Shintaro Kato

Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis.

Cyclooxygenase (COX)‐2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E2 (PGE2)–prostaglandin E2 receptor (EP)3 signaling appears critical for tumor‐associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX‐2 inhibitor NS‐398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX‐1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)‐9 and vascular endothelial growth factor (VEGF)‐A was suppressed in NS‐398‐treated mice compared with PBS‐treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3−/−) mice compared with WT mice. The expression of MMP‐9 and VEGF‐A was downregulated in metastatic lungs of EP3−/− mice. An immunohistochemical study revealed that MMP‐9‐expressing endothelial cells were markedly reduced in EP3−/− mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP‐9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP‐9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis. (Cancer Sci 2009; 100: 2318–2324)

Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice

It is known that the neural system plays a fundamental role in neovascularization. A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in the central and peripheral neuronal systems. However, it remains to be elucidated the role of CGRP in angiogenesis during ischemia. The present study examined whether endogenous CGRP released from neuronal systems facilitates revascularization in response to ischemia using CGRP knockout mice (CGRP-/-). CGRP-/- or their wild-type littermates (CGRP+/+) were subjected to unilateral hindlimb ischemia. CGRP-/- exhibited impaired blood flow recovery from ischemia and decreased capillary density expressed in terms of the number of CD-31-positive cells in the ischemic tissues compared with CGRP+/+. In vivo microscopic studies showed that the functional capillary density in CGRP-/- was reduced. Hindlimb ischemia increased the expression of pro-CGRP mRNA and of CGRP protein in the lumbar dorsal root ganglia. Lack of CGRP decreased mRNA expression of growth factors, including CD31, vascular endothelial growth factor-A, basic fibroblast growth factor, and transforming growth factor-β, in the ischemic limb tissue. The application of CGRP enhanced the mRNA expression of CD31 and VEGF-A in human umbilical vein endothelial cells (HUVECs) and fibroblasts. Subcutaneous infusion of CGRP8-37, a CGRP antagonist, using miniosmotic pumps delayed angiogenesis and reduced the expression of proangiogenic growth factors during hindlimb ischemia. These results indicate that endogenous CGRP facilitates angiogenesis in response to ischemia. Targeting CGRP may provide a promising approach for controlling angiogenesis related to pathophysiological conditions.

Vascular endothelial growth factor receptor-1 signaling promotes liver repair through restoration of liver microvasculature after acetaminophen hepatotoxicity

Vascular endothelial growth factor (VEGF) and its receptors promote liver regeneration. The objective of the present study was to examine the role of VEGF receptor 1 (VEGFR1) signaling in hepatic tissue repair after acetaminophen (N-acetyl-para-aminophenol) (APAP)-induced liver injury. To do this, we treated VEGFR1 tyrosine kinase knockout (VEGFR1 TK(-/-)) and wild-type (WT) mice with APAP (300 mg/kg, ip). In WT mice, serum levels of alanine aminotransferase (ALT) and the necrotic area peaked between 8 and 24 h and then declined. In VEGFR1 TK(-/-) mice, ALT levels remained high at 48 h and extensive hepatic necrosis and hemorrhage were observed, as well as high mortality. Downregulation of hepatic messenger RNA expression of VEGFR1 and VEGFR2 was also noted in VEGFR1 TK(-/-) mice. VEGFR1 TK(-/-) mice displayed lower expression of proliferating cell nuclear antigen and of growth factors including hepatocyte growth factor, CD31, and basic fibroblast growth factor than WT. The hepatic microvasculature in VEGFR1 TK(-/-) was compromised as evidenced by impaired sinusoidal perfusion, suppressed endocytosis in liver sinusoidal endothelial cells (LSECs), and the formation of large gaps in LSECs. In WT mice, immunofluorescence revealed that recruited VEGFR1(+) cells in the necrotic area were positive for CD11b. VEGFR1 TK(-/-) exhibited fewer VEGFR1(+) and VEGFR2(+) cells. These results suggest that VEGFR1 signaling facilitates liver recovery from APAP hepatotoxicity by preventing excessive hemorrhage and reconstituting the sinusoids through recruitment of VEGFR1-expressing macrophages to the injured area and also through affecting expression of genes including hepatotrophic and pro-angiogenic growth factors.

