Umbilical Vein Endothelial Cell Line Research Articles

Transient expression and biological activity identification of human pigment epithelium-derived factor in mammary cell line SP2/0

Objective To construct eukaryotic expression plasmid pIRESneo3-pigment epithelium-derived factor (PEDF) and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector pIRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3-(4,5-dimethylthiazol-z-y1)-2,5-diphenytetrazolium bromide(MTT) method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h [(0.92±0.04) μg/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h (P<0.05). Conclusions The eukaryotic expression plasmid pIRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity. Key words: Retinopathy of prematurity; Eye proteins; Nerve growth factors; Serpins; Eukaryotic cells

Identification of two internal signal peptide sequences: critical for classical swine fever virus non-structural protein 2 to trans-localize to the endoplasmic reticulum

BackgroundThe membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The NS2 gene was amplified by reverse transcription polymerase chain reaction, with the transmembrane region and hydrophilicity of the NS2 protein was predicted by bioinformatics analysis. Twelve cDNAs of the NS2 gene were amplified by the PCR deletion method and cloned into a eukaryotic expression vector, which was transfected into a swine umbilical vein endothelial cell line (SUVEC). Subcellular localization of the NS2 protein was characterized by confocal microscopy, and western blots were carried out to analyze protein expression.ResultsOur results showed that the -NH2 terminal of the CSFV NS2 protein was highly hydrophobic and the protein localized in the ER. At least four transmembrane regions and two internal signal peptide sequences (amino acids103-138 and 220-262) were identified and thought to be critical for its trans-localization to the ER.ConclusionsThis is the first study to identify the internal signal peptide sequences of the CSFV NS2 protein and its subcellular localization, providing the foundation for further exploration of this protein's function of this protein and its role in CSFV pathogenesis.

Anti-inflammatory Effects of Aqueous Extract from Radix Liriope muscari and Its Major Active Fraction and Component

Aim To investigate the anti-inflammatory activities of the aqueous extract from Liriope muscari (Decne.) Bailey (Lm-a), its crude saponin fraction (Lm-s) and one major component (Lm-3), and to provide some pharmacological evidence for its clinical use in inflammatory diseases. Methods The anti-inflammatory activities of Lm-a, Lm-s and Lm-3 were evaluated by xylene-induced ear swelling and paw edema induced by carrageenan or histamine in mice. Meanwhile, the activity of Lm-3 was also determined by assaying the adhesion of human pro-myelocytic leukemia cell strain (HL-60) to human umbilical vein endothelial cell line (ECV304) induced by tumor necrosis factor (TNF-α) or phorbol-12-myristate-13-acetate (PMA) in vitro. Results Lm-a significantly inhibited ear swelling caused by xylene and paw edema induced by carrageenan in mice when given orally once at doses of 336 and 672 mg·kg −1. Meanwhile, Lm-s and Lm-3 possessed similar remarkable anti-inflammatory effects in such two animal models at a single oral dose of 23.2 or 4.6 mg·kg −1 (relevant to Lm-a 672 mg·kg −1), respectively. Moreover, Lm-3 markedly suppressed acute paw edema induced by histamine in mice when given orally once at the dose of 4.6 mg·kg −1, whose potency was similar to that of Lm-a or Lm-s at a single oral dose of 672 or 23.2 mg·kg −1. Lm-3 also significantly inhibited the adhesion of HL-60 to ECV304 cells induced by TNF-α or PMA at the concentrations of 0.01, 0.1 and 1 μmol·L −1 in vitro, which suggested the involved role of protein kinase C etc. Conclusion Lm-a, Lm-s and Lm-3 have the anti-inflammatory activities, which offers pharmacological supports to the ethnomedical use of the roots of L. muscari in inflammatory diseases and reveals the major attribution of saponins-rich fraction including Lm-3.

