Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its implications to therapy.

Abstract

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.
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.
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.

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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
Abstract
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: mjin@kumamoto-u.ac.jp
Introduction
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 [1].
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 [2]. 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 [3]. A venous lake is also smooth dark bluish dome-
shaped papule/nodule that appears on the lower lip, face and ears
[4]. 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
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tumor ECs with Ki-67 and activation-related antibody in vivo and
in vitro [10]. 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
[13]. 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
expression
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.
Immunohistochemical staining
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.
sex agelocation
senile hemangioma1M 79trunk
2M 60 neck
3M45upper extremity
4M75trunk
5M57upper extremity
6 trunk
7F 69face
venous malformaiton1F66upper extremity
2F78face
3M50upper extremity
angiosarcoma1F 77 head
2M 80head
3M 86 head
4M 74head
venous lake1M 55lip
2F 63 lip
3M 54cheek
4F 44 lip
infantile hemangioma1F0 lower extremity
2M2 upper extremity
3F3face
Sample No.5 and No.6 of senile hemangioma were obtained from same patient.
doi:10.1371/journal.pone.0014334.t001
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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).
Immunofluorescence
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-
cence microscopy.
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.
doi:10.1371/journal.pone.0014334.g001
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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.
doi:10.1371/journal.pone.0014334.g002
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Table 2. The expression profiles of miRNAs as measured with
the PCR array.
DDCt
Senile
hemangioma
Venous
MalformationAngiosarcoma
let-7a
21.62 1.16
20.58
let-7b
20.661.120.66
let-7c2.95 4.342.93
let-7d2.59 1.736.79
let-7e1.263.342.35
let-7f6.925.7710.52
let-7g2.403.172.29
let-7i 2.56 2.172.88
miR-17.20 2.675.67
miR-76.875.895.29
miR-95.765.016.66
miR-10a
20.382.76 2.12
miR-10b
21.11
20.16 0.62
miR-15a 5.35 4.275.91
miR-15b1.31 3.571.18
miR-16
20.44
21.580.00
miR-173.402.344.86
miR-18a1.951.356.46
miR-18b3.132.237.01
miR-20a4.093.454.06
miR-20b3.882.52 9.64
miR-21
23.47
23.78
25.04
miR-224.302.384.41
miR-23b
21.510.38
21.09
miR-240.24 0.161.03
miR-26a
22.38
21.06
21.53
miR-33a6.786.108.13
miR-92aND*
24.35
20.70
miR-932.791.994.83
miR-966.484.045.81
miR-99a1.841.361.96
miR-100
21.240.910.89
miR-1014.122.686.02
miR-1031.961.153.99
miR-106b2.101.725.96
miR-1227.646.29 18.37
miR-1240.48
21.068.14
miR-125a-5p
21.32
20.34
20.39
miR-125b
24.01
24.06
23.12
miR-126
20.04
21.52
20.50
miR-127-5p 4.503.10 13.88
miR-128a3.99 4.424.09
miR-129-5p
22.74
24.046.80
miR-130a1.16
20.3010.36
miR-1321.680.452.05
miR-133b4.433.6017.30
miR-1342.441.225.50
miR-1375.224.34 10.05
DDCt
Senile
hemangioma
Venous
MalformationAngiosarcoma
miR-1415.37 5.286.42
miR-142-3p5.81 4.765.90
miR-142-5p4.08 2.23 10.21
miR-146a 2.33 2.392.67
miR-146b-5p3.612.790.46
miR-1501.572.201.19
miR-1555.854.674.72
miR-181a3.802.596.15
miR-1824.997.733.50
miR-1834.725.174.29
miR-1852.191.086.29
miR-1922.160.756.06
miR-1945.696.026.90
miR-195
20.61
20.45
20.55
miR-196a 2.42 1.874.80
miR-205 0.30 0.13
20.22
miR-2066.181.4710.57
miR-2086.17 5.309.37
miR-210
23.11
24.506.75
miR-214 2.223.563.22
miR-215 1.00
20.8717.88
miR-2183.852.642.21
miR-219-5p3.302.1810.09
miR-2222.852.664.53
miR-2231.280.150.87
miR-301a4.112.9015.12
miR-302a5.214.0213.05
miR-302c7.166.8116.00
miR-345
20.62
21.466.53
miR-3702.221.228.77
miR-371-3p9.748.8511.20
miR-375
21.41
22.475.18
miR-3781.851.544.21
miR-4244.962.95 3.78
miR-4525.894.888.29
miR-4885.243.9315.91
miR-4984.463.1416.82
miR-5033.692.918.80
miR-518b2.291.3020.78
miR-520g9.977.0610.90
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.
doi:10.1371/journal.pone.0014334.t002
Table 2. Cont.
mir-424 in Senile Hemangioma
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