Hydroxysafflor Yellow A Promotes Vascular Endothelial Cell Proliferation via VEGF/VEGF Receptor

Aim To study the proliferative effeet of hydroxysaftlor yellow A （HSYA） on cultured canine aortic endothelial cell （VEC） in normoxic （21% O2 ） or hypoxic （10% O2 ） culture and the underlying mechanism. Methods The endothelial cells were scratched from trypsined canine aorta endothelium. HSYA was added to the cells at final concentrations of 1 × 10^-3, 1 × 10^-4 and 1 × 10^-5 mol· L^-1, respectively. VEGF （2.6 × 10^-7 mol· L^-1 ）-treated cells were used as the positive control. The proliferative effect of HSYA on VEC was determined at 48, 72, 96, and 120 h in normoxic culture by MTI＂ assay. Similarly, the proliferation of VEC was determined at 12, 24, 48, and 72 h in hypoxic culture by MTF assay. The effects of HSYA on VEC proliferation and VEGF secretion were investigated by MTr and ELISA assays at the presence of the antibodies to VEGF and VEGF receptors. Results Pretreatment with HSYA at concentrations of 1 × 10^-3 and 1 × 10^-4 mol· L^-1 enhanced VEC proliferation in normoxic culture. The most significant enhancing effect of HSYA on VEC proliferation was achieved at 24, 48, and 72 h in hypoxic culture in concentration-dependent and time-dependent manner. HSYA at 1 × 10^-3 mol·L^-1 showed a potency similar to VEGF at 2.6 × 10^-7 mol·L^-1 . Pretreatment with the antibodies of Flt-1, KDR or VEGF blocked the proliferative effect of HSYA with similar potencies. Antibodies of Fit-1 or VEGF antagonized the promoting effect of HSYA on VEGF secretion. Conclusion HSYA promotes VEC proliferation either in normoxic or hypoxic culture, especially in the latter condition. This effect of HSYA is at least partly mediated by VEGF and VEGF receptor.

Increased protein and mRNA expression of endostatin in the ischemic brain tissue of rabbits after middle cerebral artery occlusion

Objective To explore the changes of endostatin （a strong anti-angiogenesis factor） and vascular endothelial growth factor （VEGF） in the brain tissues of rabbits following cerebral ischemia induced by middle cerebral artery occlusion （MCAO）. Methods Twenty-four New Zealand white rabbits were randomly divided into 5 groups： control （n = 5）, sham-operation （n = 4）, 2-hour ischemia （n = 5）, 24-hour ischemia （n = 5）, and 48-hour ischemia （n = 5）. The expression of VEGF and endostatin were measured by enzyme-linked immunosorbent assay （ELISA） and immunohistochemistry, respectively. In situ hybridization was used to characterize the expression of mRNA for the endostatin. Results Both the protein （at least 50%, P 〈 0.01） and mRNA （at least 70%, P 〈 0.05） of endostatin increased significantly in the ischemic brain tissues after MCAO compared with the control group. VEGF increased at least 270% in the brain after cerebral ischemia （P 〈 0.05）. Conclusion Cerebral ischemia leads to an up-regulation of endostatin in the brain, which is not associated with the increase of VEGF in the brain. The increase of endostatin may serve as a deleterious mechanism for ischemic injury through blocking angiogenesis.

1例晚期肺鳞癌的阿帕替尼单药治疗观察

目的观察阿帕替尼治疗晚期肺鳞癌的疗效及安全性。方法 1例多线治疗无效的晚期肺鳞癌患者,行阿帕替尼分子靶向治疗,对其临床疗效和安全性进行观察。结果该患者中位无进展生存时间达15个月,最佳疗效为部分缓解,主要不良反应为Ⅰ级高血压(可以控制)。结论该例晚期肺鳞癌患者使用阿帕替尼单药治疗显示出较好的疗效,不良反应较轻,阿帕替尼有可能成为晚期肺鳞癌患者可选择的靶向治疗药物之一。

Expression of vascular endothelial growth factors A and C in human pancreatic cancer