IL-22 inhibits ox-LDL-induced apoptosis and increases Bcl-2 expression in CRL-1730 cells

Objective To investigate the effect of interleukin-22(IL-22) on apoptosis of CRL-1730 cells(human umbilical vein endothelial cell line) induced by ox-LDL and the possible mechanism.Methods Expression of IL-22 receptor R1(IL-22R1) mRNA in CRL-1730 cells stimulated with ox-LDL(100 μg/ml) was detected by fluorescence quantitative RT-PCR.Flow cytometry was applied to examine the effect of exogenous IL-22(20 ng/ml) on the ox-LDL-induced apoptosis of CRL-1730 cells.The expression of Bcl-2,Bcl-xl,Bcl-w,caspase 3,and STAT3 mRNA in CRL-1730 cells treated or untreated with IL-22 and ox-LDL were measured by fluorescence quantitative RT-PCR;the phosphorylation status of STAT3 and the Bcl-2 protein level were examined by Western blotting analysis.Results The expression of IL-22R1 mRNA increased in a time-dependent manner(P0.05) and peaked at 12 h after ox-LDL stimulation.Treatment with IL-22 significantly inhibited the apoptosis of CRL-1730 cells induced by ox-LDL(P0.01),increased the expression of anti-apoptosis genes Bcl-2,Bcl-xl,and Bcl-w,and decreased expression of caspase 3 mRNA(P0.01).STAT3 phosphorylation occurred 1-2 h after IL-22 treatment and Bcl-2 protein expression increased 4 h after IL-22 treatment in ox-LDL stimulated CRL-1730 cells.Conclusion IL-22 can inhibit the pro-apoptosis effect of ox-LDL in the CRL-1730 cells,which might be associated with the increased expression of Bcl-2,Bcl-xl,and Bcl-w mRNA,decreased expression of caspase 3,and induced STAT3 phosphorylation.

Construction of human vascular endothelial growth factor 165 gene recombinant eukaryotic expression vector and its effect on the proliferation of vascular endothelial cells

Objective To construct a recombinant eukaryotic expression vector pIRES-VEGF165-tPA containing functional region of human vascular endothelial growth factor 165 (VEGF165) gene, and investigate its in vitro expression and effect on the proliferation of vascular endothelial cells in transfected human umbilical vein endothelial cell lines (EA. hy926). Methods The VEGF165 gene was inserted into the eukaryotic expression vector plRES-EGFP to construct the recombinant plasmid, which was transfected into human umbilical vein endothelial cell lines (EA. hy926) at 1.2 μg/well subsequently. The transcription and expression of VEGF165 gene were detected by reverse transcription-polymerase chain reaction ( RT-PCR, n = 3 ) and Western blotting ( n = 5 ) respectively. And the effect on the proliferation of vascular endothelial cells was evaluated by MTT assay (n =5). Results The sequence of pIRES-EGFP-VEGF165 approved that the gene of VEGF165 was inserted into the eukaryotic expression vector correctly. The transfection efficiency was about ( 11.2 ± 2. 5) %, detected by counting the positive cells of green fluorescence.The mRNA and protein levels of VEGF165 in the pIRES-EGFP-VEGF165 transfected group were significantly higher than those in the control groups respectively (P ＜ 0. 01 ). Also, MTT assay indicated the proliferation of endothelia cells was greatly improved in the pIRES-EGFP-VEGF165 transfected group. Conclusion The recombinant eukaryotic expression vector pIRES-EGFP-VEGF165 is successfully constructed and can be expressed in transfected EA. hy926 cells, and it is proved that transfection with this vector could improve the proliferation of vascular endothelial cells. Key words: Vascular endothelial growth factor; Eukaryotic expression vector; Cellular proliferation; Endothelialization

Double suicide genes selectively kill human umbilical vein endothelial cells

BackgroundTo construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells.MethodsHuman KDR promoter, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304) and KDR-negative liver cancer cell line (HepG2) were infected with the recombinant adenoviruses at different multiplicity of infection (MOI). The infection rate was measured by green fluorescent protein (GFP) expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC). The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining.ResultsRecombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs.ConclusionAdKDR-CDglyTK/double prodrog system may be a useful method for suppressing tumor angiogenesis.

Classical swine fever virus NS2 protein promotes interleukin-8 expression and inhibits MG132-induced apoptosis

Classical swine fever (CSF) caused by virulent strains of classical swine fever virus (CSFV) is a hemorrhagic disease of pigs and is characterized by disseminated intravascular coagulation, thrombocytopenia, and immunosuppression. Until now, the role of the NS2 protein produced by CSFV in the pathogenesis of CSF is not well understood. In this report, we investigated the function of CSFV NS2 by examining its effects on the pro-inflammatory CXC chemokine, interleukin-8 (IL-8) expression, and cell survival. Stable swine umbilical vein endothelial cell line (SUVEC) expressing CSFV NS2 were established and showed that CSFV NS2 expressing SUVEC cells express approximately 16-fold higher levels of IL-8 as compared to control vector GFP-expressing cells, GFP-E2 expressing cells, and untransfected cells. Further studies showed that CSFV NS2 induced endoplasmic reticulum stress and activated the nuclear transcription factor kappa B (NF-κB), which is responsible for the up-regulation of IL-8 and the anti-apoptotic protein, Bcl-2, expression. In addition, the GFPNS2-expressing SUVEC cells were resistant to MG132-induced apoptosis. This study suggested that CSFV NS2 plays an important role in the inflammatory response and in persistent CSFV infection. These findings provide novel information on the function of the poorly characterized CSFV NS2.

