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The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

Vascular endothelial growth factor (VEGF), which is known to be an angiogenetic factor, plays an important role in the inflammation of synovial tissue. To investigate the relationships between VEGF and clinical symptoms in rotator cuff disease, VEGF expression was examined using RT-PCR and immunohistochemical analysis in 50 patients with this disease (26 with full-thickness cuff tear, 12 with partial-thickness tear, and 12 with subacromial bursitis). VEGF mRNA expression was detected in 40 out of 50 patients by RT-PCR. VEGF mRNA expression was found more frequently in the patients with motion pain (39 out of 41) than in those without motion pain (1 out of 9) with statistical significance (Fisher's test, P < 0.001). Thirty-one out of 33 patients with synovial proliferation showed VEGF mRNA expression, whereas the expression of this transcript was found in 9 out of 17 patients without synovial proliferation. This association with synovial proliferation was also significant (Fisher's test, P = 0.0013). Thirty out of 41 patients with motion pain had synovial proliferation but 3 out of 9 patients without motion pain had synovial proliferation. In all these 30 patients with both motion pain and synovial proliferation, VEGF mRNA expression was detected. This association between motion pain and synovial proliferation was also significant (Fisher's test, P < 0.05). The mean vessel count and area in subacromial bursa expressing VEGF was significantly higher than in those without VEGF (Mann Whitney's U test, P < 0.01). These results suggested that VEGF expression is associated with vascularity, synovial proliferation and shoulder motion pain in the rotator cuff disease.

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.

Vascular endothelial growth factor (VEGF), a major factor mediating tumor stromal angiogenesis, is expressed as five splice variants encoded by a single gene (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206). Recently, we demonstrated that the cell-associated isoform, VEGF189, plays important roles in establishment of human colon and esophageal cancer xenografts. We have established 228 xenografts originating from various human solid tumors. In this study, we investigated the expression patterns of VEGF isoforms in those tumor xenografts by RT-PCR. The isoform patterns were VEGF121/VEGF165 in 27 xenografts (11.8%) and VEGF121/VEGF165/VEGF189 in 201 (88.2%). All human solid tumor xenografts expressed VEGF189 more frequently than primary tumors reported previously. These results suggest that VEGF189 contributes to the successful xenotransplantability of various human solid tumors via augmentation of stromal vascularization.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Two subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(-)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(-)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+)/VEGF-189(-) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189.

Xenografted neoplastic tissues are often utilized to study biology of human cancer. We studied isoform pattern (VEGF206, VEGF189, VEGF165, VEGF121) of an angiogenic factor VEGF in both esophageal primary cancers and cancer xenografts by RT-PCR analysis. Eighteen of 19 (95%) xenografts showed the cell associated aberrant isoform of VEGF189 at a significantly higher incidence than 19 of 31 (61%) primary cancers (P=0.009, Fisher's exact test). These observations suggested that the aberrant isoform of VEGF189 mRNA affects the xenotransplantability of human esophageal cancer.

Vascular endothelial growth factor (VEGF) is a well known factor that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and 121, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF121. The isoform patterns were classified into three groups: type 1, VEGF121; type 2, VEGF121 + VEGF165; type 3, VEGF121 + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence (12 of 16, 75%) than those without liver metastasis (20 of 45, 44%) (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis (P < 0.01, Cox-Mantel). The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.

Vascular endothelial growth factor (VEGF) has four isoforms (VEGF121, VEGF165, VEGF189, VEGF206). We examined the expression patterns of VEGF isoforms in non-small cell lung cancer (NSCLC) by RT-PCR. The cell-associated isoform VEGF189 was more frequently expressed in NSCLC (90.5%) than in extraneoplastic lung tissue (57.6%, p=0.00004). Immunohistochemical examination confirmed VEGF protein expression in 33 of 76 NSCLC expressing VEGF189 (V189p+ group). The V189p+ group showed significantly poorer prognosis than those without VEGF189 mRNA and protein (V189p- group, p=0.01722). These results suggest that expression of the cell-associated isoform VEGF189 is closely associated with progression of NSCLC.