VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignant mesothelioma tumours.

The vascular endothelial growth factor (VEGF) family is a novel regulator of endothelial cell proliferation. We assessed the mRNA expression of VEGF, VEGF type C (VEGF-C) and their receptors together with the microvessel density (VD) and microlymphatic vessel density (LVD) in pursuit of their connection and prognostic value in malignant pleural mesothelioma (MPM). We used four human MPM cell lines, 54 MPM tumours and five normal pleural tissues. Expression levels for receptors and ligands were assessed by semiquantitative reverse transcriptase polymerase chain reaction analysis. Microvessels were highlighted by immunohistochemical staining for factor VIII. The discrimination of lymphatics was performed by enzyme-histochemistry for 5'-nucleotidase after adequate inhibition of non-specific activity. The expression levels of VEGF, VEGF-C and VEGFRs were high in all MPM cell lines. The percentages of tumours with higher expression compared to the mean values of normal pleural tissues were 31.5% (17/54) for VEGF, 66.7% (36/54) for VEGF-C, 20.4% (11/54) for fms-like tyrosine kinase (flt)-1, 42.6% (23/54) for kinase insert domain-containing recepter (KDR) and 59.3% (32/54) for flt-4. Significant positive correlations were found between VEGF-C and flt-4, VEGF and KDR, VEGF and flt-1 in tumour tissues. The association between LVD and VEGF-C expression level was especially strong (P< 0.0001, r= 0.63). There were also significant correlations between LVD and flt-4, and VD and VEGF. No correlation, however, was found between LVD and nodal metastasis. VD was a negative prognostic indicator in this study. The associations between VEGFNEGF-C and vessel density suggest that these factors play an important role in angiogenesis and lymphangiogenesis in this tumour, and assessment of vascularity may be a useful prognostic indicator for MPM patients.

Ohta Y ，Shridhar V ，Bright RK ，Kalemkerian GP ，Du W ，Carbone M ，Watanabe Y ，Pass HI ... -  《-》

Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma.

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946+/-14 pg/ml vs. 180+/-17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase-related VEGF receptors Flt-1 and KDR. Recombinant human VEGF phosphorylated both Flt-1 and KDR and increased proliferation of all four MM cell lines in a dose-dependent fashion. Neutralizing antibodies against either VEGF, Flt-1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt-1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non-malignant pleural disease (1885.7+/-894.9 pg/ml vs. 266.9+/-180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth.

Strizzi L ，Catalano A ，Vianale G ，Orecchia S ，Casalini A ，Tassi G ，Puntoni R ，Mutti L ，Procopio A ... -  《JOURNAL OF PATHOLOGY》

Expression of the Ets-1 transcription factor in human astrocytomas is associated with Fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesis and neoangiogenesis.

Marked neovascularization and vascular endothelial proliferation are characteristic features of malignant gliomas. Vascular endothelial growth factor (VEGF), an angiogenic protein secreted by glioma cells, appears to play a crucial role for induction of neoangiogenesis. The VEGF receptors fms-like tyrosine kinase-1 (Flt-1)/VEGFR-1 and kinase insert domain-containing receptor (KDR)/ VEGFR-2 are up-regulated on the surface of endothelial cells (ECs) in gliomas. Both receptor genes contain an Ets-responsible element in their promoters. The proto-oncogene ets-1 encodes a transcription factor that has been associated with blood vessel formation in vivo under physiological and pathophysiological conditions including tumor neovascularization. Ets-1 is induced by VEGF in cultured ECs. In vitro data also point to a role of Ets-1 as a transcriptional activator of Flt-1. These properties prompted us to investigate Ets-1 expression in 32 human astroglial tumors of WHO grades I-IV and to correlate the data with the expression pattern of VEGF, Flt-1, and KDR. By in situ hybridization, high ets-1 mRNA levels were found in the glioma microvasculature with particularly prominent signals in glomeruloid vascular endothelial proliferations of glioblastomas (WHO grade IV). Semiquantitative reverse transcription-PCR identified the full-length ets-1 transcript but none of three known splice variants encoding isoforms with different functional domains. Immunohistochemical staining demonstrated Ets-1 protein preferentially in the nucleus of those ECs with an epithelioid morphology consistent with an activated state, whereas quiescent flat-shaped ECs predominantly displayed cytosolic immunoreactivity. This observation proposes nuclear translocation of Ets-1 during neoangiogenesis. VEGF synthesis by glioma cells was accompanied by Ets-1 expression in adjacent microvascular ECs. Furthermore, a highly significant correlation was observed between Ets-1 and Flt-1 (but not KDR) expression in ECs of the glioma microvasculature. Our data suggest that VEGF secreted by glioma cells induces Ets-1 in adjacent microvascular ECs, which subsequently transactivates the VEGF receptor Flt-1. This cascade may crucially promote neoangiogenesis in human gliomas.