AIM： To study the expression of vascular endothelial growth factor A （VEGF-A） and VEGF-C and to determine whether the presence of VEGF-A and VEGF-C was associated with the clinicopathologic characteristics of pancreatic cancer. METHODS： VEGF-A and VEGF-C mRNA transcripts were examined by Northern blot in 6 human pancreatic cancer cell lines and 8 normal pancreatic tissues and 8 pancreatic carcinoma specimens. The expression of VEGF-A and VEGF-C proteins was examined by Western blot in the tested cell lines and by immunohistochemical stain in 50 pancreatic carcinoma samples. RESULTS： VEGF-A and VEGF-C mRNA transcripts were present in all the 6 human pancreatic cancer cell lines. Immunoblotting revealed the presence of VEGF-A and VEGF-C proteins in all the cell lines. Northern blot analysis of total RNA revealed 3.0-fold and 3.6-fold increase in VEGF-A and VEGF-C mRNA transcript in the cancer samples, respectively. Immunohistochemical analysis confirmed the expression of VEGF-A and VEGF-C in cancer cells within the tumor mass. Immunohistochemical analysis of 50 pancreatic cancer tissue samples revealed the presence of VEGF-A and VEGF-C immunoreactivity in 50% and 80% of the cancer tissue samples, respectively. The presence of VEGF-A in these cells was associated with larger tumor size and enhanced local spread （x^2= 6.690, P= 0.035〈0.05） but was not associated with decreased patient survival. However, the presence of VEGF-C in the cancer cells was associated with increased lymph node metastasis （x^2= 5.710, P= 0.017〈0.05）,but was not associated with decreased patient survival. There was no correlation between the expression of VEGF-A and VEGF-C in the same cancer cells. CONCLUSION： VEGF-A and VEGF-C are commonly overexpressed in human pancreatic cancer and may contribute to tumor growth and lymph node metastasis, There is no relationship between the expression of VEGF-A and VEGF-C in pancreatic cancer.

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.

Suppression of intracranial glioma tumorigenesis with vascular endothelial growth factor antisense oligonucleotide in rats

Objective: To observe the inhibition of intracranial glioma tumorigenesis by vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ODN) in rats. Methods: Totally 20 μ1 Hank's liquid containing 1×106 C6 glioma cells was seeded into rat right caudate putaraen in high-flow microinfusion with stereotactic technique. VEGF antisense ODN was simultaneously used with glioma cell. Each rat of the treated group Ⅰ and the treated group Ⅱ was treated with 1 000 μmol/L VEGF antisense ODN. Each rat of the treated group Ⅲ and the treated group Ⅳ was treated with 2 000 μmol/L VEGF antisense ODN. The experimental periods of the treated group Ⅰ , the treated group Ⅲ and the control group Ⅰ were 2 weeks, those of the treated group Ⅱ , the treated group Ⅳ and the control group Ⅱ were 3 weeks. Before sacrifice, MRI was performed on each rat. Tumor magnitude and pathologic examination were detected after samples were dissected. Results: The survival state of all treated rats was better, and that of the control rats was in severe danger. The tumor volumes of the treated group Ⅰ and the treated group Ⅱ were remarkably lessened. Tumor tissue could not be found macroscopically in the brain samples of the treated group Ⅲ and the treated group Ⅳ, but tumor nest could be found with microscopy. Tumors of the treated group I and the treated group Ⅱ had weak expressions of VEGF mRNA and VEGF, while normal brains and the samples of the treated group Ⅲ and the treated group Ⅳ had negative expressions, but tumors of the control groups had strong expressions. Conclusion: VEGF antisense ODN used early in situ can suppress angiogenesis and growth of rat intracranial glioma to retard tumorigenesis.

The effect of rh-endostatin on micrangium and angiogenic factors in tumor and myocardium tissue

Objective： The aim of this study was to compare effect of rh-endostatin on microvasculature in tumor and myocardium tissue. Methods： Nude mice were randomized into 4 groups, blank control group [did not burden tumor, normalsaline （NS） 100 μL/d], drug control group （did not burden tumor, rh-endostatin 400 μg/d）, model group （mice burdened tumor, NS 100 μL/d） and treatment group （mice burdened tumor, rh-endostatin 400 μg/d）, administration was given during d1-d28. The volume of tumor and the weight of mouse were measured before and after administration. The expression of CD34, MMP-2, MMP-9, HIF-la and VEGF in myocardium and tumor were detected by immunohistochemistry. The structure of vasculature was observed by immunoenzymatic double staining with CD34 and Masson. Results： The tumor volume increase of treatment group （48.18 mm3） was less than the model group （113.80 mm3）, the change of weight was not significant among the four groups. After treated with endotar, the expression of MMP-9 and VEGF in tumor were obviously down-regulated, but the same results was not found in MMP-2, HIF-la of tumor. MVD in tumor of treatment group decreased significantly compared with model group. Proportion of tumor vessels covered by collagen in treatment group increased compared with model group. However, MVD and microvasculature in myocardium did not change significantly. Conclusion： Rh-endostatin can decrease the expression of MMP-9, VEGF and MVD to inhibit growth of tumor and normalize micrangium in tumor but cannot weaken MMPs and MVD of mature micrangium in myocardium.

