Inhibition of tumor‐induced edema by antisense VEGF is mediated by suppressive vesiculo‐vacuolar organelles (VVO) formation

Abstract.Endostatin is an anti-angiogenic agent that blocks endothelial cell proliferation, tumor growth, and metastasis. Several lines of direct evidence show that gliomas express high levels of endostatin. However, the anti-angiogenic activity and cellular mechanisms of endostatin from tumor cells have not been completely elucidated. In this study, we established C6 glioblastoma (GBM) xenografts in nude mice by subcutaneously injecting glioma cell lines, C6-null cells, and stable transfected-C6 cells overexpressing mock vector (C6-mock) and endostatin (C6-endo). We found that overexpression of endostatin in C6-endo cells significantly suppressed the expression of VEGF in tumor cells in vivo as well as in vitro. Furthermore, the tumor growth derived from C6-endo cells was inhibited. Our data demonstrate that endostatin could play a direct role in inhibiting tumor cells, and suggest that enhancing endostatin expression in gliomas could be an effective treatment strategy.

of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats. Am J Physiol Lung Cell Mol Physiol 309: L1323-L1332, 2015. First published September 4, 2015 doi:10.1152/ajplung.00016.201543 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known, and the underlying mechanism has not been described. With cecal ligation and puncture-induced sepsis in rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and its relationship to the RhoA/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs was significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. The connexin inhibitor carbenoxolone (10 mg/kg iv) and the Rock1 inhibitor Y-27632 (2 mg/kg iv) alleviated the vascular leakage of lung, mesentery, and kidney in sepsis rats. Overexpressed Cx43 increased the phosphorylation of 20-kDa myosin light chain (MLC 20) and the expression of Rock1 and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC20 and the expression of Rock1 and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor Y-27632 alleviated LPS-and overexpressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and that the mechanism is related to the Rock1-MLC20 phosphorylation pathway. connexin 43; vascular permeability; sepsis; RhoA/Rock1 pathway; MLC 20

Abstract. Zebrafish (Danio rerio) is becoming an increasingly popular vertebrate cancer model. In this study, we established a xenotransplanted zebrafish embryo glioma model to further investigate the molecular mechanisms of tumor angiogenesis. We find that the glioma cell line U87 can survive, proliferate and induce additional SIV branches in zebrafish embryos. In addition, by the means of in situ hybridization and quantitive RT-PCR analyses we find that the transplanted U87 cells can induce the ectopic zebrafish vascular endothelial growth factor A (VEGF A) and its receptor VEGFR2/KDR mRNA expression and increase their expression levels, resulting in additional SIV branches. IntroductionGlioma is the most common primary brain tumor in adults and the median survival for patients without therapy is up to 3 months (1). Currently the traditional treatment such as resection, chemotherapy and radiotherapy has not greatly improved the median survival for patients. Malignant glioma is notorious for its behavior of rapid proliferation, great invasion and metastasis and it is also among the best vascularized tumors in humans. It is well known that angiogenesis plays a critical role in tumor progression. Therefore new anti-angiogenic treatment strategies are required (2-6). Revealing of various signaling pathways that lead to activation of angiogenesis will provide molecular insight into developing therapeutic agents to treat glioma. In the past decades, researchers have discovered many molecular mechanisms related to tumor angiogenesis (7-9).Traditional rodent cancer models have disadvantages, e.g., the high cost, long developmental phase, and limited availability for high-throughput assays (10), which limited effective ways to find the new molecular mechanisms and monitor the tumor in vivo in real time.The teleost zebrafish (Danio rerio), a promising alternative vertebrate cancer model has attracted considerable attention in recent years because of compelling advantages of transparency of the embryo, rapid development, fecundity, tractable genetics, great screening efficiency, high levels of physiologic and genetic homology with higher vertebrates (11-13). Moreover, the zebrafish embryo within 1 week is immune-free, and showed no immunosuppression to transplanted human glioma cells (14). The transgenic zebrafish strains, e.g., casper transparent mutant line (15), and VEGFR2:G-RCFP line with green fluorescence specifically in blood vessels (16), offered new tools for cancer research.In this study, we microinjected human U87 glioma cells into zebrafish embryos and developed a xenograft zebrafish glioma model. The U87 cells are first labeled with a red fluorescence protein and then microinjected into perivitelline space of VEGFR2:G-RCFP transgenic zebrafish embryos at 48 h post-fertilization. Then in order to investigate the angiogenesis mechanisms in details, we make use of the staining of endogenous alkaline phosphatase, fluorescent microscopy monitoring, in situ hybridization and quantitive RT-PCR to investigate the ch...