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Thrombosis Research 129 (2012) e70–e76

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Thrombosis Research
journal homepage: www.elsevier.com/locate/thromres

Regular Article

Podoplanin expression in advanced atherosclerotic lesions of human aortas
Kinta Hatakeyama a,⁎, 1, Mika Kato Kaneko b, 1, Yukinari Kato b,⁎, Tetsunori Ishikawa c, Kensaku Nishihira d,
Yuta Tsujimoto b, Yoshisato Shibata d, Yukio Ozaki e, Yujiro Asada aaDepartment of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889–1692 Japan
Molecular Tumor Marker Research Team, The Oncology Research Center, Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi,
Yamagata 990–9585, Japanc
Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889–1692 Japand
Miyazaki Medical Association Hospital, Miyazaki, 738–1 Funato, Shinbeppucho, Miyazaki 880–0834, Japane
Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409–3898 Japanba r t i c l e

i n f oArticle history:
Received 22 November 2011
Received in revised form 28 December 2011
Accepted 3 January 2012
Available online 28 January 2012
Keywords:
podoplanin
atherosclerosis
thrombus formation
macrophagessmooth muscle cells

a b s t r a c t
Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-likereceptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression
in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicatedthat podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smoothmuscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages.
Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was signiﬁcantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanincontributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel moleculartarget for an anti-thrombus drug.
© 2012 Elsevier Ltd. All rights reserved.

Introduction
Thrombus formation on disrupted atherosclerotic plaques leads tothe onset of cardiovascular diseases such as acute myocardial infarction and arteriosclerosis obliterans [1]. It also contributes to atherosclerosis progression. The thrombogenicity of atherosclerotic lesions
is mostly dependent on plaque components. Because the process ofarterial thrombus formation is initiated by platelet adhesion and aggregation, the platelet activation in plaques is critical to the onset ofclinical events [2].
Podoplanin (Aggrus), which is expressed on the surface of several
tumor cells, is an endogenous ligand for C-type lectin-like receptor 2(CLEC-2), and is involved in tumor cell-induced platelet aggregation[3,4]. Although podoplanin is reportedly expressed in normal tissues⁎ Corresponding authors at: Molecular Tumor Marker Research Team, The Oncology
Research Center, Advanced Molecular Epidemiology Research Institute, Yamagata
University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990–9585, Japan. Tel./
fax: + 81 23 628 5295.
E-mail addresses: kpathol@fc.miyazaki-u.ac.jp (K. Hatakeyama),
yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
1
These authors equally contributed to this work.
0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2012.01.003

