血瘀证兔模型血管内皮细胞内分泌功能变化及血府逐瘀汤作用的影响

用牛血清白蛋白和去甲肾上腺素损伤而造成血瘀证兔模型，并用血府逐瘀汤治疗，观察其血管内皮细胞内分泌功能的变化。结果表明，与正常对照兔比较，造型兔血浆中内皮素（ＥＴ）含量及组织型纤溶酶原激活物抑制剂（ＰＡＩ）明显增高（０．０１），前列环素（ＰＧＩ２）含量及组织型纤溶酶原激活物（ｔＰＡ）的活性明显降低（Ｐ＜０．０１）。血府逐瘀汤治疗后，造型兔血浆ＥＴ含量明显减少（Ｐ＜０．０１），ＰＧＩ２含量显著增加（Ｐ＜０．０１），并恢复到正常水平；ｔＰＡ活性明显升高（Ｐ＜０．０１），ＰＡＩ活性明显降低（Ｐ＜０．０１）。上述结果提示血瘀证血管内皮细胞内分泌功能紊乱。

Expression of Angiopoietin-2 and Vascular Endothelial Growth Factor in Oral Squamous Cell Carcinoma and Its Significance

Objective： To investigate the expression of angiopoietin-2 （Ang-2） and vascular endothelialcell growth factor （VEGF） in oral squamous cell carcinoma （OSCC） and their correlations with clinicopathologic parameters, angiogenesis and vessel maturation of OSCC. Methods： The expression of Ang-2 and VEGF was detected in 41 speciments of human OSCC, 30 adjacent noncancerous oral tissues and 10 specimens of normal oral mucosa by conventional immumohistochemistry. Microvessel density （MVD） and vessel maturation index （VMI） were also assessed by double-labelling immumohistochemistry staining against CD34, a marker of pan-endothelial cells, and that against alpha-smooth muscle actin （α-SMA）, a marker of mural cells （pericytes/smooth muscle cells）. Results： The positive expression rate of Ang-2 and VEGF in 41 OSCC tissues was 51.22% and 63.42%, respectively. The expression of Ang-2 and VEGF was significantly higher in OSCC than in adjacent noncancerous oral tissues （all P〈0.05） and normal oral mucosa （all P〈0.05）. In the clinicopathologic parameters, the Ang-2 expression was closely correlated with tumor lymph node metastasis （P〈0.01） and the VEGF expression was correlated with tumor differentiated degree （P〈0.05）, but there was no significant correlation among the Ang-2 and VEGF expression and patients＇ sex, age and TNM stages （all P〉0.05）. The MVD of OSCC positive for both Ang-2 and VEGF was significantly higher than that of OSCC negative for both Ang-2 and VEGF （P〈0.05）. The VMI of OSCC positive for Ang-2 was significantly lower than that of OSCC negative for Ang-2 （P〈0.05）. When Ang-2 expression was combined with the staus of VEGF expression, MVD of OSCC positive for both Ang-2 and VEGF was the highest （51.08±2.99） as compared with that of other status in patient with OSCC （all P〈0.05）. Conclusion： The overexpression of Ang-2 and VEGF may play a crucial role in the development of OSCC. They are closely associated with angiogenesis and vessel maturation of tumor.

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.

Clinical significance of the expression of isoform 165 vascular endothelial growth factor mRNA in noncancerous liver remnants of patients with hepatocellular carcinoma

AIM: To investigate the prognostic role of isoform 165 vascular endothelial growth factor messenger RNA (VEGF165 mRNA)in noncancerous liver tissues from patients with primary hepatocellular carcinoma (HCC).METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in noncancerous liver tissues from 60 consecutive patients with HCC undergoing curative resection. We categorized the patients with VEGF165 mRNA over 0.500 in noncancerous liver tissues as group A, and those below 0.500 as group B.RESULTS: Among the isoforms of VEGF mRNA by multivariate analysis, a higher level of VEGF165 mRNA in noncancerous liver tissue correlated significantly with a higher risk of HCC recurrence (P = 0.039) and recurrence-related mortality (P= 0.048), but VEGF121 did not. The other significant predictors of recurrence consisted of vascular permeation (P = 0.022),daughter nodules (P = 0.033), cellular dedifferentiation (P = 0.033), an absent or incomplete capsule (P = 0.037).A significant variable of recurrence-related mortality was Vascular permeation (P= 0.012). As to the clinical manifestations of 16 patients who developed recurrence,the recurrent tumor number over 2, recurrent extent over two-liver segments, and the median survival after recurrence,all significantly correlated with group A patients (P = 0.043,0.043, and 0.048, respectively). However, the presence of extrahepatic metastasis was not (P＞0.05). The difference in recurrence after treatment between the two groups had no statistical significance (P＞0.05).CONCLUSION: The higher expression of isoform VEGF165mRNA in noncancerous liver remnant of patients with HCC may be a significant biological indicator of the invasiveness of postoperative recurrence.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo

The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of blood- borne compounds to the parenchyma. They are func- tionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understand- ing the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phe- notypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

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.

Expression of mucosal addressin cell adhesion molecule 1 on vascular endothelium of gastric mucosa in patients with nodular gastritis

AIM:The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT).Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center,strongly associated with H pylori infection.The purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG. METHODS:We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls.A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM- 1 and integrin β7.In simultaneous viewing of serial sections, the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated.We also performed immunostaining with anti-CD20,CD4,CD8 and CD68 antibodies to determine the lymphocyte subsets co- expressing integrin β7. RESULTS:Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection.Of note,the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls.Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates.Integrin β7-expressing mononuclear cells,mainly composed of CD20 and CD4 lymphocytes,were associated with vessels lined with MAdCAM-1-expressing endothelium.CONCLUSION: Our results suggest that the MAdCAM一1/ integrin a4p7 homing system may participate in gastric inflammation in response to H py/o}i-infection and contributes to MALT formation, typically leading to the development of NG.

A novel gene delivery system targeting cells expressing VEGF receptors

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-β-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) andhuman malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with theGene delivery system targeting VEGF receptors relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.

Expression of angiopoietins, Tie2 and vascular endothelial growth factor in angiogenesis and progression of hepatocellular carcinoma

AIM： TO investigate the significance of angiopoietins, Tie2 and vascular endothelial growth factor （VEGF） expression in the angiogenesis and progress of hepatocellular carcinoma （HCC）. METHODS： Fresh surgically resected specimens of HCC and noncancerous liver （NCL） tissue from 38 patients with HCC were obtained, and expression of angiopoietin-1 （Ang-1）, angiopoietin-2 （Ang-2）, Tie2, and VEGF messenger RNA （mRNA） was examined by real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Expression pattern of each gene in HCC and NCL tissue specimens was compared and the potential role and interaction in angiogenesis of HCC were analyzed. Genes＇ expression level and its relationship with tumor＇s clinicopathological parameters were also investigated. Immunohistochemical staining of CD34 was performed to determine the microvessel density （MVD） and Ang-2/Ang-1 ratio was calculated. Relationships between Ang-2/Ang-1 ratio, VEGF and MVD and clinicopathological features were also tested so as to evaluate their significance in the progression of HCC. RESULTS： Ang-2 and VEGF mRNAs in HCC were significantly higher than those in NCL tissue （P 〈 0.05）, whereas the Ang-1 and Tie2 mRNAs showed no statistical significance （P 〉 0.05）, though slightly lower level of Ang-1 mRNA in HCC was observed. Ang-2/ Ang-1 ratio and VEGF were both positively correlated to MVD. The Ang-2/Ang-1 ratio, Ang-2 and VEGF were all associated with tumor＇s clinicopathological parameters （P 〈 0.05） except for histological grades （P 〉 0.05）. Ang-1 and Tie2 levels in different clinicopathological groups were not significantly different （P 〉 0.05）. CONCLUSION： Dominant Ang-2 expression against Ang-1 through Tie2 receptor in the presence of VEGF plays a critical role in initiating early neovascularization and transformation of noncancerous liver to hepatocellular carcinoma. Its consequently constant operation in formed HCC induces further angiogenesis and progression of HCC.

