Ubiquitin-specific peptidase 20 constrains endothelial cell activation and angiogenic sprouting through NF-κB inhibition

Nuclear factor-{kappa}B (NF-{kappa}B) mediates inflammation-driven angiogenesis, which promotes the growth of atherosclerotic plaques and tumors. The deubiquitinase ubiquitin-specific peptidase 20 (USP20) suppresses NF-{kappa}B activation in vascular smooth muscle cells (SMCs) and attenuates atherosclerosis. Phosphorylation of USP20 at Ser334 increases NF-{kappa}B signaling in SMCs. However, the role of USP20 in endothelial cells (ECs) remains undefined. We therefore tested whether USP20 activity diminishes NF-{kappa}B signaling in ECs and thereby diminishes angiogenesis. Cytokine-induced NF-{kappa}B activity was elevated in primary ECs isolated from Usp20-/- mice as compared with ECs from wild-type (WT) mice. Concordantly, cytokine-induced NF-{kappa}B activity was elevated in mouse coronary ECs (MCECs) expressing dominant-negative USP20 (USP20-DN) or phospho-mimetic USP20 (USP20-S334D), but blunted in MCECs expressing WT USP20 (USP20-WT) or phospho-resistant USP20 (USP20-S334A). MCEC migration and spheroid sprouting/angiogenesis were increased with overexpression of USP20-DN or USP20-S334D, but decreased with overexpression of USP20-WT or USP20-S334A. Angiogenesis assessed by the aortic ring assay was significantly increased in Usp20-/- aortas and suppressed by TPCA-1, an inhibitor of NF-{kappa}B signaling. Angiogenesis was augmented in Usp20-S334D aortic rings but reduced in Usp20-S334A aortic rings. By screening known angiogenic factors, we identified matrix metalloproteinase 3 (MMP-3), a transcriptional target of NF-{kappa}B, as a gene that is also regulated by USP20 expression. Inhibiting MMP-3 reduced angiogenic sprouting in the Usp20-/- mouse aortic rings. We conclude that USP20 expression inversely correlates with the extent of angiogenesis, and that inhibiting USP20 Ser334 phosphorylation could be a useful strategy to constrain inflammation-driven angiogenesis in pathological conditions like solid tumor malignancies and atherosclerosis.