Simultaneous in vivo Quantitation of Vascular Endothelial Growth Factor mRNA Splice Variants

Abstract

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. In vivo expression of four different VEGF isoforms, consisting of 121, 165, 189 or 206 amino acids, has been found in the human organism, with all isoforms arising from a single gene by alternative mRNA splicing. We developed an assay for simultaneous quantitation of VEGF isoform expression using competitive polymerase chain reaction (PCR). RNA was isolated from cells, reverse transcribed to cDNA and coamplified with a synthetical competitor DNA using VEGF specific primers. Amplification products were analyzed electrophoretically and concentration of VEGF transcripts calculated. Concentration of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts was quantitated as above, VEGF gene activity is presented as ratio VEGF mRNA to GAPDH mRNA. Using this assay, we were able to detect and quantitate in vivo expression of VEGF121, VEGF165 and VEGF189 by human peripheral blood mononuclear cells (PBMC). These are the first quantitative data of in vivo VEGF expression by PBMC, suggesting a role for them in the maintenance of the vasculature.