such as lymphatic endothelial cells, podocytes, and type I alveolar epithelia [5], podoplanin is also known to be overexpressed in various
tumors such as squamous cell carcinomas, testicular seminomas, malignant brain tumors, osteosarcomas, ﬁbrosarcomas, and malignant
mesotheliomas [6–13]. Furthermore, previous reports show that
podoplanin is associated with cell migration [14], epithelial–mesenchymal transition [15], and tumor metastasis [16,17]. Moreover, increased expression of podoplanin relates to tumor malignancy andpoor clinical outcome [11,18–20]. To establish a therapy targeted to
podoplanin, we generated a rat anti-human podoplanin monoclonal
antibody (mAb), NZ-1 [8], which suppressed podoplanin-induced
pulmonary metastasis through inhibition of tumor-induced platelet
aggregation [17,21]. Furthermore, we showed that NZ-1 has not
only high speciﬁcity and sensitivity but also high binding afﬁnity
against podoplanin, making it a candidate for radioimmunotherapy
or immunotoxin therapy [22]. However, podoplanin expression inatherosclerotic lesions has not been investigated. CLEC-2 is expressedon platelets; therefore, expression of podoplanin in atherosclerotic lesions may be involved in the activation of platelets leading tothrombosis.
In this study, we investigated whether podoplanin is expressed inatherosclerotic lesions and whether it is critical to the onset ofAuthor's personal copy
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cardiovascular events. To this end, we examined podoplanin expression
in abdominal aortas using immunohistochemistry, Western-blot, andreal time-PCR analyses.
Materials and methods
Specimens
We examined the abdominal aortas of 31 patients (24 male, sevenfemale; 18–83 years of age, mean 66 years) autopsied at University ofMiyazaki Hospital and Miyazaki Medical Association Hospital(Table 1). The respective Institutional Ethics Committees approved
the study protocol. Postmortem abdominal aortas were removed as
described [23]. Several fresh aortic tissues (2 × 2 cm) were takenfrom various degrees of atherosclerotic lesions. Each tissue was cut
into two specimens. In one specimen of each tissue, the intima of theaortas were separated mechanically from the media, and were storedat −80°C until just before using for Western-blot and real time-PCR
analyses [23,24]. Another specimen was frozen in OCT compound.
Then the tissue sections were stained with hematoxylin and eosin(HE). Atherosclerotic lesions were categorized histologically in threelesions as two early lesions (diffuse intimal thickening (DIT)/initial lesion, fatty streak) and advanced lesions according to AHA classiﬁcation [25]. The DIT, that is considered almost as normal intima ofartery, is composed exclusively of proliferated smooth muscle cells
and extracellular matrix. Fatty streak lesion is DIT with an accumulation of lipid-laden macrophages. The advanced lesion is atheromatous
plaque, which contains proliferated smooth muscle cells, lipid-laden
macrophages, a large amount of extracellular matrix, and the central
necrotic core.
ImmunohistochemistryWith the primary antibodies, 4-μm thick serial sections of 31 aortic
intima were stained immunohistochemically [24]. Brieﬂy, after blocking
endogenous peroxidase activity in 3% hydrogen peroxide in methanol
for 20 min, the sections were incubated with NZ-1, anti-α-smooth muscle actin (SMA, clone 1A4; Dako, Glostrup, Denmark), and anti-CD68(clone PGM-1; Dako). Then, the sections were immunostained with
the EnVision + (Dako) or LSAB kit (Nichirei Corp., Tokyo, Japan). Horseradish peroxidase activity was visualized with 3, 3′- diaminobenzidine
containing hydrogen peroxide. The negative control contained normalmouse IgG or rat serum instead of the primary antibody. Podoplaninpositive cells were identiﬁed with immunohistochemical or immunoﬂuorescent double stain as reported previously [23,26]. To perform

Table 1Proﬁles of autopsy cases.
NumberAge (y; mean ± SD)
Male, n (%)
Cardiovascular death, n (%)
Acute myocardial infarction, nRupture of aortic aneurysm, n
Cerebral infarction, n
Cerebral hemorrhage, nSubarachnoid hemorrhage, nCardiomyopathy, nNon-cardiovascular death, n (%)
Malignancy, n
Idiopathic pulmonary ﬁbrosis, n
Gastrointestinal bleeding, nRenal failure, nPulmonary hemorrhage, nOthers, nsemiquantitative analysis of podoplanin immunoreactivity in various
stages of atherosclerosis, the slides stained with NZ-1 were evaluatedby an experienced, board-certiﬁed pathologist on a four-tiered scale(0–3) for percentage of intimal cells that were positive for podoplanin(staining in more than 30% of intimal cells was scored as 3+; staining
in more than 5% but fewer than 30% of intimal cells was scored as 2+;
staining in fewer than 5% of intimal cells was scored as 1+; lack of staining was scored as 0).
Western-blot analysis
The frozen tissues of 10 aortic intima (early lesions including three
DIT lesions and four fatty streak lesions, and three advanced lesions)were solubilized with lysis buffer (1% Triton in phosphate-bufferedsaline (PBS) and 50 mg/ml aprotinin). They were then electrophoresed under reducing conditions on 10–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd., Osaka, Japan). The separated
proteins were transferred to a nitrocellulose membrane. After blocking with 4% skim milk in PBS, the membrane was incubated with a
rat monoclonal antibody to podoplanin (1 μg/ml; clone NZ-1) [8] ormouse anti-β-actin antibody (1/5,000 dilution; Sigma-Aldrich Corp.,
St. Louis, MO); then it was incubated with peroxidase-conjugatedsecondary antibodies (1/1,000 diluted; GE Healthcare, Buckinghamshire, UK). The proteins were subsequently developed for 3 minusing ECL reagents (GE Healthcare) with X-Omat AR ﬁlm (Eastman
Kodak Co., New York, NY).
Quantitative real-time PCR analysis
Total RNAs were prepared from frozen tissues of 29 aortic intima(16 DIT lesions, three fatty streak lesions, and 10 advanced lesions)with TRIzol (Life Technologies Corp., Grand Island, NY). The initial
cDNA strand was synthesized using SuperScript III transcriptase(Life Technologies Corp.) by priming an oligo-dT primer according
to the manufacturer's instructions. We performed PCR using oligonucleotides: human podoplanin sense (5'-GGAAGGTGTCAGCTCTGCTC3') and human podoplanin antisense (5'-CGCCTTCCAAACCTGTAGTC3'); human β-actin sence (5'-GGCATCCTCACCCTGAAGTA-3') andhuman β-actin antisense (5'-AGGTGTGGTGCCAGATTTTC-3'). Realtime PCR was conducted using the QuantiTect SYBR Green PCR (Qiagen Inc., Hilden, Germany). The PCR conditions were 95 °C for 15 min(1 cycle) followed by 40 cycles of 94 °C for 15 s, 53 °C for 20 s, and
72 °C for 10 s. Subsequently, a melting curve program was appliedwith continuous ﬂuorescence measurement. A standard curve forpodoplanin templates was generated through serial dilution of PCR
products (1 × 10 8 copies/μl to 1 × 10 2 copies/μl). The expression levelof podoplanin was normalized by β-actin. The statistical signiﬁcanceof podoplanin mRNA expression in tissues was determined using
single-tailed, Student's t-tests.
Results