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM： To evaluate the effect of antisense vascular endothelial growth factor （VEGF） RNA （PCMV-FGEV） transfection on the profile of hepatocellular carcinoma （HCC） SMMC-7721 cells in vitro and in vivo.METHODS： SMMC-7721 cells were transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine as antisense group, sense group and control group respectively. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the cells were determined by RT-PCR and immunohistochemistry. Cell proliferation was observed by Mnassay. FACS analysis was used to determine the effect of PCMV-FGEV transfection on cell apoptosis. The growth of transfected cells in vivo was also observed in nude mice.RESULTS： VEGF expression was reduced in SMMC-7721 transfected with PCMV-FGEV, which was confirmed by RT-PCR and immunohistochemistry. No effect of PCMV- FGEV transfection was found on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cells transfected with PCMV-FGEV was slow in nude mice and accompanied with obvious apoptosis. The latent time of tumors in the antisense group was 25.0 ：l： 1.8 d, which was longer than that in sense and control groups （F= 19.455, P〈 0.01）. 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）.CONCLUSION： The expression of VEGF can be inhibited by antisense PCMV-FGEV. Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721 in vitro but can inhibit tumor growth and induce cell apoptosis in vivo.

Prokineticin 2/Bv8 is expressed in Kupffer cells in liver and is down regulated in human hepatocellular carcinoma

AIM： TO study the implication of prokineticin 1 （PKI/EGVEGF） and prokineticin 2 （PK2/13v8） in hepatocellular carcinoma angiogenesis.METHODS： The gene induction of PK1/EG-VEGF and PK2/Bv8 was investigated in 10 normal, 28 fibrotic and 28 tumoral livers by using real time PCR. Their expression was compared to the expression of VEGF （an angiogenesis marker）, vWF （an endothelial cell marker） and to CD68 （a monocyte/macrophage marker）. Furthermore, the rnRNA levels of PK1/EG-VEGF, PK2/Bv8, prokineticin receptor 1 and 2 were evaluated by real time PCR in isolated liver cell populations. Finally, PK2/Bv8 protein was detected in normal liver paraffin sections and in isolated liver cells by immunohistochernistry and immunocytochemistry.RESULTS： PK2/Bv8 mRNA but not PK1/EG-VEGF was expressed in all types of normal liver samples examined. In the context of liver tumor development, we reported that PK2/13v8 correlates only with CD68 and showed a significant decrease in expression as the pathology evolves towards cancer. Whereas, VEGF and vWF mRNA were significantly upregulated in both fibrosis and HCC,as expected. In addition, out of all isolated liver cells examined, only Kupffer cells （liver resident macrophages） express significant levels of PK2/Bv8 and its receptors, prokineticin receptor 1 and 2.CONCLUSION： In normal liver PK2/Bv8 and its receptors were specifically expressed by Kupffer cells. PK2/Bv8 expression decreased as the liver evolves towards cancer and did not correlate with HCC angiogenesis.

Identification of colorectal cancer metastasis markers by an angiogenesis-related cytokine-antibody array

AIM： To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer （mCRC） with the aim of identifying prognostic markers.METHODS： The expression of 44 angiogenesis- secreted factors was measured by a novel cytokine antibody array methodology. The study evaluated vas- cular endothelial growth factor （VEGF） and its soluble vascular endothelial growth factor receptor （sVEGFR）-I protein levels by enzyme immunoassay （EIA） in a panel of 16 CRC cell lines, mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction （Q-RT-PCR） and vascular en- dothelial growth factor receptor （VEGFR）-2 expressionwas analyzed by flow cytometry.

