To study the effect of VEGF fully phosporothioated antisense oligodeoxynucleotide (VEGF-ASODN) on VEGF expression in acute monocyte leukemic cell line U937 in vitro, U937 cells were incubated with VEGF-ASODN at concentrations of 10, 20 and 30 micro mol/L or scrambled sequence as compared with negative control. The expression of VEGF mRNA was measured by semi-quantitative RT-PCR. The expression of VEGF protein was measured by Western blot. The result showed that VEGF-ASODN had obviously inhibitive effect on expression of VEGF in U937 cell, as compared with scrambled sequence and negative control (P < 0.05). Scrambled sequence group had no significant difference compared with negative control group (P > 0.05). It is concluded that the expressions of VEGF mRNA and protein in leukemic cell line U937 are down-regulated by VEGF-ASODN.
Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; U937 Cells ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; genetics

OBJECTIVE: Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. METHODS: The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. RESULTS: After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4+/-8.6 microg/mL (n=3), 9.2+/-2.1 microg/mL (n=3), and 174.8+/-31.1 microg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. CONCLUSION: These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.
Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Ovarian Neoplasms/*therapy ; *Peptide Library ; Sequence Analysis, DNA ; Signal Transduction/physiology ; Vascular Endothelial Growth Factor A/metabolism ; Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors/*metabolism

OBJECTIVETo determine the effects of PCMV-FGEV transfection on the profile of SMMC-7721 hepatocellular in vitro in vivo.

METHODSSMMC-7721 hepatocellular was transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the SMMC-7721 hepatocellular were determined by in situ hybridization and immunochemical analysis. The effect of PCMV-FGEV transfection on SMMC-7721 hepatocellular proliferation was observed by MTT colorimetric assay. Flow cytometry was used to determine the effects of PCMV-FGEV transfection on cell apoptosis of SMMC-7721. The growth of transfected cells was also observed in nude mice.

RESULTSThere was reduced VEGF expression in SMMC-7721 transfected with PCMV-FGEV confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of PCMV-FGEV transfection on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cell with PCMV-FGEV transfected was slow in nude mice (vivo) and accompanied with obvious apoptosis. The latent time of tumor in the antisense mice group was 25.0+/-1.8 days, which was longer than that in sense and control group significantly (F=19.455, P<0.01). On the other hand, the average tumor weight in antisense group (0.96 g+/-0.28 g) was the smallest among the three groups (F=21.501, P<0.01).

CONCLUSIONSThe expression of VEGF was inhibited by PCMV-FGEV. There was no effect on cell proliferation and cell apoptosis of SMMC-7721 by transferring PCMV-FGEV gene into SMMC-7721 cells in vitro. But in vivo it can inhibit tumor growth and induce cell apoptosis.

Apoptosis ; Cell Division ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Liver Neoplasms ; pathology ; therapy ; RNA, Antisense ; therapeutic use ; Transfection ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; genetics

OBJECTIVETo explore the effects of vascular endothelial growth factor (VEGF) antisense phosphorothioated oligodeoxynucleotide (AS-ODN) on the expression of VEGF in human leukemic cell lines (HL-60 and K562 cells).

METHODSThe levels of VEGF mRNA and protein in leukemic cells incubated with VEGF AS-ODN were measured by RT-PCR, immunohistochemistry assay and ELISA. MTT test was used to examine the influence of the culture supernatant (CS) of VEGF AS-ODN treated leukemic cells on the proliferation of human umbilical vein endothelial cells (ECV304).

RESULTSAfter leukemic cells were treated with different concentrations (2.5 approximately 15.0 micro mol/L) of VEGF AS-ODN for 24 h, VEGF mRNA level in the cells decreased remarkably in a concentration dependent manner, no change was found in the VEGF missense ODN treated cells (MS-ODN). When the leukemic cells were treated with 5 micro mol/L VEGF AS-ODN for 24 h, VEGF protein level decreased greatly both in the cells and in the CS; and the proliferation stimulating effect of the treated CS on the ECV304 cells reduced. Meanwhile, there was no obvious change in VEGF protein and its effect in the VEGF MS-ODN treated group.

CONCLUSIONVEGF AS-ODN could inhibit VEGF expression in human leukemic cell lines in vitro.

Enzyme-Linked Immunosorbent Assay ; HL-60 Cells ; Humans ; Immunohistochemistry ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

OBJECTIVETo study the inhibition effect of HIF-1α specific siRNA expression vector pSUPERH1-siHIF-1α on retinal neovascularization in a mouse model of retinopathy of prematurity (ROP).