Regional hyperthermia combined with intrapleural chemotherapy in patients with malignant pleural effusion

Objective: The aim of our study was to assess the efficacy of regional hyperthermia combined with intrapleural chemotherapy and to evaluate the effect on the immunologic cells and vascular endothelial growth factor (VEGF) in patients with malignant pleural effusion. Methods: The 102 patients with malignant pleural effusion were included in this study: 52 patients undergoing regional hyperthermia with intrapleural chemotherapy (HICT), and 50 patients treated with intrapleural chemotherapy (ICT). Chemotherapy was administered into the thoracic cavity weekly through a tube with CDDP (dose = 40 mg/m2), and hyperthermia was performed twice a week for 60 minutes following the ICT. We evaluated the response rates and side-effects after 4 weeks. Before and after the treatment, T cell subsets and NK cells were detected by flow cytometry and VEGF was measured with ELISA kits. Results: Compared HICT to ICT, the overall response rates of the whole group, breast cancers and lung cancers were 80.8% vs 54% (P < 0.01), 86.7% vs 56.3% (P > 0.05) and 78.4% vs 52.9% (P < 0.05) respectively. The ratios of CD4+, CD4+/CD8+ and NK cells increased and the concentration of VEGF decreased more significantly after HICT. Conclusion: We concluded that combined regional hyperthermia with intrapleural chemotherapy could control the malignant pleural effusion effectively with mild toxicity. The levels of the T cell subset, NK cells and VEGF in both blood and effusion changed obviously.

Intracranial hemorrhage in patients treated with bevacizumab:Report of two cases

Treatment with bevacizumab,an antiangiogenic agent,in patients with metastatic or unresectable colorectal cancer was approved less than 4 years ago in Japan.Bevacizumab improves the survival of patients with metastatic colorectal cancer;however,it may lead to complications such as bleeding,which are sometimes fatal.Bevacizumab should be administered only after careful consideration because the potential risks of therapy outweigh its benefits.Therefore,pharma-ceutical companies do not recommend bevacizumab therapy for patients with brain metastases.While some reports support the cautious use of bevacizumab,others report that it is not always necessary to prohibit its use in patients with metastases to the central nervous system(CNS),including the brain.Thus,bevacizumab therapy in colorectal cancer patients with brain metastases is controversial,and it is unclear whether brainmetastases are a risk factor for intracranial hemor-rhage during anti-vascular endothelial growth factor(VEGF)therapy.We report a 64-year-old man and a 65-year-old man with recurrent colorectal cancer with-out brain metastases;these patients developed multifocal and solitary intracranial hemorrhage,respectively,after the administration of bevacizumab.Our findings suggest that intracranial hemorrhage can occur even if the patient does not have brain metastases prior to bevacizumab treatment and also suggest that brain metastases are not a risk factor for intracranial hemor-rhage with bevacizumab treatment.These findings also question the necessity of excluding patients with brain metastases from clinical trials on anti-VEGF therapy.

Inhibition of angiogenesis by DADAG in vivo and in vitro

1,2,5,6-Dianhydro-3,4-diacetyl-galactitol （DADAG）, an alkylating sugar alcohol derivative, has been shown effective against tumor growth. In this research, we explored the effect of DADAG on angiogenesis in chick chorioallantoic membrane （CAM） model and on the proliferation and migration of human umbilical vein endothelial cells （HUVECs）. We also studied the possible mechanism of the anti-angiogenesis effect of DADAG. The results showed that DADAG （100, 500 and 1000μnol/L） inhibited angiogenesis in CAM model dose-dependently. Sulforhodamine B （SRB） assay indicated that DADAG （45, 90, 135, 180 and 225 μmol/L） suppressed HUVECs proliferation in a dose-dependent and time-dependent manner. High Content Screening （HCS, Cellomics） assay, in which the influence of cell proliferation on migration could be excluded, indicated that DADAG （45, 135 and 225 ~xmol/L） directly inhibited the motility ofHUVECs. Immunofiuorescence assay suggested that DADAG inhibited angiogenesis possibly by decreasing vascular endothelial growth factor （VEGF） expression in HUVECs. Our findings reveal that DADAG show anti-angiogenic activity in vivo and in vitro, which is related to the downregulation of VEGF expression in endothelial cells.