Valter MM ，Hügel A ，Huang HJ ，Cavenee WK ，Wiestler OD ，Pietsch T ，Wernert N ... -  《CANCER RESEARCH》

The short form of the alternatively spliced flt-4 but not its ligand vascular endothelial growth factor C is related to lymph node metastasis in human breast cancers.

Angiogenesis is essential for tumor growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently, VEGF-C, a new VEGF family member, has been identified that binds to the tyrosine kinase receptors flt-4 [VEGF receptor (VEGFR) 3] and KDR (VEGFR2). Although the importance of VEGF has been shown in many human tumor types, the contribution of VEGF-C and its primary receptor flt-4 to tumor progression is less well understood. We have therefore measured the level of VEGF-C, flt-4, and KDR mRNA by RNase protection assay and the pattern of VEGF-C expression by immunohistochemistry in 11 normal breast tissue samples and 61 invasive breast cancers. No significant difference in VEGF-C expression was observed between normal and neoplastic breast tissues (P = 0.11). There was a significant correlation between VEGF-C and both flt-4 (P = 0.02) and KDR (P = 0.0002), but no association was seen between VEGF-C and either lymph node status (P = 0.66) or number of involved nodes (P = 0.88), patient age (P = 0.83), tumor size (P = 0.20), estrogen receptor status (P = 0.67), or tumor grade (P = 0.35). No significant relationship was present between VEGF-C and vascular invasion (P = 0.30), tumor vascularity (P = 0.21), VEGF-A (P = 0.62), or thymidine phosphorylase expression (P = 1.00). VEGF-C was expressed predominantly in the cytoplasm of tumor cells, although occasional stromal components including fibroblasts were also positive. We could demonstrate no association between lymph node metastasis and either VEGF-C (P = 0.66) or flt-4 (P = 0.4). However, we did observe a significant loss of the long but not the short isoform of flt-4 in tumors compared with normal tissues (P = 0.02 and P = 0.25, respectively), and this difference was largely accounted for by the reduction of long flt-4 in node-positive tumors. These findings strongly support a role for VEGF-C/flt-4 signaling in tumor growth by enhancement of angiogenesis and/or lymphangiogenesis and suggest that differential regulation of these processes may be controlled via flt-4 isoform transcription. They further suggest that the measurement of flt-4 isoform expression may identify a patient group that is likely to have node-positive disease and therefore benefit from additional treatment and also emphasize an additional ligand interaction that could be exploited by anti-VEGFR therapy.

Gunningham SP ，Currie MJ ，Han C ，Robinson BA ，Scott PA ，Harris AL ，Fox SB ... -  《CLINICAL CANCER RESEARCH》

Microvessel density, vascular endothelial growth factor and its receptors Flt-1 and Flk-1/KDR in hepatocellular carcinoma.

Assessment of angiogenesis may yield important information for an effective antiangiogenic treatment for hepatocellular carcinoma (HCC) because HCC is characteristically hypervascular We examined the relationship of microvessel density (MVD), vascular endothelial growth factor (VEGF), and VEGF receptors Flt-1 and Flk-1/KDR in 50 patients with HCC and in 3 hepatoma cell lines. VEGF messenger RNA (mRNA) was overexpressed in 26 tumors (52%), and the 3 VEGF isoforms (121, 165, and 189) were present in high frequencies. Flt-1 mRNA was overexpressed in 34 tumors (68%), with levels significantly increased in HCCs compared with the nontumorous livers. Tumor Flt-1 mRNA significantly correlated with tumor VEGF mRNA levels. Within the group of tumors 8.5 cm or less in diameter, tumors with intrahepatic metastasis in the form of tumor microsatellite formation had significantly higher VEGF mRNA levels. MVD assessed by immunohistochemical analysis with CD34 antibody was inversely related to tumor size. Angiogenesis as assessed by MVD and tumor VEGF expression seems to have a more important role in tumor growth and intrahepatic metastasis in smaller HCCs. The differential up-regulation of Flt-1 suggests that it may have an important role in angiogenesis in HCC.

Ng IO ，Poon RT ，Lee JM ，Fan ST ，Ng M ，Tso WK ... -  《AMERICAN JOURNAL OF CLINICAL PATHOLOGY》