METHODSThe mouse model of ROP was prepared by the method Smith described. Forty-eight ROP mice were randomly divided into two groups: an experimental group that was intravitreously injected with pSUPERH1-siHIF-1α and a control group that was injected with pSUPER retro vector. The levels of HIF-1α and vascular endothelia growth factor (VEGF) in the retina were examined by Western blot. The retinal neovascularization was evaluated by angiography using FITC Dextran and quantitated histologically.

RESULTSThe levels of HIF-1α and VEGF in the retina in the experimental group were reduced 90% and 65% respectively compared with those in the control group. Meanwhile, the number of retinal neovascular endothelial nucleus outbreaking the inner limit membrane in the experimental group was significantly reduced compared with that in the control group.

CONCLUSIONSThe development of retinal neovascularization of ROP can be markedly inhibited by RNA interference targeting HIF-1α.

Animals ; Disease Models, Animal ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; antagonists & inhibitors ; genetics ; Infant, Newborn ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; prevention & control ; Retinopathy of Prematurity ; therapy ; Vascular Endothelial Growth Factor A ; analysis

BACKGROUNDThe peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor beta1 (TGF-beta1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF), a downstream mediator of TGF-beta1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells.

METHODSRetrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-beta1 5 ng/ml, low glucose DMEM + TGF-beta1 5 ng/ml + PRS-CTGF-siRNA(1-4) and low glucose DMEM + TGF-beta1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot.

RESULTSLow levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-beta1, the levels of CTGF and VEGF were significantly upregulated (P < 0.01). Introduction of PRS-CTGF-siRNA(1-4) resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P < 0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P > 0.05).

CONCLUSIONSThe expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-beta1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.

Animals ; Base Sequence ; Cells, Cultured ; Connective Tissue Growth Factor ; Epithelial Cells ; metabolism ; Humans ; Immediate-Early Proteins ; analysis ; antagonists & inhibitors ; genetics ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Peritoneum ; cytology ; metabolism ; RNA, Messenger ; analysis ; RNA, Small Interfering ; pharmacology ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1 ; pharmacology ; Vascular Endothelial Growth Factor A ; analysis ; genetics

OBJECTIVETo study the expression of signal transducer and activator of transcription 3 (Stat3), hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in Wilms' tumor and their roles in the development of Wilms' tumor.

METHODSThe expression of Stat3, HIF-1alpha and VEGF were detected by the immunohistochemical staining in 52 specimens from Wilms' tumor tissues, 47 from adjacent kidney tissues and 8 from normal kidney tissues. The expression intensity was analyzed by computer image processing.

RESULTSThe expression of Stat3, HIF-1 and VEGF were significantly up-regulated in Wilms' tumor tissues compared to those in adjacent tissues and normal kidney tissues (P < 0.05). Stat3 and VEGF proteins in Wilms' tumor tissues of stage III-IV and high risk histopathology were significantly higher than those of stage I-II and low risk histopathology. The higher expression of HIF-1 in Wilms' tumor tissues was shown in tumors with high risk histopathology and tumor size > or = 6 cm.

CONCLUSIONSIncreased expression of Stat3, HIF-1 and VEGF were found in Wilms' tumor tissues, and may be related to the development and angiogenesis of Wilms' tumor. Stat3 may regulate the expression of HIF-1 and VEGF, so it could be an effective target for inhibiting VEGF expression and angiogenesis of Wilms' tumor.

Child ; Child, Preschool ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Immunohistochemistry ; Infant ; Kidney Neoplasms ; blood supply ; chemistry ; pathology ; Male ; Neoplasm Staging ; Neovascularization, Pathologic ; etiology ; STAT3 Transcription Factor ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; Wilms Tumor ; blood supply ; chemistry ; pathology