Glycer-AGEs-RAGE signaling enhances the angiogenic potential of hepatocellular carcinoma by upregulating VEGF expression

AIM:To investigate the effect of glyceraldehyde-derived advanced glycation end-products(Glycer-AGEs) on hepatocellular carcinoma(HCC)cells.METHODS:Two HCC cell lines(Hep3B and HepG2 cells)and human umbilical vein endothelial cells(HUVEC)were used.Cell viability was determined using the WST-8 assay.Western blotting,enzyme linked immunosorbent assay,and real-time reverse transcriptionpolymerase chain reactions were used to detect protein and mRNA.Angiogenesis was evaluated by assessing the proliferation,migration,and tube formation of HUVEC.RESULTS:The receptor for AGEs(RAGE)protein was detected in Hep3B and HepG2 cells.HepG2 cells werenot affected by the addition of Glycer-AGEs.GlycerAGEs markedly increased vascular endothelial growth factor(VEGF)mRNA and protein expression,which is one of the most potent angiogenic factors.Compared with the control unglycated bovine serum albumin(BSA) treatment,VEGF mRNA expression levels induced by the Glycer-AGEs treatment were 1.00±0.10 vs 1.92 ±0.09(P<0.01).Similarly,protein expression levels induced by the Glycer-AGEs treatment were 1.63±0.04 ng/mL vs 2.28±0.17 ng/mL for the 24 h treatment and 3.36±0.10 ng/mL vs 4.79±0.31 ng/mL for the 48 h treatment,respectively(P<0.01).Furthermore,compared with the effect of the control unglycated BSA-treated conditioned medium,the Glycer-AGEstreated conditioned medium significantly increased the proliferation,migration,and tube formation of HUVEC,with values of 122.4%±9.0%vs 144.5%±11.3%for cell viability,4.29±1.53 vs 6.78±1.84 for migration indices,and 71.0±7.5 vs 112.4±8.0 for the number of branching points,respectively(P<0.01).CONCLUSION:These results suggest that Glycer-AGEs-RAGE signaling enhances the angiogenic potential of HCC cells by upregulating VEGF expression.

Zinc Finger Protein-activating Transcription Factor Up-regulates Vascular Endothelial Growth Factor-A Expression in Vitro

Objective To construct the zinc finger protein-activating transcription factor (ZFP-ATF) plasmid and evaluate its efficacy in inducing vascular endothelial growth factor (VEGF) expression in EY.HY926 endothelial cells. Methods Firstly, we constructed the ZFP-ATF plasmid, then testified the quantity of VEGF protein in EY.HY926 endothelial cells after transfected with ZFP-ATP plasmid by Western blot, finally, we used the RT-PCR to testify whether the ZFP-ATF can stimulate expression of VEGF splice variants. Results The ZFP-ATF DNA sequences were located the multiclone sites of PVAX1 vector between the site of BamH Ⅰ and Xhol. Western blot result showed VEGF expression in EY.HY926 endothelial cells transfected with ZFP-ATF plasmid was significantly higher than that in cells transfected with VEGF165 (19.95±3.95 vs. 12.15±1.55 μg/μL, P<0.01). RT-PCR result showed VEGF-A mRNA expression level induced by ZFP-ATF was high than that induced by VEGF165. Conclusion ZFP-ATF can up-regulate the VEGF-A expression in comparison with VEGF165, which might have beneficial effects in angiogenesis process.

Altered expression of mitochondrial related genes in the native Tibetan placents by mitochondrial cDNA array analysis