31
66.4 ± 15.2
24 (77)7 (23)
21
1
1
1
1
24 (77)
14
4
2
1
1
2

Immunohistochemical analysis against human atherosclerotic lesionusing an anti-podoplanin antibody
Podoplanin possesses platelet-aggregating activities, which play
crucial roles in thrombosis/hemostasis, tumor metastasis, andTable 2
Results of podoplanin staining by NZ-1 (31 cases).
Stage

DIT
Fatty streakadvanced

No.
of
cases

Podoplanin immunostaining
0

1+

2+

3+

10
6
15

5
2
0

5
3
4

0
1
5

0
0
6

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lymphangiogenesis [3,4]. To date, no report in the literature has described a study of podoplanin expression in human atherosclerotic lesions. We previously produced NZ-1, a rat anti-human podoplanin
monoclonal antibody (mAb), to investigate the relation between podoplanin expression in human cancer and tumor-induced platelet aggregation [8]. Although other anti-podoplanin antibodies (D2-40, 18H5,
AB3) do not possess neutralizing activities, NZ-1 suppressed
podoplanin-induced pulmonary metastasis through inhibition of thetumor-induced platelet aggregation [17,21]. Furthermore, we showedthat NZ-1 has not only high speciﬁcity and sensitivity but also highbinding afﬁnity against podoplanin; KD of NZ-1 is ~1 × 10− 10 M, whereas KD of clone P2-0 is ~1 × 10-8, indicating that the binding afﬁnity of
NZ-1 is about 100 times higher than that of P2-0, although P2-0 also
possesses neutralizing activity [22,27]. Therefore, we selected NZ-1
among several anti-podoplanin mAbs (D2-40, 18H5, AB3, P2-0, NZ-1)
to analyze podoplanin expression in human atherosclerotic lesions.
We ﬁrst performed immunohistochemical analysis using NZ-1 antibody against the abdominal aortas of 31 patients (Table 2). In our

A

E

B

F

C

G

D

H

Fig. 1. Representative immunohistochemical results of advanced atherosclerotic lesions (A-D or E-H are serial sections, respectively) in abdominal aortas. Specimens were stained
by NZ-1 (A, E), anti-SMA (B, F), anti-CD68 (C, G), or rat serum (D, H). Macrophages and smooth muscle cells are positive for podoplanin in advanced lesions (atheromatous plaques). Original magniﬁcation × 400.

