UCLA

Throughout the vascular tree, endothelial cells function as the interface between circulation and underlying tissues. They act not only as gatekeepers but are also designed to gauge and adapt to circulating factors for regulation of vascular tone, development, and repair. The severity of many diseases, such as cardiovascular disease and diabetes, hinge on endothelial function and the ability to initiate vascularization of hypoxic tissues. Vascular morphogenesis requires a delicate molecular gradient of Notch signaling in endothelial cells to pattern correct angiogenic sprouting. This process is controlled, at least in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (Jag1) expression is compartmentalized in the vascular plexus remains unclear. Understanding the molecular regulators for this process can help our understanding of drivers toward endothelial dysfunction. Here, we showed that mRNA decay protein Zinc-finger protein 36 (ZFP36) is robustly induced in vitro in various human primary endothelial cells (ECs) downstream of canonical vascular endothelial growth factor (VEGF) signaling. This induction, observed at the mRNA and protein level, is uniquely specific to ZFP36 and not shared by other family members ZFP36L1 nor ZFP36L2, which have already been linked to vascular development. This induction was also observed to be primarily facilitated through VEGF receptor 2 (VR2) as receptor inhibition blunted ZFP36 induction. We identified Jag1 mRNA as direct target of zinc finger protein 36 (ZFP36) via crosslinking-immunoprecipitation and verified conserved Jag1 regulation in human primary ECs. Human ECs lacking ZFP36 achieved with CRISPR KO, displayed high levels of JAG1 and increased angiogenic sprouting in vitro. Similarly, in mice with tamoxifen induced endothelial specific vascular endothelial-cadherin Cre-mediated KO of Zfp36, endothelial cells displayed mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front altered Notch1 signaling, increasing the number of tip cells; a phenotype that was rescued by crossing the EC specific Zfp36 KO with Jag1 KO mice to achieve haploinsufficiency of Jag1. Additionally, we tested the significance of ZFP36 induction in the broad context of molecular drivers of endothelial plasticity: metabolism, kinase signaling, and shear stress. We were unable to confirm several metabolic targets identified in mouse embryonic fibroblasts (MEFs) in our human primary ECs in vitro. However, conserved sensitivity to changes in kinase activity, confirmed through inhibitor studies, suggests that these discrepancies in targets could be a result of cell specific targets for ZFP36 mRNA binding and regulation. Microenvironment can further play a role in ZFP36 mediated endothelial transcript regulation as we show that shear stress is able to suppress overall ZFP36 expression. Our findings reveal an important feedforward loop, whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.