Oxidative stress plays critical roles in airway inflammation that is usually accompanied by increased vascular permeability and plasma exudation. VEGF increases vascular permeability and leads to airway inflammation. In addition, VEGF has been shown to enhance receptor activator of NF-kappaB (RANK) expression in endothelial cells. An aim of the study was to determine the potential role of antioxidant in the regulation of RANK expression in murine model of asthma. We have used a C57BL/6 mouse model of allergic asthma to evaluate the effect of L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, which acts as an antioxidant, and VEGF receptor inhibitor on RANK mRNA expression. The mice develop the following pathophysiological features of asthma in the lungs: increased expression of RANK mRNA, increased number of inflammatory cells of the airways, increased vascular permeability, and increased levels of VEGF. Administration of OTC and VEGF receptor inhibitor markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs. We also showed that the increased RANK mRNA expression at 72 h after ovalbumin inhalation were reduced by the administration of OTC or VEGF receptor inhibitor. The results indicate that OTC and VEGF receptor inhibitor which inhibit up-regulation of VEGF expression modulate RANK expression that may be in association with the regulation of vascular permeability, and suggest that VEGF may regulate the RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other airway inflammatory disorders.
Vascular Endothelial Growth Factor A/analysis/antagonists & inhibitors/metabolism ; Thiazolidines ; Thiazoles/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors ; Receptors, Tumor Necrosis Factor/genetics/*metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Reactive Oxygen Species/metabolism ; RNA, Messenger/genetics/metabolism ; Pyrrolidonecarboxylic Acid ; Proto-Oncogene Proteins c-akt/metabolism ; Protein Kinase Inhibitors/pharmacology ; Prodrugs/pharmacology ; Phosphorylation/drug effects ; Ovalbumin/immunology ; Osteoprotegerin ; Mice, Inbred C57BL ; Mice ; Immunohistochemistry ; Glycoproteins/genetics/*metabolism ; Gene Expression/drug effects ; Female ; Capillary Permeability/drug effects ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Blotting, Western ; Asthma/*drug therapy/immunology/metabolism ; Antioxidants/*pharmacology ; Animals

We report a case of cytomegalovirus (CMV) retinitis after intravitreal bevacizumab injection. A 61-year-old woman with diabetic macular edema developed dense vitritis and necrotizing retinitis 3 weeks after intravitreal bevacizumab injection. A diagnostic vitrectomy was performed. The undiluted vitreous sample acquired by vitrectomy was analyzed by polymerase chain reaction and culture. Polymerase chain reaction of the vitreous was positive for CMV DNA. Other laboratory results did not show evidence of other infectious retinitis and systemic immune dysfunction. Human immunodeficiency virus antibodies were also negative. After systemic administration of ganciclovir, retinitis has resolved and there has been no recurrence of retinitis during the follow-up period of 12 months. Ophthalmologists should be aware of potential risk for CMV retinitis after intravitreal bevacizumab injection.
Angiogenesis Inhibitors/administration & dosage/adverse effects ; Antibodies, Monoclonal, Humanized/administration & dosage/*adverse effects ; Cytomegalovirus/genetics ; Cytomegalovirus Retinitis/diagnosis/*etiology/immunology ; DNA, Viral/analysis ; Diagnosis, Differential ; Female ; Humans ; Immunocompetence/*drug effects ; Intravitreal Injections ; Macular Edema/diagnosis/*drug therapy ; Middle Aged ; Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A/antagonists & inhibitors

To evaluate the effect of imatinib mesylate (STI571) on primitive/committed malignant progenitor cells in chronic myelogenous leukemia (CML) and to further elucidate the mechanisms involved in CML relapse and in some CML cells resistant to STI571, bone marrow-derived malignant bcr/abl-positive, Flk1(+)CD31CD34(-) cells with hemangioblastic characteristics from CML patients were grown in Methocult GF+ media with or without STI571, and inhibitory effect of STI571 on proliferation of differentiated and differentiating, bcr/abl(+), Flk1(+)CD31CD34(-) cells with hemangioblastic characteristics was investigated in vitro. The results showed that in vitro exposure to 5 micromol/L STI571 (the concentration of STI571 usually achieved in patients is 1-2 micromol/L) for 96 hours inhibited bcr/abl(+) committed progenitors (colony-forming cells, CFCs). No evident suppression of normal primitive, bcr/abl(+), and Flk1(+)CD31(-)CD34(-) cells were observed. It is concluded that CML primitive stem cells remain viable in the presence of STI571 and that inhibition of bcr/abl tyrosine kinase by STI571 restores normal hematopoiesis by removing the proliferative advantage of CML committed progenitors but that elimination of all CML progenitors may not occur. So despite dramatic short-term responses in vivo, such in vitro resistance to STI571, may translate into disease relapse after prolonged therapy.
Antigens, CD34 ; analysis ; genetics ; Benzamides ; Bone Marrow Cells ; drug effects ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Fluorescent Antibody Technique ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Hematopoietic Stem Cells ; drug effects ; metabolism ; pathology ; Humans ; Imatinib Mesylate ; In Situ Hybridization, Fluorescence ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; genetics ; metabolism ; Piperazines ; pharmacology ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; genetics ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor Receptor-2 ; analysis ; genetics