To determine the effect of andrographolide (Andro) on angiogenesis of human umbilical vein endothelial cells (HUVECs).
 Methods: HUVECs were treated with different concentrations of Andro and the cell viability was detected with Cell Counting Kit-8 (CCK-8). HUVECs were treated with half lethal dose (IC50) of Andro. Matrigel was used to make capillary formation of HUVECs and the effect of Andro on capillary formation was evaluated by calculating the percentage of capillary formation. Moreover, the effects of Andro and the supernatant from cultured A549 tumor cells on capillary formation were evaluated by calculating the percentage of capillary formation. The effect of Andro on the expression of matrix metalloproteinase-9 (MMP-9) was determined with Western blot.
 Results: The cell viability of HUVECs decreased with the increase of Andro concentrations. IC50 was 20 μmol/L. The capillary formation of HUVECs was inhibited when treated with 20 μmol/L Andro for 24 hours. Moreover, Andro was able to antagonize the promotion of the capillary formation induced by the supernatant from cultured tumor cells. Andro could suppress the expression of MMP-9 and antagonize the capillary formation.
 Conclusion: Andro inhibits the capillary formation of HUVECs and can antagonize the promotion of angiogenesis induced by the supernatant from cultured tumor cells.
Capillaries ; drug effects ; Cell Survival ; Collagen ; Culture Media ; Diterpenes ; pharmacology ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; Matrix Metalloproteinase 9 ; metabolism ; Neovascularization, Pathologic ; enzymology ; etiology ; prevention & control ; Proteoglycans ; Tumor Cells, Cultured

BACKGROUNDAngiogenesis is the formation of new blood vessels to supply nutrients to tumors. Vascular endothelial growth factor (VEGF) and cluster of differentiation 34 (CD34) are important signaling proteins involved in angiogenesis. Many studies have demonstrated that VEGF and CD34 are related to tumor progression. This study focused on the relationship between VEGF, CD34, and perioperative hemorrhage in patients with gastric cancer.

METHODSTo observe the relationship between VEGF and CD34, we tracked 112 patients with advanced gastric cancer for 5 years to assess factors related to hemorrhage, using immunohistochemistry. The results were subjected to statistical analysis using a 2 × 2 contingency table, logistic regression, and receiver operating characteristic (ROC) test.

RESULTSThe concentrations of VEGF and CD34 were critically correlated with perioperative hemorrhage and neural invasion in patients with gastric cancer (P < 0.05). Expression of VEGF and CD34 was related (P < 0.05, χ2 = 6.834). VEGF and CD34 co-expression strongly increased the risk of preoperative bleeding (area under the ROC curve >0.7, P < 0.05).

CONCLUSIONSExpression of VEGF and CD34 was critically correlated with perioperative hemorrhage in gastric cancer patients. Co-expression of VEGF and CD34 could be an effective indicator for evaluating the risk of perioperative bleeding in gastric cancer patients.

Adult ; Aged ; Aged, 80 and over ; Antigens, CD34 ; metabolism ; Female ; Gastrointestinal Hemorrhage ; etiology ; metabolism ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neovascularization, Pathologic ; complications ; metabolism ; Prognosis ; Retrospective Studies ; Risk Factors ; Stomach Neoplasms ; metabolism ; pathology ; surgery ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

OBJECTIVETo explore the effect of colon cancer cell-derived interleukin-1α on the migration and proliferation of human umbilical vein endothelial cells as well as the role of IL-1α and IL-1ra in the angiogenesis process.

METHODSWestern blot was used to detect the expression of IL-1α and IL-1R1 protein in the colon cancer cell lines with different liver metastatic potential. We also examined how IL-1α and IL-1ra influence the proliferation and migration of umbilical vascular endothelial cells assessed by PreMix WST-1 assay and migration assay, respectively. Double layer culture technique was used to detect the effect of IL-1α on the proliferation and migration of vascular endothelial cells and the effect of IL-1ra on the vascular endothelial cells.

