Inducible nitric oxide synthase expression is related to angiogenesis,bcl-2 and cell proliferation in hepatocellular carcinoma

In this study, we examined the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistochemical staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC. Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissue close to the tumor edge was stronger than that of HCC tissue, and the strongest was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84.8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P=0.000) between iNOS and VEGF expression. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but without p53 expression. DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proliferating Index) and SPF (S-phase fraction) in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group. The present findings suggested that iNOS expression was significantly associated with angiogenesis, bcl-2 and cell proliferation of HCC. Project supported in part by the National Ninth-Five-Years Project Fund (No. 96909121), China     

Inducible nitric oxide synthase expression is related to angiogenesis, bcl-2 and cell proliferation in hepatocellular carcinoma

ＩＮＴＲＯＤＵＣＴＩＯＮＮｉｔｒｉｃｏｘｉｄｅ (ＮＯ) ,ａｓｈｏｒｔｈａｌｆ ｌｉｆｅｒａｄｉｃａｌ,ｉｓｈｉｇｈｌｙｒｅａｃｔｉｖｅ ,ａｎｄｉｓｉｎｖｏｌｖｅｄｉｎｍａｎｙｂｉｏ ｌｏｇｉｃａｌｐｒｏｃｅｓｓｅｓ,Ｉｔｓｅｅｍｓｔｏｐｌａｙａｎｉｍｐｏｒｔａ     

Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmid

Objective： To investigate whether vascular endothelial growth factor （VEGF） gene plasmid carried by polytetrafluoroethylene （PTFE） vascular graft materials could transfect endothelial cells （ECs） and promote their growth. Methods： PTFE vascular graft materials carried with pCDI-hVEGF121, pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF121/pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein （GFP） expression in ECs on pEGFP-PTFE materials was examined with fluorescence mi- croscopy. Results： The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGFI21-PTFE materials were higher than those ofpCDI or pEGFP-PTFE materials （P〈0.05）. VEGF protein concentration of pCDI-hVEGF121-PTFE materials was higher than that of pC DI or pEGFP-PTFE materials at 6, 24, 72 and 120 h （P〈0.01）. GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. Conclusion： PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.     

Inhibition of expression of vascular endothelial growth factor and its receptors in pulmonary adenocarcinoma cell by TNP-470 in combination with gemcitabine

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis. The inhibition effects of O-（chloroacetyl-carbamoyl） fumagillol （TNP-470）, an angiogenesis inhibitor, and gemcitabine, a chemotherapeutic agent, on expression of growth factors were investigated using human pulmonary adenocarcinoma cell line, A549. The A549 cells were divided into four groups： control group, 10^-6 mg/ml gemcitabine treated group, 10^-4 mg/ml TNP-470 treated group and gemcitabine＋TNP-470 treated group. The mRNA and protein expression of vascular endothelial growth factor （VEGF） and its receptors, FMS-like tyrosine kinase-l （FLT-1） and kinase insert domain-containing receptor （KDR）, in different groups were measured. The growth of A549 cell cultured with gemcitabine or TNP-470 was inhibited in an almost dose-dependent manner. Although gemcitabine （10^-6 mg/ml） alone and TNP-470 （10^-4 mg/ml） alone had no effect on the mRNA and protein expression of VEGF and its receptors （FLT-1, KDR） in A549 cells compared to the control （P〉0.05）, 10^-6 mg/ml gemcitabine in combination with 10^-4 mg/ml TNP-470 had significant effect （P〈0.01）. Moreover, combination of the two drugs significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone （P〈0.05）. This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro.     

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

Objective: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAMNEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. Methods: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. Results: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. Conclusion: With a low toxicity, high safety, and high transfection rate, G4PAMAMNEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.     

自发性高血压大鼠血管内皮细胞一氧化氮合酶的观察

目的观察高血压大鼠血管内皮细胞一氧化氮合酶在高血压不同时期的变化情况.方法采用免疫组织化学方法(ABC法)及计算机图象分析方法,观察及定量分析8周、12周、24周高血压大鼠胸主动脉及其心肌微血管内皮细胞一氧化氮合酶变化.结果在高血压不同时期胸主动脉与心肌微血管内皮细胞一氧化氮合酶含量变化基本一致12周较8周时明显增多,而24周与12周时无明显差异.结论伴随高血压病程的变化,血管内皮细胞eNOS含量发生了变化,且不同部位的变化呈正相关.     