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previous study, NZ-1 detected the podoplanin protein of tumor cells
clearly in a membranous staining pattern [8]. As depicted in Fig. 1, podoplanin protein was highly detected in advanced lesions in a membranous or cytoplasmic staining pattern (Fig. 1A and E), whereas none orless than 1% of the intimal cells was immunopositive for podoplanin inearly lesions (Fig. 2A and E). In fatty streak, less than 1% of the intimalcells were immunopositive for podoplanin (Fig. 2E). In advanced lesions,many macrophages (Fig. 1C: serial section of Fig. 1A) and smooth muscle

Acells (Fig. 1F: serial section of Fig. 1E) in the plaques were stained by ananti-CD68 mAb or an anti-SMA mAb, respectively. These data demonstrated that podoplanin is expressed in advanced atherosclerotic lesions
and that it is localized in macrophages or smooth muscle cells. In serial
sections of Fig. 1A, a few smooth muscle cells were found (Fig. 1B),
whereas few macrophages were found in serial sections of Fig. 1E(Fig. 1G). Immunohistochemical and immunoﬂuorescent double staining indicated that podoplanin-positive cells are macrophages or smooth

E
20 µm

B

F

C

G

D

H

Fig. 2. Representative immunohistochemical results of early atherosclerotic lesions (A-D, diffuse intimal thickening; E-H, fatty streak) in abdominal aortas (A-D or E-H are serial
sections, respectively). Specimens were stained by NZ-1 (A, E), anti-SMA (B, F), anti-CD68 (C, G), or rat serum (D, H). Almost all of the intimal cells are negative for podoplanin(A, E) in early lesions, although less than 1% of cells show positive reaction for podoplanin (inset in E). The intima of early atherosclerotic lesions is mainly composed of smoothmuscle cells (B, F), focally with small numbers of macrophages (C, asterisk indicates macrophage) in diffuse intimal thickening/initial lesion or accumulation of macrophages(G) in fatty streak. (Arrows indicate the internal elastic laminae, Original magniﬁcation × 400).

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Fig. 3. Double immunohistochemical staining of podoplanin and macrophages in an atheromatous plaque. Macrophages (brown) are positive for podoplanin (red). Arrows show
double positive cells for anti-CD68/PGM-1 (macrophages) and NZ-1 (podoplanin). Original magniﬁcation × 400.

