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Abstract: PO0919

Nogo-B and Soluble Nogo-B Modulate VEGFA/VEGFR2 Signalling in Glomerular Endothelial Cells

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic


  • Ricciardi, Carlo Alberto, King's College London Faculty of Life Sciences and Medicine, London, London, United Kingdom
  • Hernandez, Ivan, King's College London Faculty of Life Sciences and Medicine, London, London, United Kingdom
  • Long, David A., University College London, London, London, United Kingdom
  • Gnudi, Luigi, King's College London Faculty of Life Sciences and Medicine, London, London, United Kingdom

Nogo-B is an endoplasmic reticulum protein present either as a full-length or circulating soluble isoform (sNogo-B) corresponding to the first ~200aa of the N-terminus. Nogo-B is expressed in the vasculature and in glomerular endothelial cells (GECs) and downregulated in diabetic glomeruli. Overexpression of sNogo-B ameliorates diabetic glomerulopathy, but the biological mechanisms are unknown. We hypothesise that, in GECs, Nogo-B and/or sNogo-B modulate VEGFA/VEGFR2 signalling and vascular remodelling.


Nogo-B deficient human GECs were generated with CRISPR/CAS9 technology. VEGFA signalling was studied in differentiated, serum starved (5 h, FBS 2%) GECs exposed to VEGFA (50 ng/ml) for 5, 10 and 15 min. VEGFR2 phosphorylation (Tyr1175) was assessed with western immunoblotting. Experiments were conducted in GECs transfected with adenoviral vector expressing sNogo-B or control vector. To investigate the role of Nogo-B on GECs survival, Caspase-3/7 activity was utilised as marker of apoptosis in WT and Nogo-B deficient GECs after 5 h incubation in 2% FBS. In vivo Matrigel-angiogenesis assay in wild-type (WT) and Nogo-A/B deficient mice were conducted in parallel.


When compared to WT GECs, Nogo-B deficient cells appeared more elongated with a peripheral distribution of F-actin but maintained expression of endothelial markers such as eNOS and CD31. Phosphorylated VEGFR2/total VEGFR2 ratio was similar in baseline condition in WT and Nogo-B deficient GECs. VEGFA-mediated VEGFR2 phosphorylation (15 min) was observed in WT GECs but not in Nogo-B deficient GECs (p<0.01). In the presence of sNogo-B, there was significant reduction in VEGFA-mediated VEGFR2 phosphorylation in WT GECs (p<0.05). There was no significant effect of sNogo-B on VEGFR2 phosphorylation in Nogo-B deficient GECs.
Apoptosis was higher in Nogo-B deficient GECs when compared to WT ones (p<0.04).
Preliminary work in in vivo Matrigel angiogenesis showed that Nogo-B deficient ECs were unable to form vascular structure when compared to wild-type cells (P<0.05). Presence of sNogo-B blunted the angiogenesis in WT mice.


Nogo-B is required for VEGFA-mediated VEGFR2 phosphorylation and for vascular remodelling (angiogenesis). Overexpression of sNogo-B blunts VEGFA-mediated VEGFR2 phosphorylation. sNogo-B could represent a tool to modulate VEGFA signalling in diseases.