Kazuyoshi Hirayama

Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells

Background: Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation. Results: Functions of lymphatic endothelial cells are inhibited by platelet releasates and BMP-9, which we identified as a novel releasate. Conclusion: Granule contents including BMP-9 released upon platelet activation by CLEC-2-podoplanin interaction may contribute to the separation in vivo. Significance: We proposed a novel mechanism of platelet-mediated blood/lymphatic vessel separation. The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2+/+ platelets, but not by CLEC-2−/− platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

Interleukin 17A plays a role in lipopolysaccharide/D-galactosamine-induced fulminant hepatic injury in mice.

BACKGROUND Lipopolysaccharide/d-galactosamine (LPS/GalN)-induced hepatic injury is an experimental model of fulminant hepatic failure in which tumor necrosis factor alpha (TNF-α) plays a pivotal role. Moreover, it was reported from our laboratory that interleukin (IL) 17A enhanced production of TNF-α by the Kupffer cell. OBJECTIVE The purpose of this study was to determine the role of IL-17A in LPS/GalN-induced hepatic injury in mice. METHODS LPS/GalN was injected into three mouse models: wild-type (WT) mice, IL-17A knockout (KO) mice, or IL-17A KO mice treated with recombinant mouse (rm) IL-17A homodimer (KO + rmIL-17A). Survival was assessed for 24 h after LPS/GalN injection, and histopathologic findings were evaluated at various time points after LPS/GalN injection for neutrophil and apoptosis markers. After LPS/GalN injection, expression of the inflammatory mediators TNF-α, IL-6, monocyte chemotactic protein 1, IL-17A, high-mobility group box 1, and soluble intercellular adhesion molecule 1 was assessed in serum by enzyme-linked immunosorbent assay. RESULTS Survival was higher in KO mice compared with WT mice after LPS/GalN injection. However, in KO + rmIL-17A mice, mortality was not significantly different compared to the other groups. Neutrophil infiltration and apoptosis were significantly greater in WT mice than KO mice. Furthermore, serum alanine aminotransferase, serum TNF-α, monocyte chemotactic protein 1, IL-17A, high-mobility group box 1, and soluble intercellular adhesion molecule 1 levels were also significantly greater in WT mice than KO mice. In KO + rmIL-17A mice, these levels were similar to those in WT mice. CONCLUSIONS IL-17A is a key regulator in hepatic injury caused by neutrophil-induced inflammatory responses after LPS/GalN injection.

The platelet-activating receptor C-type lectin receptor-2 plays an essential role in liver regeneration after partial hepatectomy in mice

Essentials Regeneration role of C‐type lectin receptor‐2 (CLEC‐2) after 70% hepatectomy (HPx) was investigated. Wild‐type or CLEC‐2 deleted from platelets of chimeric mice (flKO) underwent HPx. The liver/body weight ratio was significantly lower in the flKO than in the wild‐type. CLEC‐2 plays an essential role in liver regeneration after HPx.

Macrophage colony-stimulating factor plays a pivotal role in chemically induced hepatocellular carcinoma in mice

The specific purpose of this study was to investigate the role of macrophage colony‐stimulating factor (M‐CSF) in initiation and progression of hepatocellular carcinoma using M‐CSF‐deficient mice.

Interleukin-17A plays a pivotal role after partial hepatectomy in mice.

BACKGROUND Liver regeneration after partial hepatectomy (PH) is regulated by tumor necrosis factor (TNF)-α derived from the Kupffer cell. Furthermore, it was reported from our laboratory that interleukin (IL)-17A enhances the production of TNF-α by the Kupffer cell, suggesting that IL-17A may play a role in liver regeneration. OBJECTIVE The purpose was to determine the role of IL-17A and the spleen in liver regeneration after PH. METHODS Two mouse models including the wild-type (WT) mice or the IL-17A knockout (KO) mice underwent PH. Animals were killed at the designated time points; liver tissues were harvested for further investigation. Proliferation of hepatocytes was evaluated. Furthermore, the messenger RNA and protein expression of TNF-α and IL-6 were measured in the liver. In another set of experiments, the two animal models underwent splenectomy before PH. In an in vitro study, CD4-positive lymphocytes in the spleen were isolated from mice, and the number of IL-17A-positive cells was investigated. RESULTS Liver regeneration was significantly impaired in the KO mice compared with the WT mice. This was associated with suppression of cell proliferation assessed by cell proliferation markers in the KO mice. In the WT mice that underwent splenectomy, liver regeneration was significantly delayed compared with animals without splenectomy. In contrast, splenectomy did not affect liver regeneration in the KO mice. IL-17A-positive lymphocytes increased significantly in the spleen in the WT mice after PH. CONCLUSIONS These results indicate that IL-17A derived from CD4-positive lymphocytes in the spleen is a key regulator in liver regeneration after PH.

Macrophage colony-stimulating factor expressed in non-cancer tissues provides predictive powers for recurrence in hepatocellular carcinoma

AIM To investigate the role of macrophage colony-stimulating factor (M-CSF) in patients with hepatocellular carcinoma (HCC) after surgery. METHODS Expression of M-CSF, distribution of M2 macrophages (MΦs), and angiogenesis were assessed in the liver, including tumors and peritumoral liver tissues. The prognostic power of these factors was assessed. Mouse isolated hepatic MΦs or monocytes were cultured with media containing M-CSF. The concentration of vascular endothelial growth factor (VEGF) in media was assessed. Furthermore, the role of the M-CSF-matured hepatic MΦs on proliferation of the vascular endothelial cell (VEC) was investigated. RESULTS A strong correlation between the expressions of M-CSF and CD163 was observed in the peritumoral area. Also, groups with high density of M-CSF, CD163 or CD31 showed a significantly shorter time to recurrence (TTR) than low density groups. Multivariate analysis revealed the expression of M-CSF or hepatic M2MΦs in the peritumoral area as the most crucial factor responsible for shorter TTR. Moreover, the expression of M-CSF and hepatic M2MΦs in the peritumoral area had better predictable power of overall survival. Values of VEGF in culture media were significantly greater in the hepatic MΦs compared with the monocytes. Proliferation of the VEC was greatest in the cells co-cultured with hepatic MΦs when M-CSF was present in media. CONCLUSION M-CSF increases hepatocarcinogenesis, most likely by enhancing an angiogenic factor derived from hepatic MΦ and could be a useful target for therapy against HCC.

Clinical Impact of Histological Heterogeneity in the Metastatic Lymph Nodes of Patients with Colorectal Cancer

Aim: The purpose of this study was to evaluate the clinical impact of histological heterogeneity in patients with node-positive colorectal cancer (CRC). Patients and Methods: One hundred and twenty-nine patients who underwent curative surgical resection for histological node-positive CRC were enrolled. Patients were divided according to the histological heterogeneity in the primary lesion into p-hetero and p-homo groups. The p-hetero group was further divided according to histological heterogeneity in the metastatic lymph nodes into n-hetero and n-homo groups. Results: There were no significant differences between p-homo and p-hetero groups and between n-homo and n-hetero groups in prognosis. However, the recurrence-free survival rate of the n-homo group was significantly lower than that of the n-hetero group in the N2 category. Conclusion: Histological heterogeneity in metastatic lymph nodes may be useful for predicting prognosis, and prognosis in those with histological heterogeneity in a metastatic lymph node is not necessarily poor, even in those of the N2 category.