Tween 20, incubated with
secondary antibody, and washed again. The detection was
performed using the ECL system (Amersham, Arlington Heights,
IL) according to the manufacturer’s recommendations. As a
loading control, immunoblotting was also performed using
antibodies against b-actin.
HDMECs were detached from the wells by trypsin treatment
and counted using a CoulterH Particle Counter (Beckman Coulter,
Fullerton, CA) [22,23].
Statistical analysis was carried out with the Mann-Whitney test
for comparison of means. P values less than 0.05 were considered
Figure 3. The transcript mir-424 level in senile hemangioma. Box-and-whisker plots of mir-424 levels in normal skin (normal), senile
hemangioma (SH), vascular malformation (VM) and angiosarcoma (AS) by real-time PCR. Boxes represent the interquartile ranges and the lines
emanating from each box (the whiskers) extend to the smallest and largest observations in a group. Dotted bars show medians. The median
transcript level in samples of normal skin was set at 100. * P,0.05 as compared with the values in normal skin, VM or AS.
Figure 4. The immunoreactivity for mir-424 in senile heman-
gioma. In situ detection of mir-424 in paraffin-embedded, formalin-
fixed tissues of normal skin (a) and senile hemangioma (SH, b). Nucleus
was counterstained with nuclear fast red (magnification, 6200). The
immunoreactivity for mir-424 (blue) is indicated by arrows. Results are
representative of 3 normal skins and 3 SH.
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Pathologic features of senile hemangioma and venous
As shown in Fig. 1, histologically, senile hemangioma showed
clusters of proliferated small vascular channels in upper dermis.
Some of the vessels were dilated, especially in the deep dermis.
The overall appearance with proliferative nature of ECs was also
seen in infantile hemangioma and angiosarcoma but not in venous
malformation. On the other hand, histopathologic examination of
venous lake revealed thin-walled, dilated vessels accompanied with
thrombosis throughout the dermis, which is similar to venous
malformation. Thus, senile hemangioma is likely to be categorized
as vascular tumor, whereas venous lake seems to be one of the
The expression of angiogenic factors or the structure of
vessels are maintained in senile hemangioma and
Next, we tried to characterize these vascular anomalies further.
Up-regulation of HIF-1a and down-regulation of VEGFR1 is
reported to be characteristic to infantile hemangioma while down-
regulation of VEGFR1 is also seen in angiosarcoma, and such
abnormal expression of angiogenic factors are thought to play some
senile hemangioma with infantile hemangioma or angiosarcoma as
described above, we compared the expression levels of these factors
by real-time PCR using total RNA derived from sections of normal
skin and various vascular anomalies (Fig. 2a). Although the mRNA
levels of HIF-1a, VEGFR1 and VEGFR2 were increased in SH
tissue and were not increased in VL tissue compared with those in
normal skin, the values were quite variable and there was no
normal skin or other vascular anomalies. Thus, these angiogenic
factors are not likely to correlate with the formation of senile
hemangioma and venous lake.
Also, we determined whether the structure of vessels including
smooth muscle cells and the basement membranes is normally
maintained in senile hemangioma or venous lake. Immunohisto-
chemical staining showed no abnormality in the expression and
distribution of a-smooth muscle actin (SMA) and type IV collagen
on the proliferated small capillaries of senile hemangioma, while a-
SMA expression in venous malformation partly disappeared and
became irregular, causing dilated vessels (Fig. 2b), as described
previously . To note, most of the affected vessels of venous
lake, which has similar dilated vessels to venous malformation,
showed intact a-SMA expression. These results indicate that
angiogenic factors or vessel structures are not likely to be
associated with the formation of senile hemangioma and venous
lake. Although venous lake is similar to venous malformation
histologically, the etiology seems to be different.