Vascular endothelial growth factor gene transfer improves host endothelialization of xenogeneic biologic heart valve in vivo

OBJECTIVE: To investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene. METHODS: Bovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously. RESULTS: The concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P

盐酸多柔比星神经节介入对脑缺血影响的实验研究

目的研究盐酸多柔比星神经节介入对脑缺血再灌注模型大鼠血管内皮生长因子(VEGF)、过氧化物歧化酶(SOD)及丙二醛(MDA)水平的影响。方法30只Wistar大鼠按体重随机分为模型组、实验组以及空白组,每组各10只。用Pulsinelli四血管阻断法,建立脑缺血再灌注模型。实验组于术前48 h,开始于椎间孔缓慢注射盐酸多柔比星1 mg·kg-1,模型组与空白组注射同等剂量生理盐水q12 h。以酶联免疫吸附法(ELISA)测定大鼠脑组织内VEGF、SOD及MDA水平。结果在给药12,24 h的神经功能评分,与空白组的0分比较,模型组分别为(2.41±0.25),(1.98±0.23)分,明显增加,2组比较差异有统计学意义(P<0.05);与模型组比较,实验组分别为(1.58±0.19),(1.31±0.15)分,明显降低,2组比较差异有统计学意义(P<0.05)。模型组在给药12,24 h,脑组织VEGF表达分别为124.63±15.63,120.57±12.84,血清中丙二醛水平分别为(3.56±0.38),(3.85±0.35)mmol·mg^(-1);实验组在给药12,24 h,脑组织VEGF表达分别为102.26±12.38,100.47±11.91,血清中丙二醛水平分别为(2.97±0.35),(3.25±0.35)mmol·mg^(-1),较模型组明显降低,2组比较差异有统计学意义(P<0.05)。在给药12,24 h,模型组血清中SOD分别为(120.48±15.03),(119.37±12.58)U·mg^(-1);实验组血清中SOD水平分别为(182.73±19.49),(181.57±18.63)U·mg^(-1),实验组较模型组明显升高,2组比较差异有统计学意义(P<0.05)。结论盐酸多柔比星神经节介入通过改善氧化应激反应以及炎症反应,从而保护脑缺血再灌注大鼠脑组织缺血再灌注损伤。

Role of VEGF-A/C and CCR-7 in the enhanced metastasis of A549 cells induced by 2 and 4 Gy X-rays in vitro and in vivo

Radiotherapy is a common strategy in treating lung cancer.Accumulating evidence suggested that radiotherapy has the potential to promote the metastasis and invasion of carcinoma cells.In this study,we aimed to testify the role of vascular endothelial growth factor(VEGF)and C-C chemokine receptor-7(CCR-7)in the metastasis of human adenocarcinoma A549 cells in vivo and in vitro.Nude mice were injected with A549 cells irradiated by 0,2 and 4 Gy X-rays,respectively.Quantitative detections of VEGF-A/C and CCR-7 mRNA from lung sample were performed by real-time RT-PCR.Small interfering RNA(siRNA)transfection technology was further used to testify the role of the genes in the metastasis of A549 cells.VEGF and CCR-7mRNA could only be detected 10 weeks post injection in vivo when visible tumor foci scattered in lung.In addition,VEGF-A/C mRNA expressed significantly higher in mice injected with A549 cells irradiated by 2 and 4 X-rays than those with 0 Gy X-rays irradiation.On the other hand,A549 cells with or without X-rays irradiation transfected with VEGF siRAN and CCR-7 siRNA showed a dramatic decrease of invasiveness compared to normal A549 cells with or without irradiation.The migration indexes were?0.7,?0.48,?0.34 and?0.32 for A549 cells with CCR-7 siRNA,VEGF siRNA,X-rays combined CCR-7 siRNA and X-rays combined VEGF-siRNA respectively.These results demonstrated that X-rays could promote the metastasis of A549 cells,and VEGF-A/C and CCR-7 mRNA were closely related to the metastasis of A549 cells in vivo and in vitro.

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.