不同恶性程度胶质瘤中血管生成拟态的鉴定及其临床意义

目的：明确不同恶性程度胶质瘤中是否存在血管生成拟态及其临床意义。方法：应用三维培养技术、HE染色、CD 34-PA S双重染色方法观察胶质瘤中拟态血管的存在情况。结果：U87细胞培养48h后可见细胞相互融合,连接成网络样结构。对照组中,SHG 44培养72h后生长紊乱,无明显规律,但是在缺氧组中,SHG 44互相连接,形成网络样结构。CD 34-PA S双重染色可见Ⅰ级胶质瘤中未发现血管生成拟态,Ⅱ-Ⅲ级胶质瘤中发现3例血管生成拟态阳性,阳性率为8.6%（3/35）,与Ⅰ级胶质瘤组相比,两者具有显著性差异（P〈0.05）,Ⅳ级胶质瘤中发现11例血管生成拟态阳性,阳性率为28.9%（11/38）,与Ⅱ-Ⅲ级胶质瘤组（P〈0.05）及Ⅰ级胶质瘤组相比,均有显著性差异（P〈0.001）。结论：胶质瘤中存在血管生成拟态现象,血管生成拟态与肿瘤的恶性程度呈正相关。

Study of relationship between VEGF expression and vasculogenic mimicry of tumor

Objective： The aim of this study was to explore the relationship between VEGF expression and vasculogenic mimicry of tumor in vitro. Methods： We observed the ability to form vasculogenic mimicry in several cell lines follow as LO2 cell, hepG2 cell, SMMC-7721 cell and A549 cell in three-dimensional cultures; we detected the expression of VEGF in the level of mRNAand protein by polymerase chain reaction （PCR） and western blot in those cell lines. Results： HepG2 cell and A549 cell had the ability to form vasculogenic mimicry in three-dimensional cell cultures, while LO2 cell and SMMC-7721 cell hadn＇t the ability. The rates of VEGF16JGAPDH were 0.212 ± 0.011,0.208 ± 0.013, 0.117 ± 0.009 and 0.214±0.012 respectively, and the rates of VEGF121/GAPDH were 0.186 ± 0.018, 0.192± 0.014, 0.050 ± 0.010, 0.196 ± 0.017 in LO2 cell, hepG2 cell, SMMC-7721 cell and A549 cell, separately. The expression of VEGF gene in mRNA and protein levels in HepG2 cell, LO2 cell and A549 cell were significantly higher than that in SMMC-7721 cell （P 〈 0.05）. Conclusion： The cell lines which the expression of VEGF gene is low don＇t have the ability to form vasculogenio mimicry and the high expression of VEGF gene cann＇ t form vasculogenic mimicry at all, while the expression of VEGF gene in the cell lines is high in the cell lines which can form vasculogenic mimicry. VEGF has impact on the formation vasculgenic mimicry. Only the cells which the expression of VEGF is high have the potential to form vasculogenic mimicry. But, it doesn＇t play a unique key role in vasculogenic mimicry.

The effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation of glioma

Objective:To investigate the vasculogenic mimicry formation induced by hypoxia in Ⅱ-Ⅲ human glioma cell and the effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation and the mechanism.Methods:MTT,Transwell and three-dimentional culture were used to detect the proliferation,migration and tubule formation of SHG44.The expression of vascular endothelial growth factor-α(VEGF-α),erythropoietin-producing hepatocellular carcinoma-A2 (EphA2) and matrix metalloproteinases 2 (MMP2) was detected by RT-PCR and Western blotting analysis.Results:The OD 490 in hypoxia group was 0.60±0.06 and in control group was 0.46±0.05.The number of cell migration was 178.71±18.81 in hypoxia group and 85.86±17.92 in control group.The tubule formation was 56.80±12.21 in hypoxia group and 4.20±2.62 in control group.The proliferation,migration and tubule formation in hypoxia group were significantly higher than that in control group.The expression of VEGF-α,EphA2 and MMP2 was upregulated in hypoxia.When various concentrations of alphastatin (100,1 000,10 000 nmol/L) were added to hypoxia group,the numbers of cell migration were 142.57±12.12,92.71±17.68,30.00±7.72 and the tubule formation were 47.71±10.58,18.86±8.40,8.43±5.62.The cell migration and tubule formation were significantly suppressed by alphastatin in a dose-dependent manner.In alphastatin group,the phosphorylation of EphA2 protein (P=0.037,F=4.629) and activation of MMP2 protein (P=0.005,F=9.331) were significantly suppressed but there was no change in VEGF-α protein.Conclusion:Ⅱ-Ⅲ human glioma cell is able to form vasculogenic mimicry induced by hypoxia and alphastatin peptide can suppress the hypoxia-induced vasculogenic mimicry.VEGF-α induced EphA2 phospharilation and MMP2 activation maybe the key pathway to form vasculogenic mimicry.