miRNA expression profile in senile hemangioma
Then, we examined why the ECs of senile hemangioma are
proliferated. We expected miRNA as the angiogenic factor which
induce the proliferation of ECs in senile hemangioma. A mixture
of equal amounts of miRNAs from 3 senile hemangioma, 3 venous
malformation or 3 angiosarcoma were prepared, and miRNA
expression profile in each tumor in vivo was evaluated using
miRNA PCR array, consisting of 88 miRNAs involved in human
cell differentiation and development (Table 2). There were several
overexpressed or suppressed miRNAs specifically in senile
hemangioma. Among them, the expression of mir-424 in senile
hemangioma was decreased compared to that in venous
malformation (2-cycle difference in DDCT method) or angiosar-
coma (1-cycle). Real-time PCR using specific primer for mir-424
with increased number of samples (7 senile hemangioma, 3 venous
malformation and 4 angiosarcoma) revealed that the mir-424 level
in senile hemangioma was further lower than normal skin or other
vascular anomalies (Fig. 3). In addition, in situ hybridization
showed that signal for mir-424 was evident in normal vessels, but
not in the ECs of senile hemangioma (Fig. 4). To note, we
determined mir-424 expression in skin samples from young and
old healthy controls, but there was no significant difference
between young and old skin (data not shown). These results
suggest that decreased mir-424 level in vivo is specific to senile
Low mir-424 expression leads to MEK1- and cyclin E1-
dependent cell proliferation in senile hemangioma
We tried to determine the role of mir-424 in the formation of
senile hemangioma. According to miRNA target gene predictions
of mir-424 using the TargetScan (version 5.1, http://www.
targetscan.org/), a leading program in the field , we focused
Figure 5. The transcript levels of MEK1 and cyclin E1 in senile hemangioma. Mean relative transcript levels of MEK1 (white bars) and cyclin
E1 (black bars) in the tissues from normal skin (normal) or vascular anomalies such as senile hemangioma (SH), vascular malformation (VM),
angiosarcoma (AS), venous lake (VL) and infantile hemangioma (IH) by real-time quantitative PCR. The transcript levels in normal skin were set at 1.
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on MEK1 and cyclin E1 as the target genes of mir-424. MEK1
(mitogen-activated protein kinase kinase) is known as the upstream
molecule of ERK and one of the most important mitogenic
regulators . Cyclin E1 is also implicated in the regulation of
cell cycle . We expected that decreased mir-424 expression
causes increased cell proliferation via MEK1 and cyclin E1 in
Then, we examined the mRNA levels of MEK1 or cyclin E1 in
the tissue section. Although there was no significant difference in
the mRNA levels between the section of senile hemangioma and
those of other vascular anomalies (Fig. 5), protein expression of
MEK1 (Fig. 6a) or cyclin E1 (Fig. 6b) on the proliferated vessels of
senile hemangioma was increased compared to normal skin or
venous malformation. To note, because CD34 (red) is expressed in
cell surface whereas MEK1 and cyclin E1 (green) is in cytoplasm
or nucleus, they did not co-localize [30,31]. Considering that
miRNAs usually inhibit translation of their target genes and do not
cause degradation of the target transcript, our results indicate that
mir-424 down-regulates MEK1 and cyclin E1 at the translation
levels without altering mRNA levels, and that decreased mir-424
results in the overexpression of MEK1 and cyclin E1 in senile
To further investigate the association of mir-424 with MEK1 or
cyclin E1, normal human dermal microvascular ECs (HDMECs)
were transfected with mir-424 inhibitor and the expression of
MEK1 and cyclin E1 was evaluated. The inhibition of mir-424 in
Figure 6. The immunoreactivity for MEK1 and cyclin E1 in senile hemangioma. Increased expression of MEK1 (a) or cyclin E1 (b) in ECs of
senile hemangioma. Sections of normal skin or vascular anomalies were stained with antibodies against MEK1 or cyclin E1 (green) and CD34 (red). SH;
senile hemangioma, VM; vascular malformation.
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vitro resulted in the increase of protein expression of MEK1 or
cyclin E1, while mRNA levels were not significantly affected by the
inhibitor (Fig. 7a and b), which is consistent with above result in
vivo (Fig. 5 and 6).
Lastly, we determined whether mir-424 can be involved in the
regulation of proliferative activity of ECs via MEK1 and cyclin E1.
Specific inhibitor of mir-424 induced the cell proliferation
significantly, whereas mir-1, which was also decreased in senile
hemangioma by miRNA array analysis (see Table 2), did not
(Fig. 7c). On the other hand, the transfection of siRNA for MEK1
or cyclin E1 led to slight but significant decrease in cell number
(Fig. 7d). Taken together, decreased mir-424 expression and
increased levels of MEK1 or cyclin E1 in senile hemangioma may
cause abnormal cell proliferation in the tumor.
In this study, we have presented two major findings.
First, we showed that senile hemangioma and venous lake is
likely to be categorized in vascular tumor and vascular
malformation, respectively. The vessels of senile hemangioma
are reported to be characterized by both proliferation and dilation
[32–34]. Thus, it seems to be still unclear whether the tumor is
either vascular tumor or malformation. The generic term
‘hemangioma’, classically used as a synonym of ‘vascular tumor’
in the classification above, has been frequently and incorrectly
used by non-expert clinicians to describe different kinds of vascular
anomalies. Confusion due to the persistent use of inappropriate
and misleading terms still occurs: ‘‘hemangioma’’ simplex or
cavernous ‘‘hemangioma’’ are terms still in use to name a capillary
malformation or venous malformation, respectively. Our study
may contribute to further understanding of the classification of
vascular anomalies. On the other hand, venous lake are known to
consists of dilated preexisting vessels, but there has been no
obvious definition . We showed that venous lake is similar to
venous malformation, but the smooth muscle cells were main-
tained in venous lake. Further studies should be needed to clarify
the cause of vessel dilation in this tumor.
Second, we found that mir-424 level was significantly reduced in
senile hemangioma compared to normal skin and other vascular
anomalies. mir-424 regulated cell proliferation via the silencing of
the expression of MEK1 and cyclin E1. Recently, several miRNAs
including let7-f, miR-27b and mir-130a have been implicated in
angiogenesis [14,15]. This is the first report implicating mir-424 in
the angiogenic process and identifying MEK1 and cyclin E1 as the
true target genes of mir-424. MEK1 and cyclin E1 may belong to
the same signaling pathway to regulate cell cycle, because MEK1
is the up-stream molecule of ERK and cyclin E1 is down-stream
target of ERK [36,37]. Thus, mir-424/MEK1/cyclinE1 pathway
may be able to regulate cell proliferation effectively. The effect of
mir-424 inhibitor or siRNA for MEK1 or cyclin E1 on cell
proliferation was significant, but moderate (20–40%, Fig. 7).
However, for example, proliferative activity of ECs isolated from
infantile hemangioma was increased only by 30–40%, compared
with those of HDMECs , indicating that such modest increase
of cell proliferation in vitro can cause hemangioma formation in
As reported previously, Val-Bernal et al. concluded that senile
hemangiomas is a tissue overgrowth composed of mature vessels,
lined by ECs with virtually no turnover . Our results may
sound contradictory to the report. However, this difference may be
explained by the natural history of this tumor: Donsky et al.
reported that microscopic examination of senile hemangioma in
the early stage showed numerous newly formed capillaries and
later these capillaries dilate . The tumor in the previous report
by Val-Bernal et al. seems to be late stage, because of the increased
dilated vessels. Proliferative activation of ECs may be specific to
early stage and it may disappear at the late stage.
Senile hemangioma may be the good model for cutaneous
angiogenesis. Investigation of senile hemangioma and the
regulatory mechanisms of angiogenesis by miRNA in the aged
skin may lead to new treatments using miRNA by the transfection
into senile hemangioma.
We thank Ms. Junko Suzuki and Ms. Chiemi Shiotsu for their valuable
Conceived and designed the experiments: TN. Performed the experiments:
TN MJ TE SF SM KM YI TI HI. Analyzed the data: TN MJ TE SF SM
KM YI TI HI. Wrote the paper: TN.
1. Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and
maintenance. Neuro Oncol 7: 452–464.
2. Mulliken JB, Glowacki J (1982) Hemangiomas and vascular malformations in
infants and children: a classification based on endothelial characteristics. Plast
Reconstr Surg 69: 412–423.
4. Bu J, Shi H, Hu M, Liu H (2002) Oral venous lakes: a clinicopathologic analysis
of 20 cases. Zhonghua Kou Qiang Yi Xue Za Zhi 37: 33–35.
5. Bernstein EF (2009) The pulsed-dye laser for treatment of cutaneous conditions.
G of Ital Dermatol Venereol 144: 557–572.