Objective： To explore the mechanism of native Tibetan fetuses adaptation to hypoxia, we tried to find the different expression genes about mitochondrial function in the native Tibetan placents. Methods： In this study, the placents of native Tibetan and the high-altitude Hart （ha-Hart） were collected. After the total RNA extraction, the finally synthesized cDNAs were hybridized to mitochondrial array to find the altered expression genes between them. Then, the cytochrome c oxidase 17 （Coxl7）, dynactin 2 （DCTN2, also known as p50）, and vascular endothelial growth factor receptor （VEGFR, also known as KDR） were chosen from the altered expression genes to further verify the array results using the SYBR Green real-time PCR. Because the altered expression genes （such as Cybb and Cox 17） in the array results related to the activities of COXI and COXIV, the placental mitochondria activities of COXI and COXIV were measured to find their changes in the hypoxia. Results： By a standard of≥1.5 or ≤0.67, there were 24 different expressed genes between the native Tibetan and the ha-Han placents, including 3 up-regulated genes and 21 down-regulated genes. These genes were related to energy metabolism, signal transduction, cell proliferation, electron transport, cell adhesion, nucleotide-excision repair. The array results of Cox17, DCTN2 and KDR were further verified by the real-time RT-PCR. Through the mitochondria respiration measurements, the activity of COXI in the native Tibetan placents were higher than that of ha-Han, there was no difference in COXIV activity between them. Conclusion： The altered mitochondrial related genes in the native Tibetan placents may have a role in the high altitude adaptation for fetuses through changing the activity of mitochondrial COX.

VEGF-D expression correlates with colorectal cancer aggressiveness and is downregulated by cetuximab

AIM:To gain mechanistic insights into the role played by epidermal growth factor receptor (EGFR) in the regulation of vascular endothelial growth factors (VEGFs) in colorectal cancer (CRC). METHODS:The impact of high-level expression of the growth factor receptors EGFR and VEGF receptor (VEGFR)3 and the VEGFR3 ligands VEGF-C and VEGF-D on disease progression and prognosis in human CRC was investigated in 108 patients using immunohistochemistry. Furthermore, the expression of the lymphangiogenic factors in response to the modulation of EGFR signalling by the EGFR-targeted monoclonal antibody cetuximab was investigated at the mRNA and protein level in human SW480 and SW620 CRC cell lines and a mouse xenograft model. RESULTS: Human CRC specimens and cell lines displayed EGFR, VEGF-C and VEGF-D expression with varying intensities. VEGF-C expression was associated with histological grade. Strong expression of VEGF-D was significantly associated with lymph node metastases and linked to a trend for decreased survival in lymph node-positive patients. EGFR blockade with cetuximab resulted in a significant decrease of VEGF-D expression in vitro and in vivo. CONCLUSION:In conclusion, the expression of VEGF-D in colorectal tumours is significantly associated with lymphatic involvement in CRC patients and such expression might be blocked effectively by cetuximab.

The expression of HIF-1α and VEGF as well as their correlation with angiogenesis in esophageal squamous cell carcinomas

Objective： To investigate the correlations among the expressions of hypoxia-inducible factor-1α （HIF-1α）, vascular endothelial cell growth factor （VEGF） and microvessel density （MVD）, and their relationships to the clinicopathologic characteristics of esophageal squamous cell carcinomas （ESCC）. Methods： The expressions of HIF-1α, VEGF and MVD were detected by immunohistochemical method in 45 cases of ESCC, 30 intraepithelial neoplasia and 35 normal esophageal mucosal epithelia tissues. The correlations among the expressions of HIF-1α, VEGF and MVD, and their relationships to the clinicopathologic features of ESCC were analyzed. Results： The rate of positive expression of HIF-1α and VEGF which were 80% and 84% in ESCC were significantly higher than those in intraepithelial neoplasia and normal esophageal mucosal epithelium tissues （P 〈 0.01） and so did the MVD value which was71.10 ±15.02 in ESCC （P 〈 0.01）. The expression of HIF-1α and VEGF were positively correlated with the depth of tumor invasion, lymph node metastasis and TNM staging of ESCC. The expressions of HIF-1α were positively correlated with the expressions of VEGF and the value of MVD. Conclusion： Overexpression of HIF-1α is found in ESCC. HIF-1α may induce the angiogenesis in ESCC by upregulating the transcription of VEGF gene. It may play an important role in the carcinogenesis and aggression in ESCC, HIF-1α, VEGF and MVD may be a useful marker for evaluating the biological behaviors of ESCC.

Effects of warm ischemia time on biliary injury in rat liver transplantation

AIM:To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in livertransplanted rats. METHODS:Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups:a shamoperation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion. RESULTS:With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened. CONCLUSION:A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.