Shohei Yamashina

Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3−/−) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3−/−, in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3−/−, compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3−/−. These results demonstrate significance of host stromal PGE2-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.

Blockade of angiotensin AT1a receptor signaling reduces tumor growth, angiogenesis, and metastasis

It was reported that angiotensin II stimulates angiogenesis in vivo, and angiotensin-converting enzyme (ACE) inhibitors inhibit angiogenesis. We found that an AT1-receptor (AT1-R) antagonist, TCV-116, inhibited tumor growth, tumor-associated angiogenesis, and metastasis in a murine model. Tumor growth of Sarcoma 180 (S-180) cells and of fibrosarcoma (NFSA) cells was strongly inhibited by administration of TCV-116 in the diet at a dose of approximately 100 mg/kg/day. This reduction was accompanied with a marked reduction in tumor-associated angiogenesis. The same treatment also reduced the lung metastasis of intravenously injected Lewis lung carcinoma cells. These effects of TCV-116 were equivalent to those of the ACE inhibitor, lisinopril. In S-180 and NFSA tumor tissues, ACE and AT1a receptor (AT1a-R) mRNAs were expressed when assessed with RT-PCR. AT1b receptor and AT2 receptor, however, were not detected. Immunoreactive AT1-R was detected mainly on the neovascularized vascular endothelial cells in which expression was reduced by TCV-116 and lisinopril. These results suggested that TCV-116 inhibits the angiogenesis, growth, and metastasis of tumors highly dependent on AT1a-R blockade. Blockade of AT1a-R signaling may therefore become an effective novel strategy for tumor chemoprevention.

Cyclo-oxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo‐oxygenase (COX)‐2 mediated angiogenesis in chronic and proliferate granuloma. In rat sponge implants, angiogenesis was gradually developed over a 14‐day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF). In sponge granuloma, mRNA of COX‐1 was constitutively expressed, whereas that of COX‐2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF). Topical injections of bFGF increased the expression of COX‐2 mRNA. bFGF‐stimulated angiogenesis was inhibited by indomethacin or selective COX‐2 inhibitors, NS‐398, nimesulide, and JTE‐522. The levels of PGE2 and 6‐keto‐PGF1α in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS‐398. The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS‐398. Topical injections of PGE2 and beraprost sodium, a PGI2 analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement. The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti‐sense oligonucleotide. These results suggested that COX‐2 may enhance bFGF‐induced neovascularization in sponge granuloma by PG‐mediated expression of VEGF, and that a COX‐2 inhibitor would facilitate the management of conditions involving angiogenesis.

GBF1, a Guanine Nucleotide Exchange Factor for ADP-Ribosylation Factors, is Localized to the cis-Golgi and Involved in Membrane Association of the COPI Coat

Formation of coated carrier vesicles, such as COPI‐coated vesicles from the cis‐Golgi, is triggered by membrane binding of the GTP‐bound form of ADP‐ribosylation factors. This process is blocked by brefeldin A, which is an inhibitor of guanine nucleotide exchange factors for ADP‐ribosylation factor. GBF1 is one of the guanine nucleotide‐exchange factors for ADP‐ribosylation factor and is localized in the Golgi region. In the present study, we have determined the detailed subcellular localization of GBF1. Immunofluorescence microscopy of cells treated with nocodazole or incubated at 15 °C has suggested that GBF1 behaves similarly to proteins recycling between the cis‐Golgi and the endoplasmic reticulum. Immunoelectron microscopy has revealed that GBF1 localizes primarily to vesicular and tubular structures apposed to the cis‐face of Golgi stacks and minor fractions to the Golgi stacks. GBF1 overexpressed in cells causes recruitment of class I and class II ADP‐ribosylation factors onto Golgi membranes. Furthermore, overexpressed GBF1 antagonizes various effects of brefeldin A, such as inhibition of membrane recruitment of ADP‐ribosylation factors and the COPI coat, and redistribution of Golgi‐resident and itinerant proteins. These observations indicate that GBF1 is involved in the formation of COPI‐coated vesicles from the cis‐Golgi or the pre‐Golgi intermediate compartment through activating ADP‐ribosylation factors.

Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B2 receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B2 receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B2 receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10−8-10−6 m), and this stimulatory effect of BK was abolished with a B2 receptor antagonist, Hoe140 (10−5 m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B2 signaling to up-regulate VEGF production mainly in fibroblasts.

Roles of bradykinin in vascular permeability and angiogenesis in solid tumor.