6. Fodor L, Ramon Y, Fodor A, Carmi N, Peled IJ, et al. (2006) A side-by-side
prospective study of intense pulsed light and Nd:YAG laser treatment for
vascular lesions. Ann Plast Surg 56: 164.
7. Paller AS (2000) Responses to anti-angiogenic therapies. J Investing Dermatol
Symp Proceed 5: 83.
8. Neumann RA, Knobler RM (1990) Venous lakes (Bean-Walsh) of the lips–
treatment experience with the argon laser and 18 months follow-up. Clin Exp
Dermatol 15: 115–118.
9. Jay H, Borek C (1998) Treatment of a venous-lake angioma with intense pulsed
light. Lancet 351: 112.
Figure 7. The association of mir-424 with MEK1 or cyclin E1. (a, b) mir-424 inhibitor induces the protein expression of MEK1 and cyclin E1 but
not mRNA expression in human dermal microvascular ECs. Cells were transfected with the control inhibitor or mir-424 inhibitor for 48 hours. (a)
Relative amounts of transcripts (normalized with GAPDH) determined in total RNA with quantitative PCR. Error bars represent SD of +1. (b) Lysates
from cells transfected with control or mir-424 inhibitor were subjected to immunoblotting with antibody for MEK1 or cyclin E1. The same membrane
was then stripped and reprobed with anti b-actin antibody as a loading control. (c, d) mir-424/MEK1/cyclin E1 pathway altered the cell proliferation
activity. (c) HDMECs at a density of 56103cells/well in 24-well culture plates were transfected with control inhibitor or inhibitors specific for mir-424
or mir-1. After 48 hours, the number of cells was counted with a ColterH Particle Counter (Beckman Coulter, Fullerton, CA) as described in ‘Materials
and Methods’. The value in the cells transfected with control inhibitor were set at 1. The mean and SD from 3 separate experiments are shown.
* P,0.05 in comparison to the value in the cells transfected with control inhibitor. (d) HDMECs at a density of 16104cells/well in 24-well culture
plates were transfected with control siRNA or siRNA specific for MEK1 or cyclin E1. After 72 hours, the cell number was counted as described in Fig. 7c.
mir-424 in Senile Hemangioma
PLoS ONE | www.plosone.org10December 2010 | Volume 5 | Issue 12 | e14334
10. Tuder RM, Young R, Karasek M, Bensch K (1987) Adult cutaneous
hemangiomas are composed of nonreplicating endothelial cells. J Invest
Dermatol 89: 594–597.
11. Chung JH, Eun HC (2007) Angiogenesis in skin aging and photoaging.
J Dermatol 34: 593–600.
12. Kelly RI, Pearse R, Bull RH, Leveque JL, de Rigal J, et al. (1995) The effects of
aging on the cutaneous microvasculature. J Am Acad Dermatol 33: 749–756.
13. Friedman RC, Farh KK, Burge CB, Bartel DP (2009) Most mammalian
mRNAs are conserved targets of microRNAs. Genome Res 19: 92–105.
14. Kuehbacher A, Urbich C, Dimmeler S (2008) Targeting microRNA expression
to regulate angiogenesis. Trends Pharmacol Sci 29: 12–15.
15. Chen Y, Gorski DH (2008) Regulation of angiogenesis through a microRNA
(miR-130a) that down-regulates antiangiogenic homeobox genes GAX and
HOXA5. Blood 111: 1217–1226.
16. Furer V, Greenberg JD, Attur M, Abramson SB, Pillinger MH (2010) The role
of microRNA in rheumatoid arthritis and other autoimmune diseases. Clin
Immunol. In press.
17. Liston. A, Linterman M, Lu LF (2010) MicroRNA in the Adaptive Immune
System, in Sickness and in Health. J Clin Immunol. In press.
18. Davidson-Moncada J, Papavasiliou FN, Tam W (2010) MicroRNAs of the
immune system: roles in inflammation and cancer. Ann N Y Acad Sci 1183:
19. Martin MM, Buckenberger JA, Jiang J, Malana GE, Nuovo GJ, et al. (2007) The
human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates
microrna-155 binding. J Biol Chem 282: 24262–24269.