RESULTSWestern blot analysis showed that IL-1α protein was only detected in highly metastatic colon cancer HT-29 and WiDr cells, but not in the lowly metastatic CaCo-2 and CoLo320 cells.Migration assay showed that there were significant differences in the number of penetrated cells between the control (17.9±3.6) and 1 ng/ml rIL-1α group (23.2±4.2), 10 ng/ml rIL-1α group (31.7±4.5), and 100 ng/ml rIL-1α group (38.6±4.9), showing that it was positively correlated with the increasing concentration of rIL-1α (P<0.01 for all). The proliferation assay showed that the absorbance values were 1.37±0.18 in the control group, and 1.79±0.14 in the 1 ng/ml rIL-1α group, 2.14±0.17 in the 10 ng/ml rIL-1α group, and 2.21±0.23 in the 100 ng/ml rIL-1α group, showing a positive correlation with the increasing concentration of rIL-1α(P<0.01 for all). IL-1ra significantly inhibited the proliferation and migration of vascular endothelial cells (P<0.01). The levels of VEGF protein were (1.697±0.072) ng/ml, (3.507±0.064)ng/ml and (4.139±0.039)ng/ml in the control, HUVECs+ IL-1α and HUVECs+ HT-29 co-culture system groups, respectively, showing a significant difference between the control and HUVECs+ 10 pg/ml rIL-1α groups and between the control and HUVECs+ HT-29 groups (P<0.01 for both).

CONCLUSIONSOur findings indicate that colon cancer cell-derived IL-1α plays an important role in the liver metastasis of colon cancer through increased VEGF level of the colon cancer cells and enhanced vascular endothelial cells proliferation, migration and angiogenesis, while IL-1ra can suppress the effect of IL-1α and inhibit the angiogenesis in colon cancer.

Blotting, Western ; Caco-2 Cells ; Cell Line, Tumor ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Coculture Techniques ; Colonic Neoplasms ; blood supply ; metabolism ; pathology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Interleukin 1 Receptor Antagonist Protein ; metabolism ; physiology ; Interleukin-1alpha ; metabolism ; physiology ; Liver Neoplasms ; secondary ; Neovascularization, Pathologic ; etiology

OBJECTIVETo observe the change of PTEN/PI3K/AKT pathway hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF) in rats with adjuvant arthritis and to explore the mechanism of neovasculization in rheumatoid arthritis.

METHODSThirty rats were randomly divided into normal control group and model control group. The model control group were established the model of adjuvant arthritis using Freund's complete adjuvant. At 19 days after modeling, the expression of microvascular density (MVD), HIF-1α, VEGF were detected by ELISA assay and PTEN, PI3K, AKT were detected by Werstern Blotting.

RESULTSCompared with the normal control group, paw swelling, arthritic index were increased, and the expression of MVD, VEGF, HIF-1α of serum, PI3K, AKT of synovial tissue were significantly increased, PTEN was significantly decreased in model control group. PI3K, HIF-1α were positively correlated with MVD; VEGF, AKT were positively correlated with paw swelling; PTEN was negatively correlated with the arthritis index; HIF-1α was positively correlated with VEGF; PI3K was positively correlated with AKT, PTEN was negatively correlated with PI3K, AKT, VEGF.

CONCLUSIONImbalance of PTEN/PI3K/AKT pathway in rats with adjuvant arthritis is one of the mechanisms of synovial neovasculization.

Animals ; Arthritis, Experimental ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; physiology ; Neovascularization, Pathologic ; etiology ; PTEN Phosphohydrolase ; physiology ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

OBJECTIVE: The aim of this study is to investigate the expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) in laryngeal carcinoma and its relations to clinical and pathophysiological characteristics, and determine the effect of HIF-1alpha, VEGF in the processing of angiogenesis. To offer the theoretical basis for the pathogenesis of laryngeal carcinoma and the new ideas for early diagnosis of laryngeal carcinoma. METHOD: The major was divided into two groups:60 specimens of laryngeal carcinoma and 20 specimens of normal tissues beside the carcinoma. The expression of HIF-1alpha, VEGF was detected in 60 specimens of laryngeal carcinoma tissues and 20 specimens of normal tissues beside the carcinoma as controls by immunohistochemical staining technique. Microvessel was stained by antifactor CD105 antibody to analyse microvessel density. RESULT: Positive rate of HIF-1alpha and VEGF protein expression was 71.67% (43/60) and 65.00% (39/60) respectively in laryngeal carcinoma tissues, which was significantly higher than that in normal laryngeal tissues (10.00% and 20.00%, respectively). HIF-1alpha expression was closely related to P-TNM stage, the grade of cell differentiation and lymph node status; VEGF expression was closely related to P-TNM stage and lymph node status. The coincidence rate of the expression of HIF-1alpha and VEGF was 56.67% (34/60), showing a close relation between them. MVD was much higher in tumor with HIF-1alpha(+)/VEGF(+) than that in tumor with HIF-1alpha(--)/VEGF(--) (P < 0.01). CONCLUSION HIF-1alpha protein and VEGF was over-expressed in laryngeal carcinoma. The positive expression of HIF-1alpha, VEGF and MVD in laryngeal carcinoma have a synergistic effect on the neovascularization of the tumor.
Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Laryngeal Neoplasms ; blood supply ; metabolism ; Larynx ; metabolism ; Male ; Microvessels ; Neovascularization, Pathologic ; etiology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar.

