No abstract

S U M M A R Y We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC. LYMPHATIC VESSELS express not only platelet-endothelial adhesion molecule-1 (PECAM-1) but also the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in inflamed and uninflamed human small intestine and tongue, and lymphatic endothelium enhances VCAM-1 and ICAM-1 production with tumor necrosis factor-a (TNF-a) (Sawa et al. 1999(Sawa et al. ,2007Ebata et al. 2001). It is well established that VCAM-1 and ICAM-1 induction needs a signaling pathway that involves protein kinase C and p38 mitogenactivated protein kinases (MAPKs)-mediated activation of transcription factors nuclear factor-kappaB (NF-kB) and activator protein 1 (AP-1) (Voraberger et al. 1991;Ahmad et al. 1998;Ishizuka et al. 1998;Oertli et al. 1998;Kobuchi et al. 1999;Lawson et al. 1999;Roebuck 1999). It is thought that lymphatic endothelium has signal transduction pathways to induce leukocyte adhesion molecule expression through receptors for inflammatory cytokines. However, it has not been established whether lymphatic endothelium has the expression mechanisms of immunological functional molecules independent of the inflammatory cytokines.The toll-like receptor (TLR) initiates a series of innate immune mechanisms against various microorganism infections by sensing the presence of pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS), which is the major component of the outer surface of Gram-negative bacteria. The LPS stimulates leukocyte and blood endothelium through the LPS recognition systems, binding with CD14 and transferring to TLR4 and MD-2 complex, followed by a myleoid differentiation primary response protein (MyD88)-dependent or -independent pathway that culminates in the early-or late-phase activation of the IkB kinase (IKK) complex and MAPK...

We characterized the structural and immunohistological changes of sinusoidal endothelial cells that occur during cirrhosis in rats made cirrhotic with thioacetamide. Thioacetamide (200 mg/kg body wt) was injected intraperitoneally three times a week into male Wistar rats. Two, 4, 6 and 12 wk later, rat livers were observed under transmission and scanning electron microscopy and regular microscopy and immunostained with laminin and von Willebrand factor (factor VIII-related antigen) antibodies. The diameters and numbers of sinusoidal endothelial fenestrations did not change significantly after 2 wk in the thioacetamide-treated rats; however, they decreased within 4 wk after thioacetamide treatment. A basement membranelike structure in Disse's space was noted 6 wk after thioacetamide treatment. Laminin was detected in Disse's space after 4 wk. In vitro, in cultured sinusoidal endothelial cells, the diameter of sinusoidal endothelial fenestrations was significantly lower at 6 wk in thioacetamide-treated rats. von Willebrand factor was detected in the cytoplasm as granular fluorescence after 6 wk of thioacetamide treatment. These results suggest that as fibrosis develops in cirrhosis, the structural and immunohistochemical characteristics of sinusoidal endothelial cells change.

Podoplanin is a platelet aggregation-inducing factor associated with tumor metastasis, malignant progression, and cancer stem cells. We produced a rat-human chimeric anti-podoplanin mAb, NZ-8, from rat anti-podoplanin mAb (NZ-1). Although both NZ-1 and NZ-8 possess high binding affinities and high neutralizing activities of platelet aggregation, the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of NZ-8 were much higher than NZ-1. Furthermore, both NZ-1 and NZ-8 inhibited the growth of podoplanin-expressing tumors in vivo. Both NZ-1 and NZ-8 also suppressed hematogenous metastasis of podoplanin-expressing tumors. These results suggest that antipodoplanin mAbs suppressed hematogenous metastasis by both neutralization and antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activities. Targeting therapy to podoplanin-expressing tumors should be useful as a novel immunotherapy. (Cancer Sci 2012; 103: 1913-1919 P odoplanin is a platelet aggregation-inducing factor, and its expression has been reported in many tumors including malignant brain tumors, mesotheliomas, and squamous cell carcinoma.(1-10) Importantly, recent investigations have suggested that expression of podoplanin is associated with tumor metastasis, malignant progression, and epithelial-mesenchymal transition.(11-18) Podoplanin expression has also been reported to be associated with clinical outcome. (19)(20)(21) Recently, we have shown podoplanin expression in atherosclerotic lesion. (22) In solid tumors such as brain tumors, only a small and phenotypically distinct subset of cells could be responsible for generating and sustaining tumors, and thus be considered as cancer stem cells or tumor-initiating cells (TICs).(23) Because TICs are thought to be resistant to conventional therapies, and are responsible for relapse, targeting TICs could be a promising approach to cancer therapy.(24) Podoplanin has been reported to be a TIC marker; (25) therefore, immunotherapy using specific antibodies reactive to podoplanin may eradicate TICs in cancers.We previously produced an anti-podoplanin antibody, NZ-1.(5) NZ-1 should have not only high specificity and sensitivity but also high binding-affinity against podoplanin to be applied for radioimmunotherapy or immunotoxin therapy. Previous studies showed that NZ-1 is a suitable candidate for therapy against malignant gliomas because NZ-1 was highly internalized into glioma cell lines, and also well accumulated into tumors in vivo.(26) Moreover, NZ-1 inhibited tumor cellinduced platelet aggregation and tumor metastasis by its neutralizing activity.(12) However, it has not been clarified whether NZ-1 possesses antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) against podoplanin-expressing tumor cells. In this study, we produced rat-human chimeric anti-podoplanin antibody (NZ-8) from rat anti-podoplanin neutralizing antibody (NZ-1), and characterized NZ-8 activity in flow cytometry, Western blot, platelet aggregation, and AD...

This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud.