Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Conditioned media from solid tumor cell lines treated with retinoic acids both decreases and increases proliferation of capillary endothelial cells.

These studies investigated the effects of retinoic acids on endothelial cell proliferation. Three human neoplastic cell lines, U-373 MG glioblastoma, DU-145 prostate carcinoma, and TCCSUP bladder transitional cell carcinoma, were treated with all-trans, 9-cis, or 13-cis retinoic acids at 0.0001 to 10 microM. Hypoxia was used to ensure the expression of the angiogenic phenotype. Conditioned media (CM) were prepared by hypoxic culturing of the tumor cells with retinoic acids for 24 hours. Then CM were transferred to bovine capillary endothelial cells for 48 hours of normoxic culturing, counted and compared to controls. CM from U-373 MG and DU-145 cells, but not TCCSUP cells, treated with all-trans or 9-cis retinoic acids at several concentrations below 1 microM, caused significant (P<0.05) increases in endothelial cell proliferation of between 13 to 18%. Both nonconditioned and conditioned media, for retinoic acid concentrations above 1 microM, inhibited endothelial cell proliferation. All CM for 13-cis retinoic acid decreased endothelial cell proliferation. These results show that the cytotoxicity of retinoic acids and the growth promoting/inhibiting ability of the conditioned media is retinoic acid isoform, time, concentration, and cell type dependent. Most importantly, the conditioned media from tumor cells treated with low concentrations of all-trans or 9-cis retinoic acids significantly increased endothelial cell proliferation.     

Biosynthetic regulation of monobutyrin, an adipocyte-secreted lipid with angiogenic activity

1-Butyrylglycerol (monobutyrin) is a novel angiogenic compound that is synthesized and secreted during the differentiation of 3T3-F442A preadipocytes into adipocytes. To study the regulation of monobutyrin biosynthesis we have developed an assay utilizing the lgycerol kinase enzyme from Cellulomonas to quantitate the levels of this compound in cell-conditioned medium. Analysis of several cultured cell types, including tumor cell lines, indicated that monobutyrin production is detectable only from adipocytes, reaching a steady-state concentration of approximately 1.0 microM in conditioned medium. Monobutyrin synthesis was demonstrated in vitro using [14C]butyryl-CoA with total homogenate or particulate fractions from adipocytes. Similar fractions from non-adipocyte cell lines failed to synthesize monobutyrin. This biosynthetic activity was shown to be distinct by substrate competition studies from the microsomal sn-glycerol-3-phosphate acyltransferase, whose activity is known to increase during adipocyte differentiation. The production of monobutyrin was hormonally regulated, as the addition of epinephrine to adipocytes caused a 10-fold increase in the amount of monobutyrin secreted. These results indicate that monobutyrin synthesis is adipocyte specific, occurs through an apparently novel particulate enzyme system, and is regulated in a hormone-dependent manner. The implications of these results for adipose physiology and angiogenesis are discussed.     

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011. © 2010 Wiley‐Liss, Inc.     

Up‐regulated basigin‐2 in microglia induced by hypoxia promotes retinal angiogenesis

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well‐characterized mouse model of oxygen‐induced retinopathy, which recapitulated hypoxia‐induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up‐regulation of PI3K‐AKT and IGF‐1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro‐angiogenic role, providing new insights into microglia‐promoting retinal angiogenesis.     

Expression of developmentally regulated endothelial cell locus 1 was induced by tumor-derived factors including VEGF

Developmentally regulated endothelial cell locus 1 (Del1) is a new angiogenic molecules expressed specifically in early embryonic endothelial cells. We investigated the relationship between Del1 and tumor cell-derived vascular endothelial growth factor (VEGF). Dunn osteosarcoma cells and high- and low-metastatic murine sarcoma cells did not express Del1. However, the expression of Del1 was observed in these primary tumor tissues and the pulmonary metastatic tissues after subcutaneous inoculation in vivo. Every tumor cell-conditioned medium containing VEGF induced the expression of Del1 in murine lung microvascular endothelial (MLE) cells, although control MLE cells did not express Del1. The anti-mouse VEGF monoclonal antibody inhibited the induction of the Del1 expression. In addition, mouse recombinant interleukin-1alpha and tumor necrosis factor-alpha also induced Del1 in MLE cells. Del1 may play an important role in tumor angiogenesis through the effects of tumor-derived factors including VEGF.     

