Targeting NRP1 with EG00229 induces neurovascular permeability

NRP1 is a therapeutic target for inhibiting vascular endothelial growth factor (VEGF)-induced blood vessel dysfunction. The small molecule EG00229 was designed to inhibit VEGF binding to NRP1 and reduce pathological blood vessel growth. However, it is unknown whether EG00229 could also be used to reduce VEGF164-induced vascular leakage, which often exacerbates ischemic diseases due to VEGF upregulation. Here, we show that prior treatment with EG00229 prevents VEGF164-induced vascular permeability signalling, but, unexpectedly, also find that EG00229 increased rather than inhibited vascular leakage. Thus, EG00229 increased vascular leakage either when added alone or concurrently with VEGF164, both in perfused retinal explants and across primary brain EC monolayers. This EG00229-induced vascular leakage was not an off-target effect, because it relied on endothelial NRP1 expression and NRP1’s VEGF164 binding pocket, yet was independent of VEGFR1 and VEGFR2. Moreover, EG00229 activated molecular events typical of VEGF164-induced paracellular permeability, including p38 MAP kinase (p38) and SRC family kinase (SFK) phosphorylation as well as CDH5 rearrangement in endothelial junctions. Investigating EG00229-induced signalling therefore helps elucidate NRP1-dependent mechanisms of paracellular permeability induction and might help identifying new approaches to modulate the neurovascular barrier.