METHODSThe human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups.

RESULTS(1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05).

CONCLUSIONSThe ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.

Cell Movement ; Cell Proliferation ; Chromones ; pharmacology ; Cicatrix, Hypertrophic ; enzymology ; pathology ; Endothelial Cells ; cytology ; drug effects ; Humans ; Lipids ; pharmacology ; Morpholines ; pharmacology ; Neovascularization, Pathologic ; etiology ; pathology ; Protein-Serine-Threonine Kinases ; genetics ; physiology ; RNA, Messenger ; analysis ; RNA, Small Interfering ; metabolism

The feasibility of contrast-enhanced ultrasonography in the assessment of atherosclerotic plaque neovascularization and its relation to histological findings were investigated. Abdominal aortic atherosclerotic plaque model was induced in 25 New Zealand white rabbits by a combination of high cholesterol-rich diet and balloon aortic denudation. Standard and contrast-enhanced ultrasonography was performed at the 16th week of the model induction period. The plaques were classified as echogenic plaques or echolucent plaques according to their echogenicity at standard ultrasonography. The maximum thickness of plaque was measured in the longitudinal section. Time intensity curve was used to quantify the enhanced intensity of the plaque. Animals were euthanized and abdominal aortas were harvested for histological staining of CD31 to evaluate the neovascularization density of atherosclerotic plaque. The results showed that the echolucent plaques had higher enhanced intensity during contrastenhanced ultrasonography and higher neovascularization density at CD31 staining than the echogenic plaques. The enhanced intensity of atherosclerotic plaque and its ratio to lumen were well correlated with histological neovascularization density (r=0.75, P<0.001; r=0.68, P<0.001, respectively). However, the maximum thickness of plaque was not correlated with neovascularization density (r=0.235, P=0.081). These findings demonstrated that the enhanced intensity in the plaque and ratio of enhanced intensity to that in the lumen of abdominal aorta may be more accurate in the evaluation of plaque neovascularization than maximum thickness. Our study indicates that contrast-enhanced ultrasonography provides us a reliable method for the evaluation of plaque neovascularization.
Animals ; Aorta, Abdominal ; diagnostic imaging ; Image Enhancement ; methods ; Image Interpretation, Computer-Assisted ; methods ; Neovascularization, Pathologic ; diagnostic imaging ; etiology ; Phospholipids ; Plaque, Atherosclerotic ; complications ; diagnostic imaging ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity ; Statistics as Topic ; Sulfur Hexafluoride ; Ultrasonography ; methods

OBJECTIVETo investigate the expression of HBx and COX-2 in hepatitis B virus-related hepatocellular carcinoma, and Its correlation with microangiogenesis and metastasis, and possible mechanism.

METHODSImmunohistochemistry was used to detect the expression of hepatitis B virus X, cyclooxygenase-2 and CD34 in hepatitis B virus related hepatic carcinoma and 22 non-HBV related hepatic carcinoma tissues. The expression of hepatitis B virus x protein and cyclooxygenase-2 in hepatitis B virus-related hepatocellular carcinoma correlated with microangiogenesis and metastasis was tested by Spearman correlation analysis. The expression of COX-2 in HepG2-X was detected by Western blot and RT-PCR. PGE2 was detected by ELISA in clear supernatant liquid of HepG2-X. Trypan blue exclusion was performed to examine the inhibitory effects of COX-2 selective inhibitor (celecoxib).