Ds-echinoside A, a new triterpene glycoside derived from sea cucumber, exhibits antimetastatic activity via the inhibition of NF-κB-dependent MMP-9 and VEGF expressions

Ds-echinoside A (DSEA), a non-sulfated triterpene glycoside, was isolated from the sea cucumber Pearsonothuria graeffei. In vitro and in vivo investigations were conducted on the effects of DSEA on tumor cell adhesion, migration, invasion, and angiogenesis. In this study, we found that DSEA inhibited the proliferation of human hepatocellular liver carcinoma cells Hep G2, with a half-maximal inhibitory concentration (IC₅₀) of 2.65 μmol/L, and suppressed Hep G2 cell adhesion, migration, and invasion in a dose-dependent manner. DSEA also reduced tube formation of human endothelial cells ECV-304 on matrigel in vitro and attenuated neovascularization in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Immunocytochemical analysis revealed that DSEA significantly decreased the expression of matrix metalloproteinase-9 (MMP-9), which plays an important role in the degradation of basement membrane in tumor metastasis and angiogenesis. DSEA also increased the protein expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important regulator of MMP-9 activation. From the results of Western blotting, the expressions of nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) were found to be remarkably reduced by DSEA. These findings suggest that DSEA exhibits a significant antimetastatic activity through the specific inhibition of NF-κB-dependent MMP-9 and VEGF expressions.     

Homoharringtonine induces apoptosis of endothelium and down-regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. How ever, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry. The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner. Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

Homoharringtonine induces apoptosis of endothelium and down-regulates VEGF expression of K562 cells

INTRODUCTION Homoharringtonine (HHT) is a cephalotoxin alkaloid with anti-leukemic activity and had been used successfully in the treatment of acute and chronic myeloid leukemias (O払rien et al., 1995; 1999; Feldman et al., 1992). The principal mecha-nism of action by HHT is the inhibition of protein synthesis in a dose- and time-dependent manner by binding to ribosome and inhibiting polypeptide chain elongation (Tujebajeva et al., 1989; Zhou et al., 1995). HHT had been shown to indu…     

Optimal time for mesenchymal stem cell transplantation in rats with myocardial infarction

Background:Bone marrow mesenehymal stem cell(MSC)transplantation is a promising strategy in the treatment of myocardial infarction(MI).However,the time for transplanting cells remains controversial.The aim of this study was to find an optimal time point for cell transplantation.Methods:MSCs were isolated and cultured from Sprague-Dawley(SD) rats.MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery.MSCs were directly injected into the infarct berder zone at 1 h,1 week and 2 weeks after MI,respectively.Sham-operated and MI centrel groups received equal volume of phosphate buffered saline(PBS).At 4 weeks after MI,cardiac function Was assessed by echocardiography;vessel density Was analyzed on hematoxylin-eosin stained slides by light microscopy;the apoptosis of cardiomyocytes Was evaluated by terminal deoxynucleotidy1 transferase-mediated dUTP nick end-labeling(TUNEL) assay;the expressions of proteins were analyzed by Western blot.Results:MSC transplantation improved cardiac function.reduced the apoptosis of cardiomyocytes and increased vessel density.These benefits were more obvious in l-week group than in 1-h and 2-week groups.There are more obvious increases in the ratio of bc1-2/bax and the expression of vascular endothelial growth factor(VEGF)and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups.Conclusion:MSC transplantation was beneficial for the recovery of cardiac function.MSC transplantation at l week post-MI exerted the best effects on increases of cardiac function,anti-apoptosis and angiogenesis.     

Therapeutic potential of traditional Chinese medicine for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is the main regulator of physiological angiogenesis during embryonic development, bone growth, and reproductive function, and it also participates in a series of pathological changes. Traditional Chinese medicine (TCM), with a history of more than 2000 years, has been widely used in clinical practice, while the exploration of its mechanisms has only begun. This review summarizes the research of recent years on the influence of TCM on VEGF. It is found that many Chinese medicines and recipes have a regulatory effect on VEGF, indicating that Chinese medicine has broad prospects as a complementary and alternative therapy, providing new treatment ideas for clinical applications and the theoretical basis for research on the mechanisms of TCM.     

