Manipulation of VEGF-A mRNA splicing repertoire as a potential therapeutic strategy in glomerular disease

• Main Author: Stevens, Megan
• Published: University of Bristol 2015
• Subjects: 616.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.682341

Aims: Vascular endothelial growth factor (VEGF-A) is differentially spliced to give two functionally different isoform families, the pro-angiogenic, pro-permeability VEGFxxx and the anti-angiogenic, anti-permeability VEGFxxxb families. Evidence suggests that the balance of pro- and anti-angiogenic isoforms of VEGF-A is key to maintaining normal glomerular function. A switch in splicing of VEGF-A to favour pro-angiogenic, pro-permeability VEGFxxx expression has been documented in many diseases such as cancer and diabetes. Furthermore, VEGF165b has been shown to protect against increases in albuminuria in mouse models of diabetic nephropathy, reduce glomerular water permeability (LpAlVi) in vivo, and be cyto-protective on podocytes in culture. This PhD aimed to investigate the manipulation of VEGF mRNA splicing in models of glomerular injury and test the general hypothesis that constitutive over-expression of VEGF165b under a nephrin promoter is able to rescue the injury phenotype seen in two models of glomerular disease the inducible podocyte-specific VEGF-A knock-out (KO) mouse and the podocyte expressing human diphtheria toxin receptor (Pod-DTR) mouse. The mechanism of action of VEGF16sb in the glomerulus with regards to VEGFR-2 expressed by glomerular endothelial cells (GEnCs) both in vivo and in vitro was also investigated. Results: The inducible podocyte-specific VEGF-A KO mouse had a significant reduction in glomerular VEGF-A mRNA 10 weeks after induction with doxycycline, when they became albuminuric and had an increased glomerular LpAlVi, when compared to wildtype (WT) littermate controls. Constitutive podocyte over-expression of human VEGF165b rescued the inducible podocyte-specific VEGF-A KO phenotype, resulting in reductions in albuminuria and glomerular LpAlVi. Furthermore, VEGF16sb resulted in a reduction in glomerular basement membrane (GBM) thickening observed in the VEGF-A KO mouse after 14 weeks. previous characterization, Pod-DTR mice did not develop albuminuria, podocyte loss or any major glomerular structural abnormalities in our hands, upon treatment with diphtheria toxin (DT) at a range of doses. However, glomerular LpA/Vi was significantly elevated over WT littermate controls, and the pattern of nephrin expression to be altered in these mice. Constitutive podocyte over-expression of human VEGF165b rescued the increased glomerular LpA/Vi and prevented the changes in nephrin expression observed in the Pod-DTR model. VEGF165b was found to increase both the expression and phosphorylation of VEGFR- 2 on GEnCs in vivo and in cultured GEnCs. Conclusions Podocyte VEGF165b over-expression rescues the podocyte-specific VEGF-A KO model and the Pod-DTR model of glomerular disease, suggesting that VEGF165b plays a protective role and that manipulation of the VEGF-A isoform ratio may have therapeutic potential. Podocyte-expressed VEGF16sb is probably acting as a weakipaltial, qualitatively specific agonist to VEGFR-2, expressed on the GEnCs.