Hypoxia Induces FGF2 Production by Vascular Endothelial Cells and Alters MMP9 and TIMP1 Expression in Extravillous Trophoblasts and Their Invasiveness in a Cocultured Model

The role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during trophoblast invasion was assessed. The human extravillous trophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were cocultured under normal and hypoxic conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells were analyzed using quantitative RT-PCR and Western blot analyses. TEV-1 cell invasion was also examined. FGF2 expression in the HUVE-12 cells cocultured with TEV-1 cells was significantly increased under hypoxic conditions. In the TEV-1 cells cocultured with HUVE-12, hypoxia reduced MMP9 expression and increased TIMP1 expression; it also reduced cell invasion by 43%. However, the expression of MMP9 and TIMP1 and ratio of MMP9/TIMP1 were increased when the TEV-1 cells were cultured alone under hypoxic conditions. These findings suggest that FGF2 release by stressed endothelial cells of uterine spiral arteries play roles in decreasing MMP9 and increasing TIMP1 production in extravillous trophoblasts (EVT) in response to stress, resulting in reduced EVT invasion and possibly shallow implantation of the placenta.

Acylated catechin derivatives inhibitors of DNA polymerase and angiogenesis

Catechins in green tea display anti-cancer and anti-angiogenesis activities. We previously found that some catechins, such as epigallocatechin-3-O-gallate (EGCG), inhibit the activities of eukaryotic DNA polymerases (pols) (Y. Mizushina et al.: Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors. Biochem Biophys Res Commun 333, 101-109 (2005)). In this study, we discuss the effects of chemical modifications of catechin and epicatechin that enhance their anti-cancer and anti-angiogenic activities based on pol inhibition. Catechins conjugated with fatty acid (3-O-acylcatechins) are stronger inhibitors of mammalian pol than epicatechins conjugated with fatty acid (3-O-acylepicatechins). Moreover, 3-O-acylcatechins are more potent inhibitors of cultured cell growth both of the human colon carcinoma cell line (HCT116 cells) and human umbilical vein endothelial cell (HUVEC) line, as well as angiogenesis by comparison with 3-O-acylepicatechins. Catechin conjugated with stearic acid ((2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; C-C18) was the strongest inhibitor in replicative pol alpha and repair-related pol beta, as well as the cultured cell growth and angiogenesis assays in the compounds tested. C-C18 also suppressed HUVEC tube formation on reconstituted basement membrane suggesting that it affected not only pols but also signal transduction pathways in HUVECs. These data indicate that the acylated catechins target both pols and angiogenesis as anti-cancer agents. Moreover, the results suggest that acylation of catechin is an effective chemical modification to improve the anti-cancer activity of catechin.

Epigenetic modulation of human breast cancer by metallofullerenol nanoparticles: in vivo treatment and in vitro analysis

Multi-hydroxylated endohedral metallofullerenol [Gd@C(82)(OH)(22)](n) nanoparticles possess the general physico-chemical characteristics of most nanoparticles. They also exhibit uniquely low toxicity and antineoplastic efficacy. In the current study, the molecular mechanisms and epigenetic characteristics of the antineoplastic action of these nanoparticles are explored. Human breast cancer MCF-7 and human umbilical vein endothelial ECV304 cell lines were used. Cell viability assay, cell hierarchical cluster analysis by cDNA microarray, semi-quantitative reverse transcription-polymerase chain reaction and Western blot analysis were conducted to investigate the changes in molecular and cellular signaling pathways caused by [Gd@C(82)(OH)(22)](n). The results demonstrated the high antitumor activity and low cytotoxicity of [Gd@C(82)(OH)(22)](n) nanoparticles both in vivo and in vitro. Their possible anti-tumor mechanisms were also discussed. The present study may provide new insight into the mechanism of action of these nanoparticles.

