Recent development in platelet functions: roles beyond thrombosis

In addition to their roles in thrombosis and hemostasis, there is an increasing body of evidence to suggest that platelets have diverse functions in various biological reactions. Of these, synthesis of specific proteins in a timely manner, involvement in inflammation, roles in anti-bacteria and anti-parasite protection, and supportive roles in liver regeneration have attracted the attention of a number of scientists. We have recently found a novel platelet-activating receptor, CLEC-2, which reacts with a snake venom, Rhodocytin. CLEC-2 has an intracellular signal transduction pathway quite similar to that of GOVI, except for its hemi-Y-xx-L motif. The endogenous ligand for CLEC-2 was identified by us as podoplanin, which is present in renal podocytes, lung alveolar macrophages, and lymphatic endothelial cells, and some types of malignant tumors. We found that CLEC-2/podoplanin interaction plays an important role in the metastasis of tumor cells with podoplanin expression. We have also found that hemophilic interaction between CLEC-2 molecules contributes to thrombus formation in vivo. CLEC-2 interaction with podoplan expressed on lymphatic endothelial cells appears to play an important role in the separation between veins and lymphatic vessels during the stage of fetal development.     

C型凝集素受体CLEC-2的研究进展

C型凝集素样受体-2(CLEC-2)是一个非经典型的C型凝集素受体,主要表达在血小板、中性粒细胞等细胞表面,在马来西亚蝮蛇蛇毒蛋白rhodocytin和跨膜唾液酸糖蛋白poloplanin等相关配体的刺激下,能通过细胞质尾部的YXXL结构域形成二聚体,从而激活syk依赖的信号传导.最近有很多研究显示:CLEC-2在血小...     

Clinicopathology significance of podoplanin immunoreactivity in esophageal squamous cell carcinoma

Backgroud and aim: Podoplanin (D2-40) is a specific marker for lymphatic endothelium. The vast majority of previous studies on podoplanin immunostaining in esophageal squamous cell carcinoma (ESCC) focused on identifying lymphatic vessel invasion (LVI) and counting lymphatic vessel density (LVD) and had contradictory results. Recent studies show podoplanin expression on cancer cells or tumor stroma in several cancers, which have specific significance; but the status in ESCC remains unclear. Therefore, the aim of this study was to further study and summarize the clinicopathological significance of podoplanin immunoreactivity in ESCC. Materials and methods: We examined podoplanin expression in tissue specimens from 107 patients with ESCC by immunohistochemistry. Podoplanin positive lymphatic vessels in intratumoral and peritumoral tissues and podoplanin positive expression in cancer cells and tumor stroma were analyzed, and correlated with clinicopathologic parameters and three-year overall and free-disease survival. Results: 34 (31.8%) and 28 (26.2%) of 107 specimens had podoplanin positive expression in cancer cells and tumor stroma, respectively. Logistic regression analysis showed high intratumoral lymphatic vessel density (I-LVD) and podoplanin positivity in cancer cells were increased risks of lymph node metastasis (LNM) (OR = 2.45, P = 0.03; OR = 0.35, P = 0.01, respectively). Survival analysis showed that I-LVD was a significant factor related to poor three-year overall and free-disease survival (P = 0.04, P = 0.03, respectively). Conclusions: Previous data and our results show that podoplanin seems to be a useful marker to predict LNM, recurrence, and worse prognosis in ESCC; in particular, LVI, high I-LVD, and podoplanin positivity in cancer cells are associated with LNM, recurrence and overall survival.     

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors

Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.     

The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary metastasis

Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression.We studied herein the role of Aggrus in tumor growth, metastasis , and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus expressing cells , which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus(Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis.     

Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.     

Podoplanin-Rich Stromal Networks Induce Dendritic Cell Motility via Activation of the C-type Lectin Receptor CLEC-2

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe?a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T?cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.     