大肠癌组织中血管内皮细胞生长因子的表达

应用免疫组化方法,比较性地研究了大肠癌组织及其同一患者形态正常大肠粘膜组织中血管内皮细胞生长因子的表达,分析血管内皮细胞生长因子表达与大肠癌发生的年龄、性别、病理分期等因素的关系。结果显示,癌组织与正常大肠粘膜的血管内皮细胞生长因子表达存在明显差异,血管内皮细胞生长因子的表达与大肠癌患者年龄、性别、分化程度等因素不存在相关性,而与Ducks分期存在明显相关性。表明肿瘤血管的生成在大肠癌的生长发展方面起着至关重要的作用,血管内皮细胞生长因子的检测可以作为大肠癌诊断的重要参考指标。     

CHOP及GRP78在复发性多形性胶质母细胞瘤患者表达显著增加

目的:研究CHOP及GRP78与复发性GBM的相关性。方法:采用Western blot检测CHOP及GRP78在初发及复发GBM组织中的表达;Real-time PCR检测GRP78在初发以及复发GBM组织中的表达。结果:CHOP及GRP78在初发GBM中表达上调,而在经过原发性肿瘤切除后接受放疗和替莫唑胺化疗的复发性GBM中增加尤为显著。结论:CHOP及GRP78在GBM发病机制中起重要作用,该分子有望成为GMB潜在的诊断及预后评估标志物。     

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods: Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination. Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α(tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.     

Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to wound healing

Objective:To explore the feasibility of using regenerated silk fibroin membrane to construct artificial skin substitutes for wound healing, it is necessary to evaluate its cytocompatibility. Methods:The effects of regenerated silk fibroin film on cytotoxicity, adhesion, cell cycle, and apoptosis of L929 cells, growth and vascular endothelial growth factor (VEGF) expression of ECV304 cells, and VEGF, angiopoietin-1 (Ang-1), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2) expressio...     

Inhibitive effects of anti-oxidative vitamins on mannitol-induced apoptosis of vascular endothelial cells

Objective： Study blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins. Methods： Healthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was per- formed with normal saline （group B）, vitamin C （group C） and vitamin E （group D）. Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline （sample B）, vitamin C （sample C） and vitamin E （sample D）. Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression. Results： In the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes. Conclusion： Vitamin C can protect vascular endothelial cells from mannitol-induced injury.     

A preliminary evaluation of VEGF-A,VEGFR1 and VEGFR2 in patients with well-controlled type 2 diabetes mellitus

Objective

Decompensated chronic hyperglycemia often leads to late microvascular complications such as retinopathy, diabetic foot syndrome, and diabetic kidney disease. The aim of this study was to determine the concentration of vascular endothelial growth factor A (VEGF-A) and its receptors in patients with well-controlled diabetes.

Methods

The study was conducted on 31 patients with well-controlled type 2 diabetes without micro-or macroangiopathy. Thirty healthy volunteers were enrolled in a control group. Serum concentrations of VEGF-A, VEGF receptors 1 and 2 (VEGFR1 and VEGFR2), fasting glucose, and lipid profiles were measured, and the plasma concentration of glycated hemoglobin (HbA1c) was determined.

Results

No significant differences were observed between the concentration of VEGF-A, VEGFR1 or VEGFR2 in the subject group and that in the control group. Positive correlations were noted between the levels of VEGF-A, VEGFR2, and triglyceride, and there was a negative correlation between the levels of VEGFR2 and high-density lipoprotein (HDL)-cholesterol in the study group.

Conclusions

The concentrations of VEGF-A and its receptors 1 and 2 in patients with well-controlled diabetes are comparable to those of healthy individuals, which may indicate that appropriate control of glucose levels delays the occurrence of vascular complications. A negative correlation between VEGFR2 and HDL-cholesterol levels, and positive correlations between VEGF-A, VEGFR2, and triglyceride levels, suggest that lipid abnormalities occurring in diabetes may be involved in the modulation of angiogenesis.