RESULTSIn Hepatitis B carcinoma tissue, HBx and COX-2 were expressed at high level. The positive rate of COX-2 expression was 88.87% (55/62) in HBx positive expression group, which was significantly higher than that of the positive expression 31.82% (7/22, x2=27.188, P<0.01) in HBx negative expression group and 40.91% (9/22, x2=20.453, P<0.01) in non-HBV related hepatic carcinoma tissues, but it had no statistical difference (x2=0.393, P=0.531) between the HBx negative expression group and non-HBV related hepatic carcinoma tissue group. The expressions of HBx and COX-2 in metastasis group were higher than that in non-metastasis group (P<0.01), MVD in HBx or COX-2 positive expression group was significantly higher than that in negative expression group and non-HBV related hepatic carcinoma tissues (P is less than 0.01). MVD with metastasis was higher than that without metastasis (P<0.01) and MVD with portal vein invasion was higher than that without invasion (P<0.05). Spearman correlation analysis showed that the expression of COX-2 was significantly correlated with the expression of HBx (Rs=0.568, P<0.01). Celecoxib suppressed the growth of both cells in a dose-dependent manner. HepG2-X was significantly susceptible to celecoxib as compared to the HepG2-PC cells. COX-2 protein and mRNA were upregulated in HepG2-X cells than in HepG2-PC. Moreover, PGE2 was upregulated in clear supernatant liquid of HepG2-X than in HepG2-PC.

CONCLUSIONThe expressions of HBx and COX-2 were higher in HBV-related hepatocellular carcinoma. COX-2 was significantly correlated with HBx in HCC and it could be a key factor involved in HBx contributed hepatocellular carcinoma's microangiogenesis and metastasis. The possible mechanism of the dual effects might be through HBx, COX-2 and PGE2 pathways in infiltration involved metastasis and microangiogenesis involved metastasis.

Carcinoma, Hepatocellular ; blood supply ; etiology ; metabolism ; virology ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Hepatitis B ; complications ; Hepatitis B virus ; metabolism ; Humans ; Liver Neoplasms ; blood supply ; metabolism ; virology ; Neoplasm Metastasis ; Neovascularization, Pathologic ; Trans-Activators ; metabolism

BACKGROUND/AIMS: Angiogenesis, which is a critical step in the initiation and progression of rheumatoid arthritis (RA), involves pro-angiogenic factors, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). We investigated the role of Toll-like receptor 3 (TLR3) in the regulation of pro-angiogenic factors in RA fibroblast-like synoviocytes (FLS). METHODS: FLS were isolated from RA synovial tissues and stimulated with the TLR3 ligand, poly (I:C). The levels of VEGF and IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assays, and the mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction. The expression patterns of VEGF and IL-8 in the RA synovium and osteoarthritis (OA) synovium were compared using immunohistochemistry. RESULTS: The expression levels of TLR3, VEGF, and IL-8 were significantly higher in the RA synovium than in the OA synovium. VEGF and IL-8 production were increased in the culture supernatants of RA FLS stimulated with poly (I:C), and the genes for these proteins were up-regulated at the transcriptional level after poly (I:C) treatment. Treatment with inhibitors of nuclear factor-kappaB (NF-kappaB), i.e., pyrrolidine dithiocarbamate and parthenolide, abrogated the stimulatory effect of poly (I:C) on the production of VEGF and IL-8 in RA FLS. CONCLUSIONS: Our results suggest that the activation of TLR3 in RA FLS promotes the production of proangiogenic factors, in a process that is mediated by the NF-kappaB signaling pathway. Therefore, targeting the TLR3 pathway may be a promising approach to preventing pathologic angiogenesis in RA.
Arthritis, Rheumatoid/drug therapy/*etiology/metabolism ; Cells, Cultured ; Fibroblasts/metabolism ; Humans ; Interleukin-8/analysis/*biosynthesis/genetics ; NF-kappa B/physiology ; Neovascularization, Pathologic/etiology ; RNA, Messenger/analysis ; Synovial Membrane/cytology/*metabolism ; Toll-Like Receptor 3/analysis/*physiology ; Vascular Endothelial Growth Factor A/analysis/*biosynthesis/genetics

OBJECTIVETo review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors.

DATA SOURCESThe data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells".

STUDY SELECTIONArticles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected.

RESULTSEndothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notch signal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer.

CONCLUSIONSEndothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

Animals ; Endothelial Cells ; physiology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Genetic Therapy ; Hematopoietic Stem Cell Mobilization ; Humans ; Neoplasms ; blood supply ; therapy ; Neovascularization, Pathologic ; etiology ; Neovascularization, Physiologic ; Receptors, CXCR4 ; physiology ; Stem Cells ; physiology