OBJECTIVE: To investigate the expression and possible regulatory mechanism of miR-34a in the lung tissue of neonatal rat model of bronchopulmonary dysplasia (BPD) induced by hyperoxia. METHODS: In the study, 80 newborn SD rats were randomly divided into hyperoxia group (FiO₂=60%) and air group (FiO₂=21%) within 2 hours after birth, 40 rats per group. Lung tissue samples of the SD rats in each group were extracted on the 1st, 7th, 14th and 21st days after birth, and the pathological changes of lung tissue were observed under light microscope after HE staining. The number of radial alveolar counts (RAC) and the mean alveolar diameter (MAD) and the thickness of alveolar septal thickness (AST) were measured to evaluate the development of alveoli. Real-time fluorescence quantitative PCR was used to detect the expression of miR-34a, angiopoietin-1 (Ang-1) and tyrosine kinase receptor-2 (Tie-2) in lung tissue of rats in hyperoxia group and air group at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect the proteins expression of Ang-1 and Tie-2 in the lung tissues of the two groups at different time points. RESULTS: The weight of rats in the hyperoxia group on the 7th, 14th and 21st days after birth was significantly lower than that in the air group (P all < 0.05). With the prolongation of oxygen exposure, the number of alveoli decreased, the volume increased, the structure simplified, the alveolar cavity enlarged obviously and the alveolar septum thickened in the hyperoxia group. On the 7th, 14th and 21st days after birth, the RAC in the hyperoxia group was significantly lower than that in the air group (P all < 0.05). Compared with the air group, MAD and AST increased significantly on the 7th, 14th and 21st days after birth in the hyperoxia group, and the difference was statistically significant (P all < 0.05). The expression level of miR-34a in lung tissue of hyperoxia group was significantly higher than that of air group on the 7th, 14th and 21st days after birth, and the difference was statistically significant (P all < 0.05). Compared with the air group at the same time point, the expression levels of Ang-1 and Tie-2 mRNA and protein in the hyperoxia group were lower than those in the air group on the 14th and 21st days after birth (P all < 0.05). CONCLUSION The new BPD model of newborn SD rats can be successfully established by continuous exposure to 60% hyperoxia. The expression of miR-34a was up-regulated in the lung tissue of the new BPD model of neonatal rats. MiR-34a may play an important role in the occurrence and development of BPD by regulating Ang-1/Tie-2 signal pathway.
Animals ; MicroRNAs/metabolism* ; Bronchopulmonary Dysplasia/genetics* ; Hyperoxia/metabolism* ; Rats, Sprague-Dawley ; Animals, Newborn ; Rats ; Angiopoietin-1/genetics* ; Disease Models, Animal ; Receptor, TIE-2/genetics* ; Lung/pathology* ; Male

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-β-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Carbolines ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Neovascularization, Physiologic ; drug effects ; Picrasma ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Receptor, TIE-2 ; genetics ; metabolism ; Zebrafish ; embryology

The tyrosine kinase system angiopoietin (Ang)/Tie interacts with vascular endothelial growth factor pathway and regulates vessel quiescence in adults as well as later steps of the angiogenic cascade related to vessel maturation. Since all Angs are able to bind to Tie-2 but none binds to Tie-1, the function of Tie-2 and its ligands have captured attention. However, emerging evidence indicates unique roles of the orphan receptor Tie-1 in angiogenesis under physiological and pathological conditions. It is required for maintaining vascular endothelial cell integrity and survival during murine embryo development and in adult and may be involved in modulating differentiation of hematopoietic cells in adult. Tie-1 exhibits poor tyrosine kinase activity and signals via forming heterodimers with Tie-2, inhibiting Tie-2 signaling mediated by Angs. This inhibition can be relieved by Tie-1 ectodomain cleavage mediated by tumor- and inflammatory-related factors, which causes destabilization of vessels and initiates vessel remodeling. Up-regulated Tie-1 expression has been found not only in some leukemia cells and tumor related endothelial cells but also in cytoplasm of carcinoma cells of a variety of human solid tumors, which is associated with tumor progression. In addition, it has pro-inflammatory functions in endothelial cells and is involved in some inflammatory diseases associated with angiogenesis. Recent research indicated that Tie-1 gene ablation exhibited significant effects on tumor blood- and lymph-angiogenesis and improved anti-Ang therapy, suggesting Tie-1 may be a potential target for tumor anti-angiogenesis treatment.
Angiogenesis Inhibitors ; therapeutic use ; Angiopoietins ; genetics ; metabolism ; Animals ; Embryo, Mammalian ; Embryonic Development ; genetics ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Neovascularization, Pathologic ; drug therapy ; genetics ; metabolism ; pathology ; Protein Binding ; Receptor, TIE-1 ; antagonists & inhibitors ; genetics ; metabolism ; Receptor, TIE-2 ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo observe the effect of Taohong Siwu decoction (THSWD) on micro-vascular density (MVD) in rat uterus, the content of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in serum, and the expression of tyrosine kinasa receptor (Tie-2) in uterus.

