Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma

Tumor ascites fluids from guinea pigs, hamsters, and mice contain activity that rapidly increases microvascular permeability. Similar activity is also secreted by these tumor cells and a variety of other tumor cell lines in vitro. The permeability-increasing activity purified from either the culture medium or ascites fluid of one tumor, the guinea pig line 10 hepatocarcinoma, is a 34,000- to 42,000-dalton protein distinct from other known permeability factors.

Vascular endothelial growth factor (VEGF)mRNA undergoes alternative splicing events that generate four different homodimeric isoforms, VEGF121, VEGF165, VEGF189, or VEGF206. VEGF121 is a nonheparin-binding acidic protein, which is freely diffusible. The longer forms, VEGF189 or VEGF206, are highly basic proteins tightly bound to extracellular heparin-containing proteoglycans. VEGF165 has intermediate properties. To determine the localization of VEGF isoforms, transfected human embryonic kidney CEN4 cells expressing VEGF165, VEGF189, or VEGF206 were stained by immunofluorescence with a specific monoclonal antibody. The staining was found in patches and streaks suggestive of extracellular matrix (ECM). VEGF165 was observed largely in Golgi apparatus-like structures. Immunogold labeling of cells expressing VEGF189 or VEGF206 revealed that the staining was localized to the subepithelial ECM. VEGF associated with the ECM was bioactive, because endothelial cells cultured on ECM derived from cells expressing VEGF189 or VEGF206 were markedly stimulated to proliferate. In addition, ECM-bound VEGF can be released into a soluble and bioactive form by heparin or plasmin. ECM-bound VEGF189 and VEGF206 have molecular masses consistent with the intact polypeptides. The ECM may represent an important source of VEGF and angiogenic potential.

Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.