Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.
Animals ; Animals, Suckling ; CHO Cells ; Cloning, Molecular ; Cricetulus ; DNA, Complementary/genetics/metabolism ; Disease Susceptibility/virology ; Foot-and-Mouth Disease/*genetics/virology ; Foot-and-Mouth Disease Virus/*physiology ; Integrin alphaVbeta3/*genetics/metabolism ; Mice

OBJECTIVETo observe the effect of Yiqixue Buganshen recipe(, YBR) on the expression of integrin ανβ3 in the endometrium of controlled ovarian hyperstimulation mice.

METHODSA total of 180 mice were divided into three groups: model group, treatment group and control group. The treatment and model groups were intraperitoneally injected with gonadotropin-releasing hormone analogue for 7 days; pregnant mare serum gonadotropin was also injected on the 7th day. After 48 h, human chorionic gonadotropin was injected. The control group was injected with an equal volume of saline at the same time. From the start of the experiment, the treatment group was intragastrically administered Jinghouzengzhi Recipe() and Cuhuangti Recipe(). The model group and the control group were intragastrically administered an equal volume of saline. Real-time reverse transcription polymerase chain reaction and Western blotting were used to detect the mRNA and protein expression of integrin ανβ3 in mouse endometrium.

RESULTSIntegrin ανβ3 was expressed in mouse endometrium in all groups. Integrin αββ3 expression increased gradually along with pregnancy, progressing from pregnant day (Pd) 1. Integrin ανβ3 expression significantly increased on Pd 4, then began to decrease on Pd 6. Integrin ανβ3 expression in the treatment group was higher than in the model group, and the difference was statistically significant (P <0.05). The difference between the treatment group and the control group was not statistically significant (P >0.05).

CONCLUSIONYBR improves endometrial receptivity, and may play an important role in embryonic implantation.

Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; metabolism ; Female ; Gene Expression Regulation ; drug effects ; Horses ; Humans ; Integrin alphaVbeta3 ; genetics ; metabolism ; Mice ; Ovulation Induction ; Pregnancy ; RNA, Messenger ; genetics ; metabolism

BACKGROUNDPeriostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway.

METHODSA human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.

RESULTSThe transfection efficiency of periostin/pGCsi to U2OS cells was about 70% - 80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F = 564.71, P < 0.001) and 58% (F = 341.51, P < 0.001), respectively. Meantime, the earlier apoptosis value increased by 417% (F = 28.69, P < 0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F = 47.00, P < 0.001), however, that of G0 - G1 phase cells increased by 12% (F = 14.50, P < 0.001). The capability of migration and invasion reduced by 41% (F = 17.79, P < 0.001) and 72% (F = 197.08, P < 0.001), respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-β and vascular endothelial growth factor decreased by 17% (F = 73.99, P < 0.001) and 47% (F = 30.25, P < 0.001), respectively. A tendency of lower focal adhesion kinase (FAK) was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21% (F = 16.81, P < 0.001).

CONCLUSIONSRNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.

Apoptosis ; Bone Neoplasms ; etiology ; pathology ; Cell Adhesion Molecules ; antagonists & inhibitors ; genetics ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alphaVbeta3 ; physiology ; Neoplasm Invasiveness ; Osteosarcoma ; etiology ; pathology ; Phosphorylation ; RNA Interference ; Transfection

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF), integrin alphavbeta3, and tissue factor (TF) in choroidal neovascularization (CNV). METHODS: CNV was induced in 25 Brown Norway (BN) rats by diode laser with 532 nm wave length. In every BN rat, one eye was induced to produce CNV, and the other eye served as the normal control eye. Fundus photography and fundus fluorescein angiography (FFA) were performed just before euthanasia on 3, 7, 14, 21, and 28 d after laser photocoagulation. The retina was processed for histopathology and immunohistochemical analysis to detect the expressions of VEGF, integrin alphavbeta3, and TF. RESULTS: There was no CNV, no expression of intergrin alphavbeta3 and TF in the normal control eyes. Only a few VEGFs were expressed in the ganglion cell layer of the retina, inner nuclear layer, retinal pigment epithelium, and vascular endothelial cell of the retina and choroid in normal eyes. FFA revealed disc-like leakage of fluorescein 7 days after the photo-coagulation, meaning there was CNV. VEGF, intergrin alphavbeta3, and TF were all expressed in the ganglion cell layer of the retina, inner nuclear layer, retinal pigment epithelium, and vascular endothelial cell of the retina and choroids 3 days after the photo-coagulation. With the development of CNV, expressions of integrin alphavbeta3, VEGF, and TF were gradually increasing (P<0.01). The expression of integrin alphavbeta3 in the retina was at peak on 7th day, VEGF on 14th day, and TF on 21st day. CONCLUSION Expressions of VEGF, integrin alphavbeta3, and TF in CNV were found at the early, middle and late stage of CNV formation. It is important to determine the time of anti-neovascularization.
Animals ; Choroidal Neovascularization ; genetics ; metabolism ; Integrin alphaVbeta3 ; genetics ; metabolism ; Male ; Rats ; Rats, Inbred BN ; Thromboplastin ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