Thromboxane A 2 receptor signaling facilitates tumor colonization through P-selectin-mediated interaction of tumor cells with platelets and endothelial cells

Thromboxane A2 (TXA2) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product, prostaglandin H(2), as the substrate. TXA2 was shown to enhance tumor metastasis, but the underlying mechanism remains unclear. B16F1 melanoma cells were intravenously injected into TXA2 receptor (TP) knockout mice (TP−/−) and wild‐type littermates (WT). TP−/− showed a reduction in B16F1 lung colonization and mortality rate, which were associated with a decreased number of platelets. Platelet activation as assessed by P‐selectin expression was suppressed in TP−/−. A selective P‐selectin neutralizing antibody decreased the lung colonization in WT mice, but not in TP−/−. The expression of P‐selectin glycoprotein ligand‐1 in B16F1 and HUVEC were enhanced by treatment with U46619, a thromboxane analog. The plasma levels of vascular endothelial growth factor (VEGF) and stromal‐derived factor (SDF)‐1 were lower in TP−/−. In TP−/−, the mobilization of progenitor cells expressing CXCR4+VEGFR1+ from bone marrow and the recruitment of those cells to lung tissues were suppressed. These results suggest that TP signaling plays a critical role in tumor colonization through P‐selectin‐mediated interactions between platelets‐tumor cells and tumor cells‐endothelial cells through the TP signaling‐dependent production of VEGF and SDF‐1, which might be involved in the mobilization of VEGFR1+CXCR4+ cells. Blockade of TP signaling might be useful in the treatment of tumor metastasis. (Cancer Sci 2012; 103: 700–707)

Thromboxane A2 induces blood flow recovery via platelet adhesion to ischaemic regions

AIMS Thromboxane A2 (TXA2) induces platelet adhesion through thromboxane prostanoid (TP) receptor. Platelets contain many pro-angiogenic factors and are recruited to the site of vascular injury. However, the cellular and molecular mechanisms of platelet-dependent angiogenesis, especially the involvement of TP signalling, have not been fully elucidated. The present study hypothesized that TP-dependent platelet adhesion would contribute to angiogenesis in a mouse hindlimb ischaemic model. METHODS AND RESULTS Blood flow recovery was suppressed by the TXA2 receptor antagonist (S-1452) and the TXA2 synthase inhibitor (OKY-046) compared with control mice. TP knockout mice (TP(-/-)) showed delayed blood flow recovery from ischaemia and impaired angiogenesis compared with wild-type (WT) mice and prostacyclin receptor knockout mice (IP(-/-)). Selective platelet adhesion to ischaemic endothelial cells (ECs) via P-selectin was identified in WT and IP(-/-), but not in TP(-/-), via in vivo microscopy. IF analysis showed that P-selectin glycoprotein ligand-1 (PSGL-1) co-localized with endothelial CD31 in ischaemic muscle in WT and IP(-/-) but not diminished in TP(-/-). Platelet-rich plasma levels of stromal cell-derived factor-1 and VEGF were increased after ischaemia in WT, and suppressed by antibody against P-selectin in WT but not in TP(-/-). Furthermore, the blood flow recovery was suppressed by neutralizing antibodies against VEGF or C-X-C chemokine receptor type 4 in WT and IP(-/-) but not in TP(-/-). CONCLUSION These results indicated that TP signalling facilitates ischaemia-induced angiogenesis via P-selectin-mediated platelet adhesion to PSGL-1 on the ECs at ischaemic sites and the supply of pro-angiogenic factors by the adherent platelets.