Therapeutic angiogenesis using tumor cell‐conditioned medium

Stem cell‐conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow‐derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life‐time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456–464, 2016     

Improved high-performance liquid chromatography of the new antineoplastic agents bisantrene and mitoxantrone

Bisantrene and mitoxantrone are two new anthracene derivatives which have shown significant antitumor activity against a wide variety of animal tumors and in human phase I and II clinical trials. We have developed a rapid, simple and sensitive sample cleanup procedure and high-performance liquid chromatographic (HPLC) assay for both drugs. This method uses a commercially available mini-cartridge with C₁₈ reversed-phase packing to isolate the drugs from the biological matrix prior to HPLC. For both drugs the average recovery of the assay was 98 ± 6% with a coefficient of variation (C.V.) of less than 7%. Using this new method our assay sensitivity has improved to less than 10 ng/ml for bisantrene and 1 ng/ml for mitoxantrone, allowing us to document a prologned terminal phase plasma half-life for both bisantrene and mitoxantrone. Equilibrium dialysis studies showed that both drugs are highly protein bound. Mitoxantrone appears less stable in human plasma than bisantrene. Recoveries from plasma after a 24-h incubation at 25 and 37°C were 40 and 20% for mitoxantrone and 90 and 85% for bisantrene, respectively. Addition of ascorbic acid prior to incubation of mitoxantrone in human plasma at 37°C resulted in less than a 10% decrease in the latter's concentration over a 24-h period. To maintain sample integrity, all plasma samples should be fortified with ascorbic acid and kept frozen prior to analyses.     

An assay for cancer stem cell-induced angiogenesis on chick chorioallantoic membrane

Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.     

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.     

Overexpression of osteopontin induces angiogenesis of endothelial progenitor cells via the avβ3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells

Angiogenesis, a hallmark of tumor growth, is regulated by various angiogenic factors. Recent studies have shown that osteopontin (OPN) is a secreted, integrin-binding protein that contributes to glioma progression. However, its effect on the angiogenesis of gliomas is not fully understood. To elucidate the role of OPN in the process of glioma angiogenesis, endothelial progenitor cells (EPCs) were treated with conditioned media of human glioma SHG44 cells overexpressing OPN. Here, we identified that OPN secreted by glioma cells accelerated EPCs angiogenesis in vitro, including proliferation, migration, and tube formation. OPN also induced the activation of AKT and endothelial nitric oxide synthase (eNOS) and increased NO production without affecting the expression of VEGF, VEGFR-1, or VEGFR-2. Moreover, the avβ3 antibody, the PI3-K inhibitor LY294002 and the eNOS inhibitor NMA suppressed the OPN-mediated increase in NO production and angiogenesis in EPCs. Taken together, these results demonstrate that OPN directly stimulates angiogenesis via the avβ3/PI3-K/AKT/eNOS/NO signaling pathway and may play an important role in tumorigenesis by enhancing angiogenesis in gliomas.     

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.     

Inhibition of production of macrophage-derived angiogenic activity by the anti-rheumatic agents gold sodium thiomalate and auranofin

We have investigated the effect of gold sodium thiomalate and auranofin, gold compounds employed in the treatment of rheumatoid arthritis, on production of macrophage-derived angiogenic activity. Elicited mouse peritoneal macrophages were cultured in the presence or absence of gold compounds or thiomalic acid, and the macrophages or their conditioned media were then assayed for their angiogenic activity in rat corneas. Control macrophage conditioned medium was potently angiogenic. In contrast, conditioned medium from gold or thiomalic acid treated macrophages was not. Addition of gold compounds or thiomalic acid to control macrophage conditioned medium did not inhibit its angiogenic activity. Drug treatments did not significantly affect macrophage lactate dehydrogenase release, lysozyme release, or protein synthesis. We conclude that gold sodium thiomalate and auranofin potently reduce the detectable angiogenic activity produced by macrophages.     

Human endomucin is an endothelial marker

The identification of novel endothelial markers is important in the study of angiogenesis, and may have potential uses in cancer diagnosis and treatment. We have isolated potential markers of tumor angiogenesis by screening human umbilical vein endothelial cells (HUVECs) treated with tumor conditioned media. Using suppression subtractive hybridization (SSH), we found endomucin, a potential cell surface marker upregulated in this system. Human endomucin is predicted to encode a 261-aa, 27.5-kDa protein with a transmembrane sequence and multiple glycosylation sites. Northern and in situ hybridization studies show that human endomucin expression is largely, if not uniquely, endothelial cell-specific. Human endomucin is present abundantly in highly vascular tissues such as heart, kidney, and lung. It is seen in human aortic endothelial cells (HAECs) as well as in human microvascular endothelial cells (HMVECs). Furthermore, its expression is increased when endothelial cells are proliferating or are stimulated by tumor-conditioned media or specific angiogenic factors such as bFGF (basic fibroblast growth factor) and TNFalpha (tumor necrosis factor), suggesting that endomucin may have a role in tumor angiogenesis.     

Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model

Endothelial cells lining blood vessels are exposed to various hemodynamic forces associated with blood flow. These include fluid shear, the tangential force derived from the friction of blood flowing across the luminal cell surface, tensile stress due to deformation of the vessel wall by transvascular flow, and normal stress caused by the hydrodynamic pressure differential across the vessel wall. While it is well known that these fluid forces induce changes in endothelial morphology, cytoskeletal remodeling, and altered gene expression, the effect of flow on endothelial organization within the context of the tumor microenvironment is largely unknown. Using a previously established microfluidic tumor vascular model, the objective of this study was to investigate the effect of normal (4 dyn/cm²), low (1 dyn/cm²), and high (10 dyn/cm²) microvascular wall shear stress (WSS) on tumor-endothelial paracrine signaling associated with angiogenesis. It is hypothesized that high WSS will alter the endothelial phenotype such that vascular permeability and tumor-expressed angiogenic factors are reduced. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability relative to mono-cultures. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, gene expression analysis revealed that high WSS (10 dyn/cm²) significantly down-regulates tumor-expressed MMP9, HIF1, VEGFA, ANG1, and ANG2, all of which are important factors implicated in tumor angiogenesis. This result was not observed in tumor mono-cultures or static conditioned media experiments, suggesting a flow-mediated paracrine signaling mechanism exists with surrounding tumor cells that elicits a change in expression of angiogenic factors. Findings from this work have significant implications regarding low blood velocities commonly seen in the tumor vasculature, suggesting high shear stress-regulation of angiogenic activity is lacking in many vessels, thereby driving tumor angiogenesis.     

Thrombospondin-2 plays a protective role in multistep carcinogenesis: a novel host anti-tumor defense mechanism

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro- angiogenic and anti-angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, we subjected TSP-2-deficient and wild-type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP-2 expression was strongly upregulated in the mesenchymal stroma of wild-type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP-2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre-malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP-2-deficient mice. While TSP-2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was significantly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP-2 expression as a novel host anti-tumor defense mechanism.     

Platelet microparticle delivered microRNA-Let-7a promotes the angiogenic switch

Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.     

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.     

Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.     

γ-Tocotrienol inhibits angiogenesis of human umbilical vein endothelial cell induced by cancer cell

Antiangiogenic therapy mediated by food components is an established strategy for cancer chemoprevention. Growth factors play critical roles in tumor angiogenesis. A conditioned medium containing growth factors from human gastric adenocarcinoma SGC-7901 cell conditioned medium was used as an angiogenic stimulus in this study. The purpose of this study was to evaluate the inhibitory effect and possible mechanism of γ-tocotrienol on tumor angiogenesis. The results showed that γ-tocotrienol (10-40 μmol/L) significantly suppressed proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by SGC-7901 cell conditioned medium in a dose-dependent manner. γ-Tocotrienol (800-1200 μg/egg) also inhibited new blood vessel formation on the growing chick embryo chorioallantoic membrane in a dose-dependent manner. Moreover, the inhibitory effects of γ-tocotrienol on HUVECs were correlated with inducing the apoptosis and arresting cell cycle at the G₀/G₁ phase at a dose of 40 μmol/L γ-tocotrienol. In addition, γ-tocotrienol inhibited angiogenesis in HUVECs by down-regulation of β-catenin, cyclin D1, CD44, phospho-VEGFR-2 and MMP-9. The antiangiogenic effects of γ-tocotrienol on HUVECs may be attributable to regulation of Wnt signaling by decreasing β-catenin expression. Thus, our results suggest that γ-tocotrienol has a potential chemopreventive agent via antiangiogenesis.