In mankind, the circulation system is a closed pressure-loaded system; the pressure in circulation flow field would change with the variation of natural or pathological geometry of the local bloodvessel, and the pressure shift induced by the variation of vascular geometry would lead to a series of physiological and pathological changes in the endothelial cells (ECs). This experiment is designed to elucidate the effects of different pressure shift on F-actin alignment and expression in cultured endothelial cells in vitro, and to investigate the relationship between the altered pressure shift and the expression intensity of Vascular adhesion molecule (VCAM) and Integrin alphaVbeta3. Non-activated cultured ECs and single shear stress loaded ECs as control group were set, the double-immuno-fluoro-cytochemistry, laser confocal scanning microscopy and image analysis system were used to observe the expression of VCAM, Integrin alphaVbeta3 and F-actin in endothelial cells which were exposed to levels of pressure shift in an improved parallel plate flow chamber. When exposed to different decreased pressure shift, the expression intensity of VCAM, Integrin alphaVbeta3 and F-actin showed regular changes. The decreased pressure shift resulted in changes in cell alignment and cytoskeleton F-actin, and also affected ECs adhesion function and transmembrane mechanotransduction function which were represented by VCAM and Integrin alphaVbeta3 respectively.
Actins ; genetics ; metabolism ; Cell Adhesion ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Hemodynamics ; Humans ; Integrin alphaVbeta3 ; genetics ; metabolism ; Pressure ; Umbilical Veins ; cytology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo study the anti-invasion effect of SEPT7 gene on U251MG glioma cells and its possible molecular mechanism.

METHODSRecombinant adenovirus vector carrying SEPT7 gene (rAd5-SEPT7) was transduced to human glioma cell line U251MG, and empty adenovirus vector was used as control. Tumor invasion was examined by Transwell method and 3 D-Matrigel assay, and tumor cell migration by wound-healing method and 2 D-Matrigel assay. Three major molecular events associated with cell motility and migration, including changes of expression in MMP2, MMP9, MT1-MMP, TIMP1 and TIMP2, the alteration of integrin alpha(v)beta(3) expression, and the structural change of cytoskeleton protein, tubulin-alpha, in U251 cells transduced with rAd5-SEPT7 were studied by Western blotting, immunofluorescence and laser scanning confocal microscope, respectively.

RESULTSThe invasive and migratory capabilities of cells transduced with rAd5-SEPT7 were inhibited. The expression of extracellular matrix metalloproteinases MMP-2, MMP-9, MT1-MMP and integrin alpha(v)beta(3) was significantly decreased, while the expression of matrix metalloproteinase inhibitor TIMP1, TIMP2 was upregulated. Intracellular cytoskeleton protein-tubulin-alpha in U251 cells exhibited prominent morphological changes which including the appearance of distortion and aggregation resulting from redistribution of tubulin-alpha, and this feature of alteration was similar to the tubulin-alpha structure in normal non-tumor cells.

CONCLUSIONSEPT7 gene can inhibit the invasion and migration ability of U251 glioma cells. Its molecular mechanism may include that SEPT7 gene reverses the imbalanced state of MMPs/TIMPs, downregulates the expression of integrin alpha(v)beta(3) and alters the structure of tubulin-alpha of U251MG glioma cells. It is suggested that SEPT7 gene could be a good candidate for gene therapy of gliomas.

Adenoviridae ; genetics ; Blotting, Western ; Cell Cycle Proteins ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; Genetic Vectors ; genetics ; Glioma ; metabolism ; pathology ; Humans ; Integrin alphaVbeta3 ; metabolism ; Matrix Metalloproteinase 14 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Microscopy, Confocal ; Neoplasm Invasiveness ; genetics ; Septins ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVETo investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro.

METHODSThe expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture.

RESULTSThe expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5.

CONCLUSIONalphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.

Antibodies ; immunology ; Cell Adhesion ; Cell Hypoxia ; Cell Line, Tumor ; Endothelial Cells ; cytology ; metabolism ; Humans ; Integrin alphaVbeta3 ; genetics ; immunology ; metabolism ; Intercellular Adhesion Molecule-1 ; immunology ; metabolism ; Ligands ; Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, Vitronectin ; genetics ; immunology ; metabolism

OBJECTIVETo analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.

METHODSEndothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.

CONCLUSIONTumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.

Antigens, CD34 ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cell Shape ; Cells, Cultured ; Endothelial Cells ; metabolism ; pathology ; ultrastructure ; Gene Expression ; Humans ; Integrin alphaVbeta3 ; metabolism ; Integrins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipoproteins, LDL ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Lung ; blood supply ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Microscopy, Electron, Scanning ; Neovascularization, Pathologic ; metabolism ; pathology ; Phenotype ; Plasminogen Activator Inhibitor 1 ; metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Receptors, Vitronectin ; metabolism ; Tissue Plasminogen Activator ; metabolism ; Tumor Cells, Cultured ; Tumor Necrosis Factor Decoy Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; von Willebrand Factor ; metabolism

To develop a new fusion protein (RGD)3/tTF for the therapy of the selective thrombosis of tumor blood vessels. The fused gene (RGD) 3/tTF was reconstructed by PCR, was cloned into vector pET22 b(+), and expressed in E. coli BL21 (DE3). The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was detected by clotting assay and F X activation assay. The specific binding of (RGD) 3/tTF to alphavbeta3 was analyzed by indirect ELISA. The recombinant plasmid pET22 b(+)/(RGD)3/tTF was obtained and expressed in E. coli BL21 (DE3). The purified fusion protein could induce blood coagulation, activiate F X. The ability of (RGD) 3/tTF binding specifically to alphavbeta3 was increased by 32%, compared with RGD/tTF. A new fusion protein (RGD) 3/tTF was successfully expressed in E. coli BL21 (DE3). The expressed proteins retained tTF activity and showed a higher binding to alphavbeta3 than that of RGD/tTF.
Animals ; Blood Coagulation ; drug effects ; Chromatography, Affinity ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Factor Xa ; metabolism ; Gene Expression ; Humans ; Integrin alphaVbeta3 ; metabolism ; Mice ; Oligopeptides ; genetics ; metabolism ; Polymerase Chain Reaction ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Thromboplastin ; genetics ; metabolism

OBJECTIVETo investigate the inhibitory effect of recombinant polypeptide CH50 of fibronectin on invasion and angiogenesis of tumors, and analyze the possible molecular mechanism of the therapeutic effect of polypeptide CH50 on tumors.

METHODSThe tumor model was established by inoculation of H22 hepatocarcinoma cells in mice. The tumor gene therapy was performed by in vivo gene transfection with a method based on hydrodynamics to express polypeptide CH50. After treatment, the inhibitory effect on tumor invasion and angiogenesis was observed by histotology with HE staining of tumor tissues. The expresison of MMP-9 mRNA and protein at the edge of tumor tissue was evaluated by RT-PCR and gelatin zymography, respectively. RT-PCR was used to detect the expression of the related genes in H22 cells treated with polypeptide CH50. Cell adhesion assay was used to analyze the influence of polypeptide CH50 on the binding of cells to fibrinogen.

RESULTS(1) Eukaryotic expression plasmid pCH510 was expressed in vivo in a non-targeting manner and produced a significant inhibitory effect on tumor growth. The therapy with polypeptide CH50 resulted in pronounced necrosis of tumor cells in pCH510 group, compared with that in control groups at histological level. (2) Polypeptide CH50 could inhibit the growth, invasion and angiogenesis of the tumor, and interfere the formation of new collateral circulation in the tumor. (3) The expression level of MMP-9 protein at the edge of tumor tissue was significantly decreased after treatment, especially the activation of pro-MMP-9 was inhibited significantly, whereas the expression level of MMP-9 mRNA was not influenced. (4) The expression of alphav, 33 and cdc2 mRNAs in H22 cells treated with polypeptide CH50 was down-regulated. (5) Cell adhesion assay manifested that polypeptide CH50 can affect the adhesion ability of H22 cells.

CONCLUSIONPolypeptide CH50 can inhibit tumor growth and angiogenesis by suppressing the functions of MMP-9 and integrin alphavbeta3.

Animals ; CDC2 Protein Kinase ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; metabolism ; pathology ; therapy ; Cell Adhesion ; genetics ; physiology ; Cell Line, Tumor ; Fibronectins ; biosynthesis ; genetics ; physiology ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; methods ; Humans ; Integrin alphaVbeta3 ; biosynthesis ; genetics ; Liver Neoplasms, Experimental ; metabolism ; pathology ; therapy ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Neovascularization, Pathologic ; genetics ; metabolism ; therapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction