Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein.
Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF165b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123-4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF165 (stimulatory) and VEGF165b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF189 mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF165 to VEGF165b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF165b siRNA to 20+/-11% of the level of mock-transfected cells (P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109+/-8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF165b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.
Cui
Tai-Gen
TG
Microvascula Research Laboratories, Dept. of Physiology, Preclinical Veterinary School, Univ. of Bristol, Southwell St., Bristol BS2 8EJ, UK. Dave.Bates@bris.ac.uk
Foster
Rebecca R
RR
Saleem
Moin
M
Mathieson
Peter W
PW
Gillatt
David A
DA
Bates
David O
DO
Harper
Steven J
SJ
D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D007536:Isomerism; D007678:Kidney Glomerulus / Q000166:cytology* / Q000502:physiology*; D018919:Neovascularization, Physiologic / Q000502:physiology; D010641:Phenotype; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A