Interaction of coagulation factors and tumor‐associated macrophages mediates migration and invasion of gastric cancer

Abundant macrophage infiltration and increased expression of coagulation factors have been observed in cancer patients. The aim of the present study was to determine how the interaction between activated coagulation factors and monocytes/macrophages contributes to gastric cancer (GC) cell migration and invasion. We assessed cytokine/chemokine production of coagulation-factor-treated macrophages by ELISA. The effects of the interaction between coagulation factors and tumor-associated macrophages (TAM) on GC cell migration and invasion were determined by in vitro migration and invasion assay. In addition, we used an in vitro co-culture system of GC cells/TAM treated by coagulation factors to evaluate the effect of coagulation factor/TAM interaction on the human umbilical vein endothelial cell line (HUVEC). We found that the M2-like phenotype of interleukin (IL)-4(high), IL-10(high), transforming growth factor (TGF)-β(high), tumor necrosis factor (TNF)-α(high) was exhibited when the human monocytic cell line THP-1 was stimulated by coagulation factors III (TF), VIIa (FVIIa) and XIIa (FXIIa). For the migration assay, the GC cells (BGC-823 or SGC-7901) that were co-cultured with activated coagulation factor/TAM both showed increased migration. For the invasion assay, both BGC-823 and SGC-7901 cells co-cultured with TF/TAM showed increased invasion. We also found that TAM activated by coagulation factors could induce vascular endothelial growth factor/MMP-9 expression, which could promote invasion of GC cells. The HUVEC co-cultured with TAM (PMA-treated THP-1 macrophages co-cultured with GC cells) expressed high levels of FXIIa. In conclusion, coagulation factors might facilitate GC cell migration and invasion by transforming macrophages toward TAM-like cells. Interaction of coagulation factors and TAM mediates migration and invasion of GC.

Effect of pregnancy-associated plasma protein-A on the function of endothelial cells

To determine the effect of pregnancy-associated plasma protein A (PAPP-A) on the function of vascular endothelial cells (VEC). Human umbilical vein endothelial cell (HUVEC) line, derived from human umbilical vein, was cultured in vitro with PAPP-A at 0, 50, 100, and 200 ng/mL for 0, 12, 24, and 48 hours, respectively. Nitric oxide (NO) levels and endothlin-1 (ET-1) levels were determined by spectrophotometer and immunehistory. The NO levels of HUVECs in the PAPP-A groups were significantly lower than those in the control group (P<0.05). The ET-1 levels of HUVECs in the PAPP-A groups were significantly higher than those in the control group (P<0.05). The changes were all dose-dependent. PAPP-A may affect the function of vascular endothelial cells by reducing the secretion of NO and increasing the level of ET-1.

Study on the injury of vascular endothelial cells affected by dust particles from workplaces in pottery factories and tungsten mines

to assess direct adverse effects of occupational dusts from pottery factories and tungsten mines on vascular endothelial cells in vitro test. human umbilical vein endothelial cell (HUVEC) line HUV-EC-C were used as target cells. HUVEC were then treated with respirable dust particles from workplaces in pottery factories and tungsten mines in concentrations of 25, 50, 100, 200 and 400 microg/ml for 24 h. Standard quartz was used as control. LDH activity, cell viability, the release of NO and TNF-α levels were determined to assess the biological responses of the dust particles. dose-response relationships between the dust concentrations and the enhancement of the LDH activity, the release of NO and TNF-α were found in both dust particles from pottery factories and tungsten mines. The cell viability decreased with the increase of dust concentration from 25 to 400 microg/ml. Compared with the dust particles from workplaces, the quartz dust induced significantly higher LDH activity (P < 0.05) after cultured with HUVEC. No significant difference of releases of NO were observed among the dust particles from workplaces and standard quartz. However, significantly higher levels of TNF-α were induced by standard quartz compared with dust samples from workplaces at concentrations of 200, 400 microg/ml. occupational dust particles from workplaces and quartz could induce the injury and the releases of TNF-α from HUVEC.

Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-kappaB pathway, but not through the p38 MAPK pathway

Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule-1 (ICAM-1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. We examined the expression of ICAM-1, IκBα, phospho-p38 MAPK and nuclear factor-kappaB (NF-κB) p65 in an umbilical vein endothelial cell line (ECV-304) treated with ATCC 33277 and W83, with or without the NF-κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real-time PCR, western blotting and immunofluorescence. Both strains could induce ICAM-1 expression; additionally W83 was able to increase ICAM-1 expression more significantly than ATCC 33277. In P. gingivalis-infected endothelial cells, both p38 MAPK and NF-κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF-κB. It was found that ICAM-1 production in endothelial cells was abrogated by inhibition of the NF-κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. The induction of ICAM-1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF-κB pathway, but not by the p38 MAPK pathway.