20. Nuovo GJ (2008) In situ detection of precursor and mature microRNAs in
paraffin embedded, formalin fixed tissues and cell preparations. Methods 44:
21. Jinnin M, Medici D, Park L, Limaye N, Liu Y, et al. (2008) Suppressed NFAT-
dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile
hemangioma. Nat Med 14: 1236–1246.
22. Makino T, Jinnin M, Muchemwa FC, Fukushima S, Kogushi-Nishi H, et al.
(2010) Basic fibroblast growth factor stimulates the proliferation of human
dermal fibroblasts via the ERK1/2 and JNK pathways. Br J Dermatol. In press.
23. Jinnin M, Ihn H, Asano Y, Yamane K, Trojanowska M, et al. (2006) Platelet
derived growth factor induced tenascin-C transcription is phosphoinositide 3-
kinase/Akt-dependent and mediated by Ets family transcription factors. J Cell
Physiol 206: 718–727.
24. Kleinman ME, Greives MR, Churgin SS, Blechman KM, Chang EI, et al.
(2007) Hypoxia-induced mediators of stem/progenitor cell trafficking are
increased in children with hemangioma. Arterioscler Thromb Vasc Biol 27:
25. Brown LF, Tognazzi K, Dvorak HF, Harrist TJ (1996) Strong expression of
kinase insert domain-containing receptor, a vascular permeability factor/
vascular endothelial growth factor receptor in AIDS-associated Kaposi’s
sarcoma and cutaneous angiosarcoma. Am J Pathol 148: 1065–1074.
26. Mulliken JB, Fishman SJ, Burrows PE Vascular anomalies., Mosby, St. Louis,
MO, USA. pp 517–584.
27. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by
adenosines, indicates that thousands of human genes are microRNA targets. Cell
28. Greulich H, Erikson RL (1998) An analysis of Mek1 signaling in cell
proliferation and transformation. J Biol Chem 273: 13280–13288.
29. Mazumder S, DuPree EL, Almasan A (2004) A dual role of cyclin E in cell
proliferation and apoptosis may provide a target for cancer therapy. Curr
Cancer Drug Targets 4: 65–75.
30. Zheng CF, Guan KL (1994) Cytoplasmic localization of the mitogen-activated
protein kinase activator MEK. J Biol Chem 269: 19947–19952.
31. Jackman M, Kubota Y, den Elzen N, Hagting A, Pines J (2002) Cyclin A- and
cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm. Mol
Biol Cell 13: 1030–1045.
32. Sheth S, Lai CK, Dry S, Binder S, Fishbein MC (2008) Benign vascular tumors
and tumor-like proliferations. Semin Diagn Pathol 25: 1–16.
33. Braverman IM, Ken-Yen A (1983) Ultrastructure and three-dimensional
reconstruction of several macular and papular telangiectases. J Invest Dermatol
34. Wolf IH (2002) Dermoscopic diagnosis of vascular lesions. Clin Dermatol 20:
35. Requena L, Sangueza OP (1997) Cutaneous vascular anomalies. Part I.
Hamartomas, malformations, and dilation of preexisting vessels. J Am Acad
Dermatol 37: 523–549.
36. Keenan SM, Bellone C, Baldassare JJ (2001) Cyclin-dependent kinase 2
nucleocytoplasmic translocation is regulated by extracellular regulated kinase.
J Biol Chem 276: 22404–22409.
37. Ugland H, Boquest AC, Naderi S, Collas P, Blomhoff HK (2008) cAMP-
mediated induction of cyclin E sensitizes growth-arrested adipose stem cells to
DNA damage-induced apoptosis. Mol Biol Cell 19: 5082–5092.
38. Boye E, Yu Y, Paranya G, Mulliken JB, Olsen BR, et al. (2001) Clonality and
altered behavior of endothelial cells from hemangiomas. J Clin Invest 107:
39. Val-Bernal JF, Martino M, Mayorga M, Garijo MF (2007) Prichard’s structures
of the fossa ovalis are age-related phenomena composed of nonreplicating
endothelial cells: the cardiac equivalent of cutaneous senile angioma. APMIS
40. Donsky HJ (1968) Vascular tumours of the skin. Can Med Assoc J 99: 993–1000.
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