Vasculogenic mimicry in non-small cell lung cancer and its relationship with tumor stage

Objective： The purpose of the study was to study the mechanism of vasculogenic mimicry （VM） and its relationship with tumor stage in non-small cell lung cancer （NSCLC）. Methods： Forty-two patients with NSCLC were collected, 19 belonged to the early stage （stages Ⅰ ＋Ⅱ） while 23 were late stage （stages Ⅲ ＋ Ⅳ）. Moreover, 20 patients got surgical treat ment and 22 got chemotherapy. We studied the relationship of VM with stage, chemotherapeutic effect, HIF-la, microves sel density （MVD） and clinicopathologic features. Results： VM in patients of early stages were significantly more than late stages （68.4% vs 26.1%, P = 0.006）, and the positive rate of VM was proportional to HIF-la （P = 0.034）. But no correlation was found between VM and chemotherapeutic effect （14.3% vs 26.7%, P = 1.00） or MVD （P 〉 0.05）. Furthermore, we found VM also showed a negative correlation with distant metastases and lymph nodes metastases （P 〈 0.05） while no correlation was found with other clinicopathologic. Conclusion： VM was generated during the early stage in NSCLC and correlated with lymph nodes metastases. As the disease progressed, VM may be replaced by vascular endothelial cells, so the late-stage patients especially people with distant metastases had fewer VM. As the main factor produced by hypoxia, HIF-la may make a difference in VM formation. Thus we inferred VM might be a new target for targeted therapy, and could provide help for clinical staging and treatment.

Expression of HIF-1α in hepatocellular carcinoma and its relationship with vasculogenic mimicry and clinical pathology

Objective： The aim of this study was to discuss HIF-la expression and vasculogenic mimicry （VM） in hepatocel- lular carcinoma （HCC） and their relationship with the clinical pathological features and clinical significance. Methods： Two hundred and seven specimens from patients in The Affiliated Hospital of North Sichuan Medical College who received hepatic cell carcinoma resection were tested by immunohistochemistry and double staining of CD31 and PSA. Then detected the expression of HIF-la, VM, and analysed the relationship between clinical pathology. Results： The HIF-la positive rate was 71.01% and its expression was associated with liver cirrhosis, tumor size and TNM stage （P 〈 0.05）. HIF-la protein expres- sion was positively associated with the VM （y = 0.1988, P = 0.0041）. Conclusion： Hypoxia may be the reason for VM in high invasive HCC, regulating the tumor microenvironment may have great significance in inhibiting invasion and metastasis of HCC.

Effect of grape seed proanthocyanidins on tumor vasculogenic mimicry in liver cancer xenograft model

Objective: As a novel blood supply pattern, vasculogenic mimicry(VM) has attracted increasingly attention in recent years, which may partly compensate for the absence of feeding and facilitate tumor perfusion. However, anti-angiogenic drugs have little effect on VM. The grape seed proanthocyanidins(GSPs), a kind of promising bioactive phytochemical, has shown anti-carcinogenesis and anti-angiogenic in several tumor models. However, GSPs regulation of VM and its possible mechanisms in a H22 hepatoma carcinoma model remain not clear. The aim of this study was to examine the effects of GSPs on proliferation and VM in a H22 hepatoma carcinoma model and to investigate the underlying mechanism. Methods: Seventy-five mice were divided into the control group and experimental groups treated with different concentration of GSPs. CD34-PAS dual staining was employed to identify the VM structure. The immunohistochemical staining for investigating the expression of VEGF, Eph A2 and MMP-2 protein was performed. Results: Treatment of the H22 model with Endostar(4 mg/kg), 50, 100, 200 mg/kg of the GSPs resulted in 6.87%, 17.81%, 27.43%, 53.52% inhibition in tumor growth, respectively. The mean weight of tumors were significantly lower in GSPs(100 mg/kg) and GSPs(200 mg/kg) groups than in the control group(all P < 0.01). Similarly, compared with the control group, the number of VM channels were significantly reduced in GSPs(100 mg/kg) and GSPs(200 mg/kg) groups(all P < 0.01). Immunohistochemistry showed significant decreases in the expression levels of VEGF, Eph A2 and MMP-2 protein in GSPs(100 mg/kg) and GSPs(200 mg/kg) groups when compared with control group(all P < 0.001). Conclusion: This is the first report providing evidence that GSPs inhibit the VM structure by regulation of the VEGF/Eph A2/MMPs signaling pathway. Therefore, we concluded that GSPs has the potential of being a clinical anti-VM inhibitor.