Anti-angiogenic effects of the fruit of Alpinia oxyphylla

The fruit of Alpinia oxyphylla, an herb commonly used in East Asian medicine, is variously used for the treatment of cancer and inflammatory conditions, which may possibly be mediated through anti-angiogenesis. This study aims to check for anti-angiogenic functions in the herb. The 95% ethanol extract and four subsequent fractions (n-hexane, ethyl acetate, n-butanol and aqueous fractions) of the fruit of A. oxyphylla were tested on zebrafish model by quantitative endogenous alkaline phosphatase assay; then the active fractions were further tested on wild type and Tg(fli1a:EGFP)y1 zebrafish embryos and human umbilical vein endothelial cells and tumor cell lines for the anti-angiogenic effects. The n-hexane and ethyl acetate fractions showed anti-angiogenic potentials in both in vivo and in vitro models. The use of A. oxyphylla for cancer and inflammation diseases may be partly due to its effects against vessel formation.

The effect of magnesium sulfate on the activity of matrix metalloproteinase-9 in fetal cord plasma and human umbilical vein endothelial cells

Clinical evidence suggests that magnesium sulfate may reduce the risk of fetal neurologic injury in preterm delivery. Matrix metalloproteinase-9 (MMP-9) levels are elevated in preterm labor patients. There is evidence that MMP-9 may break down the blood-brain barrier in humans, causing cytokine mediated cell injury. Our objective was to determine whether the addition of magnesium sulfate attenuates activity of MMP-9, a complex zinc-dependent enzyme, in fetal cord plasma. We collected cord plasma in 6 term, unlabored patients. Using enzyme-linked immunosorbent assay, we measured the activity of MMP-9 with varying concentrations of magnesium sulfate added in vitro. Results were verified using a human umbilical cord vein endothelial cell (HUVEC) line. Addition of physiologic doses of magnesium sulfate (0.07 mg/mL) resulted in a 25% decrease in active MMP-9 (P = .03). In a HUVEC line, magnesium sulfate resulted in a 32% decrease in MMP-9 activity (P = .00012). The addition of magnesium sulfate attenuated MMP-9 activity in cord plasma and in a HUVEC line.

Influence of Recombinant Human Growth Hormone (rhGH) on Proliferation of Hepatocellular Carcinoma Cells with Positive and Negative Growth Hormone Receptorsin Vitro

Recombinant human growth hormone (rhGH) is increasingly used in the clinic because it promotes the synthesis of proteins. However, rhGH is able to increase malignant transformation and tumor recurrence. The aim of this study was to investigate the effects of rhGH on hepatocellular carcinoma (HCC) cells with positive and negative growth hormone receptors (GHR) in order to guide its clinical application. Cells of the human HCC cell lines Bel-7402 (GHR+) and SMMC-7721 (GHR-) as well as human umbilical vein endothelial cell line ECV304 cells in the exponential growth phase were harvested and divided into experimental and control groups. After the human HCC cells were cultured alone or co-cultured with ECV304 cells under the different treatments, cell cycle phase, proliferation index, and expression levels of vascular endothelial growth factor (VEGF) mRNA and proteins were determined. In the Bel-7402 GHR+ cells treated with rhGH, both the percentage of cell in G2-M phase and the proliferation index were higher than those of controls (P < 0.05); this was not the case in the SMMC-7721 GHR- cells treated with rhGH (P > 0.05). Although there was no difference in the cell doubling times between ECV304 cells co-incubated with Bel-7721 GHR-cells treated with rhGH and without rhGH, the doubling times of ECV304 cells co-incubated with Bel-7402 GHR+ cells, when treated with rhGH, were significantly shortened compared to those of controls (P < 0.05). The cell doubling times of ECV304 cells co-incubated with Bel-7721 GHR- or Bel-7402 GHR+ cells which were treated with bevacizumab were longer than those of controls and of cells with rhGH (P < 0.05). The VEGF mRNA and protein expression levels were higher in Bel-7402 GHR+ cells treated with different doses of rhGH than controls (P < 0.05 or P < 0.01); however, there was no statistically significant difference in the expression levels of VEGF mRNA and proteins between SMMC-7721 GHR- cells treated with rhGH and controls. rhGH can induce VEGF secretion and stimulate proliferation of Bel-7402 GHR+ cells in vitro, but has little effect on the proliferation of SMMC-7721 GHR-cells, suggesting that rhGH may be applied safely to treatment for the catabolic state in patients with GHR-negative HCC.

Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress

BackgroundClassical swine fever (CSF) caused by virulent strains of Classical swine fever virus (CSFV) is a haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopoenia and immunosuppression, and the swine endothelial vascular cell is one of the CSFV target cells. In this report, we investigated the previously unknown subcellular localization and function of CSFV NS2 protein by examining its effects on cell growth and cell cycle progression.ResultsStable swine umbilical vein endothelial cell line (SUVEC) expressing CSFV NS2 were established and showed that the protein localized to the endoplasmic reticulum (ER). Cellular analysis revealed that replication of NS2-expressing cell lines was inhibited by 20-30% due to cell cycle arrest at S-phase. The NS2 protein also induced ER stress and activated the nuclear transcription factor kappa B (NF-κB). A significant increase in cyclin A transcriptional levels was observed in NS2-expressing cells but was accompanied by a concomitant increase in the proteasomal degradation of cyclin A protein. Therefore, the induction of cell cycle arrest at S-phase by CSFV NS2 protein is associated with increased turnover of cyclin A protein rather than the down-regulation of cyclin A transcription.ConclusionsAll the data suggest that CSFV NS2 protein modulate the cellular growth and cell cycle progression through inducing the S-phase arrest and provide a cellular environment that is advantageous for viral replication. These findings provide novel information on the function of the poorly characterized CSFV NS2 protein.

Up-regulation of integrin β3 expression in porcine vascular endothelial cells cultured in vitro by classical swine fever virus

Classical swine fever (CSF) caused by virulent strains of classical swine fever virus (CSFV) is a haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopenia and immunosuppression. The cell adhesion molecule, integrin β3, plays a central role in maintaining and regulating vascular permeability. In view of the haemorrhagic pathology of the disease, the effect of CSFV infection on integrin β3 expression was investigated using the swine umbilical vein endothelial cell (SUVEC) line, in conjunction with quantitative PCR and Western blotting techniques. Following infection, the expression levels of integrin β3 were significantly up-regulated along with corresponding transcription levels. The infected endothelial cells adhered onto immobilized extracellular matrix (ECM) with more extensive spreading than that of the control, and such interaction was strongly inhibited by an anti-integrin β3 monoclonal antibody (mAb). This study revealed the up-regulation of integrin β3 in vascular endothelial cells by CSFV infection, and cell adhesion molecules of this kind possibly play an important role in the changes of haemostatic balance in haemorrhagic pathology of CSF.

TH‐D‐BRD‐04: Increased Tumor Radioresistance by Imaging Doses From Volumetric Image Guided Radiation Therapy

Purpose: The radiation dose delivered from volumetric imaging guidance is normally between 1–10 cGy, depending on the imaging modalities, tumor sites and patient thickness. To correctly compensate for such doses, their biological effects on tumor and endothelial cells are investigated. Methods and materials: A lung carcinoma cell line (H460), a breast cancer cell line (MCF‐7) and a prostate cancer cell line (PC3) and a human umbilical vein endothelial cell line (HUVEC) were studied for their responses to small doses of radiation ranging from 5 cGy to 50 cGy. Specifically, a MTT assay was used to quantify their proliferation over time. To measure the impact of image guidance doses on cell survival, 5 cGy or 10 cGy were delivered to H460 cells before a 200 cGy therapeutic dose. The tumor cell survival was measured by clonogenic assay and compared with cells that received 200 cGy only. Results: Accelerated proliferation was observed among all tumor cell lines but not the HUVEC to low dose irradiation. The acceleration was statistically significant (p&lt;0.05) for H460 and MFC7 cells but not for the PC3 cells. A 12.6% increase in the H460 cell survival was observed for cells receiving 5cGy before 200 cGy radiation compared to those that received 200 cGy alone (p&lt;0.02), while cells receiving 10 cGy prior to 200 cGy had the same survival as cells receiving 200 cGy alone. Conclusions: Small pre‐treatment doses of radiation may increase the radioresistance of tumor cells. In this study, we demonstrated that the imaging dose from image guided radiation therapy is sufficient to trigger accelerated proliferation when delivered alone and increased tumor survival when delivered prior to a therapeutic dose. Our results suggest that subtracting the imaging dose from the therapeutic dose may adversely affect the tumor control probability.