Concordant podoplanin expression in cancer-associated fibroblasts and tumor cells is an adverse prognostic factor in esophageal squamous cell carcinoma

There is growing evidence that cancer-associated fibroblasts (CAFs) interact with tumor cells and play important roles in tumor progression and invasion. Podoplanin is a type-1 transmembrane glycoprotein expressed in a variety of normal human tissues, including lymphatic endothelium. Tumor cell expression of podoplanin correlates with nodal metastasis and poor prognosis in squamous cell carcinoma (SCC) of oral cavity and esophagus. Recently, podoplanin-positive CAFs have been shown to exert adverse or beneficial prognostic effect on different cancer types. However, the significance of podoplanin-positive CAFs in esophageal SCC has not been investigated. This is the first study to investigate podoplanin expression in CAFs and tumor cells by immunohistochemistry in 59 cases of surgically resected esophageal SCC. We found significant association of podoplanin expression between CAFs and tumor cells (P = 0.031). Although the abundance of podoplanin-positive CAFs per se had no prognostic effect, concordant podoplanin expression in CAFs and tumor cells (both high or both low) was strongly associated with short survival (P = 0.00088). Multivariate analysis showed that concordant podoplanin expression was the strongest independent adverse prognostic factor (hazard ratio: 3.62; 95% confidence interval: 1.69-7.77; P = 0.00094). Our data suggest that interaction between podoplanin-positive CAFs and tumor cells is important in tumor biology of esophageal SCC.     

Low podoplanin expression in pretreatment biopsy material predicts poor prognosis in advanced-stage squamous cell carcinoma of the uterine cervix treated by primary radiation.

Lymphatic invasion and nodal metastasis are predictors of poor outcome in cervix carcinoma. We have recently found that low podoplanin immunoreactivity in cervix carcinoma correlated with the presence of lymphatic invasion and nodal metastasis. In the current study, we examined whether podoplanin expression in pretreatment cervical biopsies can predict the presence lymphatic invasion, nodal metastasis, and outcome in advanced-stage tumors treated by nonsurgical means. Podoplanin expression was analyzed by immunohistochemistry in 48 cervical biopsies and corresponding hysterectomy specimens of early-stage invasive squamous cell carcinoma and in 74 pretreatment biopsies from advanced-stage tumors treated with primary radiation. We found a highly significant correlation between podoplanin expression obtained in biopsy and corresponding hysterectomy materials (r = 0.8962, P < 0.0001). Low podoplanin expression showed a significant correlation with lymphatic invasion (P < 0.0001) and nodal metastasis (P = 0.0058). Low podoplanin expression in pretreatment biopsy material showed a significant correlation with poor disease-free (P = 0.0009) and overall (P = 0.0002) survival in advanced-stage tumors. Our results suggest that in advanced-stage cervix carcinomas treated by radiation, when traditional prognostic indicators are not available and treatment decisions are based on biopsy material and clinical staging parameters, examination of podoplanin expression in pretreatment biopsy material may be a useful marker to predict lymphatic metastasis and patient outcome. Prospective studies involving larger numbers of patients are needed to further evaluate the clinical utility of examination of podoplanin expression in patients with cervix carcinoma.     

Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium

Angiosarcomas apparently derive from blood vessel endothelial cells; however, occasionally their histological features suggest mixed origin from blood and lymphatic endothelia. In the absence of specific positive markers for lymphatic endothelia the precise distinction between these components has not been possible. Here we provide evidence by light and electron microscopic immunohistochemistry that podoplanin, a approximately 38-kd membrane glycoprotein of podocytes, is specifically expressed in the endothelium of lymphatic capillaries, but not in the blood vasculature. In normal skin and kidney, podoplanin colocalized with vascular endothelial growth factor receptor-3, the only other lymphatic marker presently available. Complementary immunostaining of blood vessels was obtained with established endothelial markers (CD31, CD34, factor VIII-related antigen, and Ulex europaeus I lectin) as well as podocalyxin, another podocytic protein that is also localized in endothelia of blood vessels. Podoplanin specifically immunolabeled endothelia of benign tumorous lesions of undisputed lymphatic origin (lymphangiomas, hygromas) and was detected there as a 38-kd protein by immunoblotting. As paradigms of malignant vascular tumors, poorly differentiated (G3) common angiosarcomas (n = 8), epitheloid angiosarcomas (n = 3), and intestinal Kaposi's sarcomas (n = 5) were examined for their podoplanin content in relation to conventional endothelial markers. The relative number of tumor cells expressing podoplanin was estimated and, although the number of cases in this preliminary study was limited to 16, an apparent spectrum of podoplanin expression emerged that can be divided into a low-expression group in which 0-10% of tumor cells contained podoplanin, a moderate-expression group with 30-60% and a high-expression group with 70-100%. Ten of eleven angiosarcomas and all Kaposi's sarcomas showed mixed expression of both lymphatic and blood vascular endothelial phenotypes. By double labeling, most podoplanin-positive tumor cells coexpressed endothelial markers of blood vessels, whereas few tumor cells were positive for individual markers only. From these results we conclude that (1) podoplanin is a selective marker of lymphatic endothelium; (2) G3 angiosarcomas display a quantitative spectrum of podoplanin-expressing tumor cells; (3) in most angiosarcomas, a varying subset of tumor cells coexpresses podoplanin and endothelial markers of blood vessels; and (4) all endothelial cells of Kaposi's sarcomas expressed the lymphatic marker podoplanin.     

Podoplanin expression profiles characteristic of odontogenic tumor-specific tissue architectures

Podoplanin, a representative immunohistochemical marker for lymphatic endothelial cells, is also expressed in many other kinds of cancer cells, although its pathophysiological function is largely unknown. Our aim was to determine immunolocalization modes of podoplanin among odontogenic tumors to discuss possible roles of podoplanin in their characteristic tissue architecture formation. Immunohistochemical profiles of podoplanin were investigated in 40 surgical specimens from ameloblastoma (AM), adenomatoid odontogenic tumor (AOT), calcifying cystic odontogenic tumor (CCOT), and keratocystic odontogenic tumor (KCOT) in comparison with those of proliferating cell nuclear antigen (PCNA), integrin β1, fibronectin, and matrix metalloproteinase 9 (MMP-9). Podoplanin was localized in the basal cell layer or in the peripheral zone of AM foci. It was found in spindle-shaped tumor cells of AOT, in both the basal and polyhedral cells of CCOT, and in the basal and parabasal cells of KCOT linings. Podoplanin-positive (+) cells were located within areas of PCNA+ cells, and integrin β1 was localized in the cell membrane of podoplanin+ cells in the intercellular space where fibronectin and MMP-9 were deposited. In conclusion, podoplanin+ cells and areas in odontogenic tumors are in close associations with extracellular matrix signalings as well as cell proliferation.     

High podoplanin expression in cancer cells predicts lower incidence of nodal metastasis in patients with lung squamous cell carcinoma

Podoplanin is expressed in a variety of malignant cells, and is generally regarded as a factor promoting tumor progression in conventional studies. Conversely, a recent clinicopathological study has revealed that low podoplanin in cancer cells was correlated with poor prognosis of patients with stage IB lung squamous cell carcinoma (LSCC).We here evaluated the clinicopathological relationship between cancer-cell podoplanin expression and clinicopathological parameters in 40 cases of LSCC (stage I-III).Immunohistochemical podoplanin expression significantly correlated with N classification and pathological stage, but not with other clinicopathological parameters. Notably, all 16 cases with high podoplanin expression unexceptionally exhibited pathological N0 status. Cases without nodal metastasis showed a significantly higher podoplanin-positive score. Furthermore, patients with high podoplanin expression exhibited a significantly longer survival time and disease-free time.These findings suggest that immunohistochemical analysis for podoplanin may serve as a marker of risk of nodal metastasis and prognosis in patients with LSCC.     