The Role of Vascular Endothelial Growth Factor Receptor-1 Signaling in the Recovery from Ischemia

Vascular endothelial growth factor (VEGF) is one of the most potent angiogenesis stimulators. VEGF binds to VEGF receptor 1 (VEGFR1), inducing angiogenesis through the receptor’s tyrosine kinase domain (TK), but the mechanism is not well understood. We investigated the role of VEGFR1 tyrosine kinase signaling in angiogenesis using the ischemic hind limb model. Relative to control mice, blood flow recovery was significantly impaired in mice treated with VEGFA-neutralizing antibody. VEGFR1 tyrosine kinase knockout mice (TK-/-) had delayed blood flow recovery from ischemia and impaired angiogenesis, and this phenotype was unaffected by treatment with a VEGFR2 inhibitor. Compared to wild type mice (WT), TK-/- mice had no change in the plasma level of VEGF, but the plasma levels of stromal-derived cell factor 1 (SDF-1) and stem cell factor, as well as the bone marrow (BM) level of pro-matrix metalloproteinase-9 (pro-MMP-9), were significantly reduced. The recruitment of cells expressing VEGFR1 and C-X-C chemokine receptor type 4 (CXCR4) into peripheral blood and ischemic muscles was also suppressed. Furthermore, WT transplanted with TK-/- BM significantly impaired blood flow recovery more than WT transplanted with WT BM. These results suggest that VEGFR1-TK signaling facilitates angiogenesis by recruiting CXCR4+VEGFR1+ cells from BM.

An Aptamer-Based Biosensor for Direct, Label-Free Detection of Melamine in Raw Milk

Melamine, a nitrogen-rich compound, has been used as a food and milk additive to falsely increase the protein content. However, melamine is toxic, and high melamine levels in food or in milk can cause kidney and urinary problems, or even death. Hence, the detection of melamine in food and milk is desirable, for which numerous detection methods have been developed. Several methods have successfully detected melamine in raw milk; however, they require a sample preparation before the analyses. This study aimed to develop an aptamer-DNAzyme conjugated biosensor for label-free detection of melamine, in raw milk, without any sample preparation. An aptamer-DNAzyme conjugated biosensor was developed via screening using microarray analysis to identify the candidate aptamers followed by an optimization, to reduce the background noise and improve the aptamer properties, thereby, enhancing the signal-to-noise (S/N) ratio of the screened biosensor. The developed biosensor was evaluated via colorimetric detection and tested with raw milk without any sample preparation, using N-methylmesoporphyrin IX for fluorescence detection. The biosensor displayed significantly higher signal intensity at 2 mM melamine (S/N ratio, 20.2), which was sufficient to detect melamine at high concentrations, in raw milk.

Hishot Display—A New Combinatorial Display for Obtaining Target-Recognizing Peptides

Display technologies are procedures used for isolating target-recognizing peptides without using immunized animals. In this study, we describe a new display method, named Hishot display, that uses Escherichia coli and an expression plasmid to isolate target-recognizing peptides. This display method is based on the formation, in bacteria, of complexes between a polyhistidine (His)-tagged peptide including random sequences and the peptide-encoding mRNA including an RNA aptamer against the His-tag. When this system was tested using a sequence encoding His-tagged green fluorescent protein that included an RNA aptamer against the His-tag, the collection of mRNA encoding the protein was dependent on the RNA aptamer. Using this display method and a synthetic library of surrogate single-chain variable fragments consisting of VpreB and Ig heavy-chain variable domains, it was possible to isolate clones that could specifically recognize a particular target (intelectin-1 or tumor necrosis factor-α). These clones were obtained as soluble proteins produced by E. coli, and the purified peptide clones recognizing intelectin-1 could be used as detectors for sandwich enzyme-linked immunosorbent assays. The Hishot display will be a useful method to add to the repertoire of display technologies.