Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2)

C-type lectin family 14, member A (CLEC14A) is a single-pass transmembrane glycoprotein that is overexpressed on tumour endothelial cells. CLEC14A promotes sprouting angiogenesis and modulates endothelial function through interactions with extracellular matrix proteins. Here we show CLEC14A is cleaved by Rhomboid like 2 protein (RHBDL2), one of three catalytic mammalian rhomboid like (RHBDL) proteases, but is not cleaved by RHBDL1 or 3. Site directed mutagenesis of CLEC14A identified the precise cleavage site by RHBDL2 and targeted small interfering RNAs to knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2 fold, whereas addition of recombinant cleaved CLEC14A inhibited sprouting of human and murine endothelial cells 3 fold in several in vitro models. The in vivo role of cleaved CLEC14A in angiogenesis was assessed using the rodent subcutaneous sponge implant model and we found that CLEC14A protein inhibited vascular density by more than 50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data shows that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.