muscle cells (Figs. 3 and 4). Furthermore, macrophages and smoothmuscle cells do not always express podoplanin in these specimens(Fig. 1). Therefore, small populations of macrophages or smooth muscle
cells might be activated and express podoplanin.
Western-blot and quantitative real-time PCR analyses against humanatherosclerotic lesions
We then performed Western-blot and real-time PCR analyses toinvestigate the podoplanin expression in atherosclerotic lesions. As
presented in Fig. 5, podoplanin was detected in seven of 10 atherosclerotic lesions, which include four fatty streak lesions and three advanced lesions, although no podoplanin expression was observed in
DIT. Furthermore, the expression level of podoplanin in three advanced lesions is much higher than that of four fatty streak lesions,suggesting that podoplanin expression is associated with the progression stage of atherosclerosis. In human cancers, podoplanin expression is also associated with malignant progression and poor clinicaloutcome [11,18–20]. It is particularly interesting that the molecularweight of podoplanin in advanced lesions is apparently larger thanthat in streak lesions, indicating that podoplanin in advanced lesions
is highly glycosylated. Our previous studies showed that glycosylationof podoplanin, especially the disialyl-core 1 structure at Thr52, is critical for its platelet-aggregating activity [28,29]. Therefore, not only
high expression levels of podoplanin, but also a high glycosylationlevel of podoplanin in advanced lesions might be involved in the increased thrombogenicity of atherosclerotic plaques and progressionof atherosclerosis. Next, we conﬁrmed the expression level of podoplanin using quantitative real-time PCR analysis. As presented inFig. 6, the podoplanin mRNA level in advanced lesions (n = 10) was
signiﬁcantly higher (p b 0.05) than that of DIT (n = 16).
Discussion
Acute cardiovascular event usually involves thrombus formation
at the site of a disrupted atherosclerotic plaque. Therefore, thrombogenicity of exposed plaque constituents is critical to the onset of clinical events. Atherosclerotic lesions show predominant expression oftype I and III collagens, which are potent platelet activators [30],and signiﬁcant decrease of CD39, a major metabolic enzyme of extracellular adenosine triphosphate (ATP) and adenosine diphosphate(ADP) [31]. Those data indicate that advanced atherosclerotic lesions
have high platelet-aggregating activity. In addition, tissue factor, atrigger of extrinsic coagulation pathway, is also abundantly expressedin advanced atherosclerotic lesions [23].
In this study, we showed that the podoplanin expression might berelated to thrombogenicity of advanced atherosclerotic lesions. Because podoplanin is a platelet aggregation factor, the evidence suggests that disruption of advanced atherosclerotic lesions promotes
thrombus formation, which leads to the onset of cardiovascular
events. Recent reports of some studies have described that podoplanin is also expressed in stromal myoﬁbroblasts and that it might promote cell migration and invasion [20,32], suggesting that podoplanin
expression in atherosclerotic plaques is associated with vascularremodeling and the progression of atherosclerosis. However, thepodoplanin expression mechanisms in advanced atherosclerotic lesions remain unclear in this study. Results of recent studies showedthat inﬂammatory cytokines such as transforming growth factor-β(TGF-β) and interleukin-3 (IL-3), both of which are known to participate in plaque progression, up-regulate podoplanin expression instromal cells and endothelial cells [12,33]. Therefore, inﬂammatory
responses might play a critical role in podoplanin expression in atherosclerotic plaques, although further studies are necessary to conﬁrm this speculation.
Although many anti-platelet drugs have been developed, direct antiplatelet drugs might leads to bleeding side effects. Identiﬁcation of thefunction and the mechanism of podoplanin expression might support
the use of podoplanin as a molecular target to prevent atherothrombosis.
Conﬂict of interest statement
We have no conﬂict of interest to declare.
Acknowledgements
This work was supported in part by Grants-in-Aid for ScientiﬁcResearch in Japan (20390102, 20590344, 23390084, 23701043,
23791584) from the Ministry of Education, Culture, Sports, ScienceAuthor's personal copy
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DITM

fatty streak advanced

1

2

3

4

5

6

7

8

9 10

1

2

3

4

5

6

7

8

9 10

66-

Podoplanin
45-

M
66-

β-actin45-

Podoplanin

Fig. 5. Western-blot analysis of podoplanin in atherosclerotic lesions of abdominal aortas. Tissue homogenates from DIT lesions (lanes 1–3), fatty streak lesions (lanes 4–7),
and advanced lesions (lanes 8–10) were electrophoresed under reducing conditions
using 10–20% gels, and were Western-blotted with NZ-1 (upper; about 36 kDa) andanti-β-actin (lower).

Podoplanin/β-actin

100

10-1

10-2

10-3

10-4

DIT

Smooth muscle cellfatty streakadvanced

Fig. 6. Quantitative real-time PCR analysis of podoplanin transcripts in atherosclerotic
lesions of abdominal aortas. First-strand cDNA samples derived from 29 aortic intima(16 DIT lesions, three fatty streak lesions, and 10 advanced lesions) were used as
real-time PCR templates. The respective expression levels of podoplanin were normalized to β-actin, as described in Materials and Methods. The statistical signiﬁcance of
podoplanin mRNA expression in tissues was determined using single-tailed, Student's t-tests. *p b 0.05.

(M.K.K.) and by grants from the Global COE Program for Medical Sciences, Japan Society for Promotion of Science, from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (Y.K.,
M.K.K.).

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Fig. 4. Representative immunoﬂuorescent micrographs of an atheromatous plaque.
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staining with Cy3-labeled smooth muscle actin (red). Lower: merged immunoﬂuorescent image. Co-localized area of podoplanin and smooth muscle actin is stained yellow.

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