Podoplanin expressing cancer associated fibroblasts are associated with unfavourable prognosis in adenocarcinoma of the esophagus

Overexpression of the mucin-type sialoglycoprotein podoplanin in cancer associated fibroblasts (CAFs) was recently shown to be associated with tumor progression, metastasis and poor prognosis in lung and breast cancer. Here we investigate the role of podoplanin expressing CAFs in esophagal adenocarcinoma (AC), its precursor lesions and metastases. Podoplanin expression was investigated immunohistochemically in 200 formalin-fixed, paraffin embedded specimens of invasive esophagal ACs, their corresponding metastases and 35 precursor lesions. Podoplanin expressing CAFs (CAF+) were observed in 22 % of patients with invasive AC, but not in precursor lesions. CAF+ correlated with tumor stage (p = 0.004), lymphovascular tumor invasion (p = 0.018) and lymph node metastasis (p = 0.0016). Patients with CAF+ had a significant shorter disease free and overall survival (p < 0.05, Cox regression). Podoplanin expressing CAFs were only rarely observed in lymph node and distant metastases, as well as in local recurrences of ACs. Podoplanin expression in AC tumor cells was seen in only four cases. In around 20 % of patients with esophagal AC, podoplanin expressing CAFs are evident, defining a high risk subgroup. In these patients, podoplanin expressing CAFs might represent new therapeutical targets.     

Podoplanin: a novel diagnostic immunohistochemical marker

Podoplanin is a transmembrane mucoprotein recognized by the recently commercially available D2-40 monoclonal antibody. Recent investigations have shown that podoplanin is selectively expressed in lymphatic endothelium as well as lymphangiomas, Kaposi sarcomas, and in a subset of angiosarcomas with probable lymphatic differentiation. Podoplanin has also been shown to be strongly expressed in seminomas, epithelioid mesotheliomas, and hemangioblastomas, and immunostaining for this marker can assist in the diagnosis of these tumors. This article reviews the current information on the applications of podoplanin immunostaining in surgical pathology.     

Directed expression of transgenes to alveolar type I cells in the mouse

Podoplanin (RTI40, aggrus, T1alpha, hT1alpha-2, E11, PA2.26, RANDAM-2, gp36, gp38, gp40, OTS8) is a type I cell marker in rat lung. We show that a bacterial artificial chromosome vector containing the rat podoplanin gene (RTIbac) delivers a pattern of transgene expression in lung that is more restricted to mouse type I cells than that of the endogenous mouse podoplanin gene. RTIbac-transgenic mice expressed rat podoplanin in type I cells; type II cells, airways, and vascular endothelium were negative. A modified bacterial artificial chromosome containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) sequences in the podoplanin 3'UTR expressed rat podoplanin and transgenic GFP in type I cells. RTIbac transgene expression was absent or reduced in pulmonary pleura, lymphatic endothelium, and putative lymphoid-associated stromal tissue, all of which contained abundant mouse podoplanin. Rat podoplanin mRNA levels in normal rat lung and RTIbac transgenic lung were 25-fold higher than in corresponding kidney and brain samples. On Western blots, transgenic rat and endogenous mouse podoplanin displayed very similar patterns of protein expression in various organs. Highest protein levels were observed in lung with 10- to 20-fold less in brain; there were low levels in thymus and kidney. Both GFP and rat podoplanin transgenes were expressed at extrapulmonary sites of endogenous mouse podoplanin gene expression, including choroid plexus, eye ciliary epithelium, and renal glomerulus. Because their pulmonary expression is more restricted than endogenous mouse podoplanin, RTIbac derivatives should be useful for mouse type I cell-specific transgene delivery.     

Rac1 is essential for phospholipase C-γ2 activation in platelets

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cβ or PLCγ2. Active PLCs trigger Ca²⁺ mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCβ isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCγ2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP₃) and intracellular calcium mobilization suggesting inappropriate activation of PLCγ2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ^−/− platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA₂ analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ^−/− mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCγ2 activation in platelets and that this is critical for thrombus formation in vivo. Irina Pleines, Margitta Elvers, Amrei Strehl: these authors contributed equally to this work.     

Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells

Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.