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 investigate the influence of macrophages on the expression of the vascular endothelial growth factor (VEGF) receptor (KDR) mRNA, homeobox B2 (HOXB2) mRNA, and integrin alpha nu beta3 in vitro in vascular endothelial strain.

METHODSHuman umbilical vein cells (ECV304) were cultured in vitro and divided into 4 groups, i.e. (1) ECV304 group, (2) ECV304 + conA group [with conA (25 microg/ml in culture) added to ECV304], (3) ECV304 + U937 group (with 1 x 10(5)/ml of U937 cells added to 1 x 10(5)/ml ECV 304), (4) ECV304 + U937 + conA group [with 1 x 10(5)/ml of U937 cells and conA (25 microg/ml in culture)] groups. Forty-eight hours after culturing, the expression of integrin receptor alpha nu beta3 and the changes in the expression of KDR mRNA and HOXB2 mRNA in each group were determined by immunofluorescent technique and RT-PCR, respectively.

RESULTSThe expression of integrin receptor alpha nu beta3, KDR mRNA, and HOXB2 mRNA in ECV304 group were 6.7 +/- 1.5, 0.633 +/- 0.012, and 0.674 +/- 0.004, respectively, while those in ECV304 + U937 + conA group (10.2 +/- 1.7, 0.879 +/- 0.003, 0.947 +/- 0.003) were obviously more upregulated when compared with those in ECV304 group (P < 0.01). No difference in the above indices was found between ECV304 and ECV304 + conA, ECV304 + U937 groups (P > 0.05).

CONCLUSIONMacrophages activated by ConA can accelerate the proliferation, migration and adhesion to the basement membrane matrix of vascular endothelial cells through the influence on the expression of KDR mRNA, HOXB2 mRNA and integrin alpha nu beta3, and through this pathway the angiogenesis is modulated.

Cells, Cultured ; Endothelium ; cytology ; Gene Expression ; Homeodomain Proteins ; genetics ; Humans ; Integrin alphaVbeta3 ; genetics ; Macrophages ; metabolism ; RNA, Messenger ; genetics ; Receptors, Vascular Endothelial Growth Factor ; genetics ; Transcription Factors ; genetics

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

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

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 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

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the alpha v beta 3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of alpha v mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the alpha v beta 3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the alpha v beta 3 protein. No staining was evident in control aortas for the alpha v beta 3 antigen, tenascin or alpha v mRNA. The upregulation of av mRNA and the alpha v beta 3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.
Adult ; Aorta, Abdominal/immunology/pathology ; Aortic Aneurysm, Abdominal/*pathology ; Biological Markers/analysis/metabolism ; Female ; Humans ; Integrin alphaVbeta3/analysis/genetics/metabolism ; Male ; Neovascularization, Pathologic/genetics/*metabolism ; Nitric-Oxide Synthase/analysis/metabolism ; RNA, Messenger/analysis/metabolism ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, P.H.S. ; Tenascin/analysis/metabolism