METHODEarly pregnancy rats were intragastrically administrated with misoprostol (100 microg x kg(-1)) and mifepristong (8.3 mg x kg(-1)) to established the incomplete-abortion model. The incomplete-abortion rats were randomly divided into the model group (the same volume of distilled water), the positive control group (at the daily dose of 4.3 g x kg(-1) Motherwort Particles), and THSWD-treated groups (at the daily dose of 18.0, 9.0 and 4.5 g x kg(-1)). Pregnant rats were taken as the control group (the same volume of distilled water). After the successive oral administration for 7 days, blood was collected from aorta abdominalis, and rat uterine tissues were collected. The content of serum Ang-1 and Ang-2 were detected by ELISA; And the levels of Tie-2 and MVD in uterine tissues were detected by SP immunohistochemistry.

RESULTTHSWD remarkably increased the levels of MVD in uterus of medicine-induced abortion rats, the content of Ang-1 and Ang-2 in serum, and the expression of Tie-2 in uterine tissues.

CONCLUSIONTHSWD has the effect in markedly promoting angiogenesis in incomplete-abortion rats. Its mechanism may be related to the regulation of concentrations of Ang-1 and Ang-2 in serum and Tie-2 in uterine tissues.

Abortion, Incomplete ; blood ; drug therapy ; genetics ; Angiopoietin-1 ; blood ; genetics ; Angiopoietin-2 ; blood ; genetics ; Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression ; drug effects ; Humans ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-2 ; genetics ; metabolism ; Uterus ; blood supply ; drug effects ; metabolism

Psoriasis is a chronic inflammatory disease related to genome-wide and surroundings, it is important to develop a suitable animal model to research psoriasis pathogenesis and evolve pharmacotherapeutics. With the development of transgenetic technology in the past few years, psoriasis virulence gene animal model become a hotspot. Research of animal model of human psoriasis genes is reviewed in the paper.
Aminoquinolines ; toxicity ; Amphiregulin ; Animals ; Disease Models, Animal ; EGF Family of Proteins ; genetics ; metabolism ; Humans ; Keratin-14 ; genetics ; metabolism ; Keratin-5 ; genetics ; metabolism ; Keratinocytes ; metabolism ; Membrane Glycoproteins ; agonists ; Mice, Transgenic ; Psoriasis ; etiology ; genetics ; metabolism ; Receptor, TIE-2 ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Toll-Like Receptor 7 ; agonists ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells.

METHODSRecombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency.

RESULTSThe recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi.

CONCLUSIONSLentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Melanoma ; genetics ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, TIE-2 ; genetics ; Transfection

OBJECTIVETo evaluate the endothelial cell damage by detecting the circulating Tie2 mRNA level in a rat model of sepsis.

METHODSThe model of sepsis was established by cecal ligation and puncture (CLP) in 90 rats which were divided into 6 groups: normal, sham, CLP-3 h, CLP-6 h, CLP-12 h and CLP-24 h. Serum biochemical markers were detected by automatic biochemical analyzer. Serum IL-6 was measured with enzyme linked immunosorbent assay. The vascular permeability of liver, kidney, lung and heart was detected with Evans blue. Circulating endothelial cells (CEC) were separated using density gradient separation and counted. Total RNA of whole blood were extracted and the mRNA levels of two endothelial specific genes, Tie2 and vascular endothelial growth factor receptor 2 (VEGFR2), were measured by quantitative real-time PCR.

RESULTSThe level of serum biochemical indexes increased after CLP. The amount of serum IL-6 in CLP-6 h, 12 h, and 24 h group was increased 6.5-fold (P <0.05), 8.4-fold (P < 0.01), and 13.3-fold (P < 0.001) compared with normal group (170.68 pg/ml ± 42.46 pg/ml) respectively (F = 14.319, P < 0.001). Significantly increased organ vasopermeability of liver, kidney, lung and heart was observed after CLP respectively. The number of CEC peaked (11.83 ± 1.94) 3 hours after CLP compared with normal control (5.33 ± 1.21, P < 0.05), and then decreased gradually (F = 54.183, P < 0.001). The mRNA level of Tie2 in CLP-3 h group (3.47 ± 1.47) was also markedly higher than that in other groups (F = 10.640, P < 0.001).

CONCLUSIONUsing quantitative real-time PCR to measure the level of Tie2 mRNA in peripheral blood is a simple and relatively sensitive method to evaluate the damage of endothelial cells.

Animals ; Biomarkers ; blood ; Capillary Permeability ; Endothelial Cells ; pathology ; Interleukin-6 ; blood ; Male ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-2 ; genetics ; Sepsis ; pathology

OBJECTIVETo investigate the mechanisms of Buyang Huanwu Decoction (BYHWD) by observing its effects on expressions of angiopoietin-1 (Ang-1) and the endothelial-specific receptor tyrosine kinase (Tie-2) mRNA in damaged region of rats' brain after intracerebral hemorrhage (ICH).

METHODSOne hundred and sixty Sprague-Dawley rats were randomly divided into four groups, 10 in the normal control group, 60 in the sham-operative group, 60 in the ICH model group, and 30 in the BYHWD-treated group. The ICH model was established by injecting collagenase type VII 0.5 U stereotaxically into right globus pallidus. Animals in the BYHWD-treated group were administered orally with BYHWD, while animals in the sham-operative group and the ICH model group were administered orally with equal volume distilled water, and those in the normal control group drank water freely. The positional variations of the expression of Ang-1 and Tie-2 in the sham-operative group and the model group were assayed by immunohistochemistry on dayl, 4, 7, 14, 21 and 28 after modeling, in the meantime, the dynamic changes of Ang-1 and Tie-2 mRNA expressions in all groups were assayed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSNo significant expression of Ang-1 and Tie-2 in brain of rats in the normal or the sham-operative group was found during the experiment. In the model group, the Ang-1 and Tie-2 positive micrangio-segments appeared at the edge of clot on day 1 to day 4, they gradually penetrated to hematoma area from day 7; with Ang-1 and Tie-2 mRNA expressed from day 1, but very weak until day 4, showing no significant difference to that on day 1; thereafter, they increased gradually, and reached the peak on day 28 (P <0.05). While the two expressions in the BYHWD treated group reached the peak on day 21, and from day 7 to day 28, they were all significantly higher than those in ICH model group at the corresponding time points (P <0.01).

CONCLUSIONBYHWD can promote the up-regulation of Ang-1 and Tie-2mRNA expressions in brain of intracerebral hemorrhagic rats, which might accelerate the angiogenesis in the reconstruction of microvascular network in the damaged zone, and thus facilitating the repairing of damaged tissue.

Angiopoietin-1 ; genetics ; metabolism ; Animals ; Brain ; drug effects ; metabolism ; Cerebral Hemorrhage ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gene Expression ; drug effects ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, TIE-2 ; genetics ; metabolism

OBJECTIVETo explore the effects of angiopoietins (Ang-1 and Ang-2) and Tie-2 expression on microvessel density (MVD) in gastric cancers.

METHODSBy using semiquantitative RT-PCR, immunohistochemistry and image analysis system, the expression of Ang-1, Ang-2, Tie-2 mRNA and their proteins were detected in 68 primary gastric cancers and their adjacent normal tissues. Microvessel density (MVD) was figured out based on CD34 immunohistochemical staining.

RESULTSThe expression of all Ang-1, Ang -2, Tie-2 mRNA and their proteins was detected in gastric cancers and their paired adjacent gastric mucosa tissues. A negative correlation between Ang-1 protein, Tie-2 mRNA and MVD in gastric cancers was observed (r = -0.440, r = -0.267; P < 0.05), while the relation between Ang-2 mRNA and its protein, Ang-2/Ang-1 protein ratio with MVD were positive (r = 0.319, r = 0.729, r = 0.739; P < 0.05). It was found that MVD in groups with Ang-2 mRNA T/N ratio over 1.2 (the ratio of Ang-2 mRNA in gastric cancers and its adjacent normal mucosa) was higher than that in those with a ratio under 1.2, revealed by analysing the effects of Ang-1 and Ang-2 mRNA T/N ratio on MVD in gastric cancers.

CONCLUSIONAng-1 activates Tie-2 receptor, whereas Ang-2 antagonizes Ang-1 in the angiogenesis, and the Ang-2/Ang-1 ratio determines angiogenesis and tumor growth in gastric cancers. When the expression of Ang-2 is high and Ang-1 is low, the angiogenesis in gastric cancers is promoted, otherwise oppositely. The role of Ang-2 is dominant in the effect of Angs and their receptor on angiogenesis in gastric cancers.

Angiopoietin-1 ; biosynthesis ; genetics ; Angiopoietin-2 ; biosynthesis ; genetics ; Female ; Humans ; Male ; Microcirculation ; pathology ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, TIE-2 ; biosynthesis ; genetics ; Stomach Neoplasms ; blood supply ; metabolism ; pathology

Angiogenesis, formation of new microvessels providing oxygen and nutrient supply, is essential for tumor growth. It is dependent on the production of angiogenic growth factors by tumor cells. Angiopoietin 1 (Ang-1) and 2 (Ang-2) and their common receptor, Tie2, are thought to be critical regulators of tumor angiogenesis. We examined expression of Ang-1, Ang-2, and their common receptor Tie2 mRNAs and proteins in gastric cancers using in situ hybridization and immunohistochemistry. We also investigated the relationship between their expression and differentiation of cancer cells, lymph node metastasis, tumor size, depth of cancer cell invasion, TNM staging and microvessel density (MVD). The expression of Ang-1, Ang-2, and Tie2 mRNA in cancer cells significantly correlated with the MVD (p<0.001, <0.001 and =0.019, respectively). Ang-1 and Tie2 positivity correlated with advanced gastric cancers (p<0.05) and larger cancers had higher positive rates of Ang-1, Ang-2, and Tie2 mRNA expression (p<0.001, =0.010 and =0.039, respectively). Significant positive correlations were also found between mRNA expression of Tie2 and those of Ang-1 and Ang-2 (p<0.01 and <0.001, respectively). These findings indicate that the expression of Ang-1 and Ang-2 is important for tumor angiogenesis, and suggest a possible role of autocrine/paracrine function of angiopoietin/Tie2 system in gastric cancer progression.
Stomach Neoplasms/blood supply/*genetics/*metabolism/pathology ; Receptor, TIE-2/*genetics/*metabolism ; RNA, Neoplasm/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Neovascularization, Pathologic ; Middle Aged ; Male ; In Situ Hybridization ; Immunohistochemistry ; Humans ; Gene Expression ; Female ; Carcinoma, Signet Ring Cell/blood supply/genetics/metabolism/pathology ; Angiopoietin-2/*genetics/*metabolism ; Angiopoietin-1/*genetics/*metabolism ; Aged ; Adult ; Adenocarcinoma/blood supply/genetics/metabolism/pathology