Bradykinin (BK) is involved in tumor angiogenesis. To elucidate the mechanism underlying BK-induced angiogenesis, we evaluated the roles of BK in tumor-associated vascular permeability and angiogenesis in the different phases of tumor development in mice bearing sarcoma 180 cells. The vascular permeability was significantly enhanced in the early growth phase (which peaked at day 5), and was thereafter markedly reduced. By contrast, tumor angiogenesis increased gradually over a 20-day experimental period. Oral administration of a B2 receptor antagonist, FR173657 (30 mg/kg/day), significantly suppressed the vascular permeability, but a B1 antagonist, desArg10-Hoe140 (1 mg/kg/day) did not. An immunohistochemical study revealed the presence of immunoreactive B2 receptor in the endothelial cells in the early phase, whereas B2 receptors were also observed in the stromal fibroblasts in the late phase. We also found that VEGF was detected exclusively in the stromal fibroblasts only in the late phase. Furthermore, VEGF immunoreactivity was attenuated by the treatment with FR173657. Tumor angiogenesis was significantly reduced by treating the tumor tissues with FR173657 both in the early phase (days 1-6, 30 mg/kg/day, oral administration) and in the late phase (days 7-12, 30 mg/kg/day, oral administration), whereas it was inhibited by neutralization with anti-VEGF antibody (1 microg/site/day, local injection) only in the late phase. These results suggest that BK would promote angiogenesis by increasing vascular permeability in the early phase via B2 receptor in the endothelial cells and by promoting up-regulation of VEGF via B2 receptor in the stromal fibroblasts in the late phase.

Role of thromboxane derived from COX‐1 and ‐2 in hepatic microcirculatory dysfunction during endotoxemia in mice

Although thromboxanes (TXs), whose synthesis is regulated by cyclooxygenase (COX), have been suggested to promote inflammation in the liver, little is known about the role of TXA2 in leukocyte endothelial interaction during endotoxemia. The present study was conducted to investigate the role of TXA2 as well as that of COX in lipopolysaccharide (LPS)‐induced hepatic microcirculatory dysfunction in male C57Bl/6 mice. We observed during in vivo fluorescence microscopic study that LPS caused significant accumulation of leukocytes adhering to the hepatic microvessels and non‐perfused sinusoids. Levels of serum alanine transaminase (ALT) and tumor necrosis factor alpha (TNFα) also increased. LPS raised the TXB2 level in the perfusate from isolated perfused liver. A TXA2 synthase inhibitor, OKY‐046, and a TXA2 receptor antagonist, S‐1452, reduced LPS‐induced hepatic microcirculatory dysfunction by inhibiting TNFα production. OKY‐046 suppressed the expression of an intercellular adhesion molecule (ICAM)‐1 in an LPS‐treated liver. In thromboxane prostanoid receptor‐knockout mice, hepatic responses to LPS were minimized in comparison with those in their wild‐type counterparts. In addition, a selective COX‐1 inhibitor, SC‐560, a selective COX‐2 inhibitor, NS‐398, and indomethacin significantly attenuated hepatic responses to LPS including microcirculatory dysfunction and release of ALT and TNFα. The effects of the COX inhibitors on hepatic responses to LPS exhibited results similar to those obtained with TXA2 synthase inhibitor, and TXA2 receptor antagonist. In conclusion, these results suggest that TXA2 is involved in LPS‐induced hepatic microcirculatory dysfunction partly through the release of TNFα, and that TXA2 derived from COX‐1 and COX‐2 could be responsible for the microcirculatory dysfunction during endotoxemia. (HEPATOLOGY 2004;39:139–150.)

MARCH-XI, a novel transmembrane ubiquitin ligase implicated in ubiquitin-dependent protein sorting in developing spermatids

A mechanism by which ubiquitinated cargo proteins are sorted into multivesicular bodies (MVBs) from plasma and trans-Golgi network (TGN) membranes is well established in yeast and mammalian somatic cells. However, the ubiquitin-dependent sorting pathway has not been clearly defined in germ cells. In this study we identified a novel member of the transmembrane RING-finger family of proteins, termed membrane-associated RING-CH (MARCH)-XI, that is expressed predominantly in developing spermatids and weakly in brain and pituitary. MARCH-XI possesses an E3 ubiquitin ligase activity that targets CD4 for ubiquitination. Immunoelectron microscopy of rat round spermatids showed that MARCH-XI is localized to TGN-derived vesicles and MVBs. Fluorescence staining of rat round spermatids and immunoprecipitation of rat testis demonstrated that MARCH-XI forms complexes with the adaptor protein complex-1 and with fucose-containing glycoproteins including ubiquitinated forms. Furthermore, the C-terminal region of MARCH-XI mediates its interaction with μ1-adaptin and Veli through a tyrosine-based motif and a PDZ binding motif, respectively. Our data suggest that MARCH-XI acts as a ubiquitin ligase with a role in ubiquitin-mediated protein sorting in the TGN-MVB transport pathway, which may be involved in mammalian spermiogenesis.

Dual Wavelength Imaging Allows Analysis of Membrane Fusion of Influenza Virus inside Cells

ABSTRACT Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.

Laminin α1 chain G domain peptide, RKRLQVQLSIRT, inhibits epithelial branching morphogenesis of cultured embryonic mouse submandibular gland

Active sequences from the laminin α1 and α2 chain carboxyl‐terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583–20590; Nomizu et al. [1996] FEBS Lett. 396:37–42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin α1 chain peptide, RKRLQVQLSIRT (residues 2719–2730 and designated AG‐73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin α1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG‐73, a homologue of AG‐73 from the laminin α2 chain, did not inhibit the branching. The α2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin α2 chain‐specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E‐cadherin and α6 integrin, as detected by immunofluorescence, were unchanged in both AG‐73 and control scramble peptide‐treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG‐73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG‐73 sequence of the laminin α1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG‐73‐treated explants may indicate an important role for this region in basement membrane assembly. Dev. Dyn. 1998;212:394–402.