Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

Abstract

SUMMARYThe formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa affected endothelial cell migration and proliferation. Surprisingly, mutations in vegfab specifically reduced endothelial cell proliferation. Analysis of downstream signaling revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutants. The cell cycle inhibitor cdkn1a/p21 was upregulated in vegfab deficient embryos. Accordingly, reducing cdkn1a/p21 restored endothelial cell proliferation. Together, these results suggest that extracellular matrix bound Vegfa acts through PI3K signaling to specifically control endothelial cell proliferation during angiogenesis independently of MAPK (ERK) regulation.

Temporally and regionally distinct morphogenetic processes govern zebrafish tail fin blood vessel network expansion

“…Embryos were transferred to tanks in the zebrafish facility and raised until further analysis. Previously described zebrafish lines were Tg(-0.8flt1:RFP) hu5333 (Bussmann et al, 2010), TgBAC(pdgfrb:gal4ff) ncv24 (Ando et al, 2016), Tg(TP1bglob:VenusPEST) s940 (Ninov et al, 2012), Tg(TP1bglob:H2B-mCherry) s939 (Ninov et al, 2012), Tg3(fli1:LIFEACT-EGFP) mu240 (Hamm et al, 2016), Tg(fli1a:nEGFP) Y7 (Roman et al, 2002), Tg(gata1:dsRed) sd2 (Traver et al, 2003), TgBAC(cxcr4a:YFP) mu104 (Xu et al, 2014), Tg (flt4:citrine) hu7135 (van Impel et al, 2014), Tg(kdrl:EGFP) s843 (Jin et al, 2005), kdrl hu5088 (Bussmann et al, 2010), vegfaa mu128 (Lange et al, 2022). Some of the transgenic lines were kept in a casper (roy, nacre) double mutant background (White et al, 2008) to increase clarity for imaging due to loss of pigment cells.…”