Elsevier

Thrombosis Research

Volume 129, Issue 4, April 2012, Pages e70-e76
Thrombosis Research

Regular Article
Podoplanin expression in advanced atherosclerotic lesions of human aortas

https://doi.org/10.1016/j.thromres.2012.01.003Get rights and content

Abstract

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-like receptor 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 indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle 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 podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug.

Introduction

Thrombus formation on disrupted atherosclerotic plaques leads to the 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 of arterial thrombus formation is initiated by platelet adhesion and aggregation, the platelet activation in plaques is critical to the onset of clinical 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 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], [7], [8], [9], [10], [11], [12], [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 and poor clinical outcome [11], [18], [19], [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 in atherosclerotic lesions has not been investigated. CLEC-2 is expressed on platelets; therefore, expression of podoplanin in atherosclerotic lesions may be involved in the activation of platelets leading to thrombosis.

In this study, we investigated whether podoplanin is expressed in atherosclerotic lesions and whether it is critical to the onset of cardiovascular events. To this end, we examined podoplanin expression in abdominal aortas using immunohistochemistry, Western-blot, and real time-PCR analyses.

Section snippets

Specimens

We examined the abdominal aortas of 31 patients (24 male, seven female; 18–83 years of age, mean 66 years) autopsied at University of Miyazaki 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 taken from various degrees of atherosclerotic lesions. Each tissue was cut into two specimens. In one specimen of each

Immunohistochemical analysis against human atherosclerotic lesion using an anti-podoplanin antibody

Podoplanin possesses platelet-aggregating activities, which play crucial roles in thrombosis/hemostasis, tumor metastasis, and 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

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 of type 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

Conflict of interest statement

We have no conflict of interest to declare.

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research in Japan (20390102, 20590344, 23390084, 23701043, 23791584) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.K., Y.K., M.K.K., Y.A.), by Mitsubishi Pharma Research Foundation (Y.K., M.K.K.), by Children Cancer Association of Japan (Y.K.), by Intelligent Cosmos Academic Foundation (Y.K.), by Office for Gender Equality of Yamagata University (M.K.K.) and by grants from the Global COE

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