The Alternatively Spliced Anti-Angiogenic Family of VEGF Isoforms VEGF_xxxb in Human Kidney Development

Abstract

Background/Aim: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGF_xxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGF_xxxb, generated by distal splicing in exon 8 (exon 8b). VEGF₁₆₅b, the first described exon 8b-containing isoform, antagonises VEGF₁₆₅ and is anti-angiogenic in vivo. Methods: Using VEGF_xxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF₁₆₅b on renal endothelial and epithelial cells. Results: VEGF_xxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF₁₆₅b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGF_xxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. Conclusion: These results show that the anti-angiogenic VEGF_xxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGF_xxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGF_xxx isoforms.

Overexpression of VEGF165b in Podocytes Reduces Glomerular Permeability

“…This is true for VEGF 165 5,22 and VEGF 165 b, 20 which, in the context of glomerular epithelial cell survival, have similar properties. 5,20 There is mounting evidence that podocyte-derived VEGF-A plays a crucial role in maintaining the filtration barrier, and although its specific role in an established glomerulus is incompletely understood, multiple roles of glomerular VEGF and multiple VEGF isoforms with widely contrasting properties 14 perhaps go some way toward explaining the seeming contradictions in the literature. Eremina et al 6 were the first to support the notion that an optimal "dose of glomerular VEGF" was required to underpin the normal glomerular phenotype (for review, see reference 40).…”

“…We previously showed that at least some of the VEGF expression at the S-shape stage of glomerular development is VEGF xxx b. 20 Of note, in rodent development at least, the earliest fenestrations have diaphragms 24 that may reform in association with injury. 24 The lack of an effect on PV-1 suggests that the podocyte-VEGF 165 b phenotype may not simply relate to changes in the expression of this endothelial glycoprotein, but, clearly, further studies including immunogold may clarify the situation.…”

“…16,20 The contrasting properties of the two VEGF isoform families is striking. VEGF 165 b has been shown in receptor-binding studies, in in vitro endothelial proliferation and migration assays, in ex vivo isolated resistance vessel myograph studies, and in in vivo neovascular and tumor growth models to inhibit the actions of VEGF 165 .…”

“…Replacement of 8a by exon 8b in the VEGF xxx b family produces peptides that are antiangiogenic, inhibit permeability chronically, and reduce rather than promote tumor growth. 13, [15][16][17][18][19][20] Alternative splicing of ex- The gDNA is digested with EcoRI, and a probe made from the cDNA is used to generate the transgene. There was a single EcoRI site in the 5Ј end of the insert, so multiple insertions should generate multiple different-sized bands in the transgene.…”

“…VEGF 165 , for example, is neuroprotective, 21 and both VEGF 165 and VEGF 165 b have human podocyte cytoprotective properties. 5,20,22 In the context of established proangiogenic, propermeability properties of VEGF 165 (and the murine equivalent VEGF 164 ), the characterized phenotype of constitutive podocyte-specific VEGF 164 transgenic overexpressing animals, 6 and recent in vitro data suggesting that VEGF 165 b reduces VEGF 165 -induced human endothelial monolayer permeability, 20 in addition to being antiangiogenic in vivo, 14 here we describe the derivation of heterozygous and homozygous transgenic animals that constitutively overexpress VEGF 165 b in podocytes to address the hypothesis that sustained expression of exon 8b containing VEGF peptides will produce a distinct phenotype from transgenic animals overexpressing exon 8a-containing peptides.…”