Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific...

Nuclear factor-κB (NFκB) mediates inflammation-driven angiogenesis, which promotes growth of atherosclerotic plaques and tumors. The deubiquitinase ubiquitin-specific peptidase 20 (USP20) suppresses NFκB activation in vascular smooth muscle cells (SMCs) and attenuates atherosclerosis, but the role of USP20 in endothelial cells (ECs) was undefined. We tested whether USP20 activity diminishes NFκB signaling in ECs and thereby diminishes angiogenesis. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 ^-/- mice as compared with ECs from wild-type (WT) mice. Similarly, cytokine-induced NFκB activity was elevated in mouse coronary endothelial cells (MCECs) expressing catalytically inactive USP20 (USP20-DN) or phospho-mimetic USP20(S334D). In contrast, cytokine stimulation of MCECs expressing WT USP20 (USP20-WT) or phospho-resistant USP20(S334A) produced blunted NFκB activity. Assessed by scratch-wound healing and spheroid assays, migration and angiogenesis of MCECs, respectively, were (a) increased by USP20-DN or USP20(S334D), and (b) decreased by USP20-WT or USP20(S334A). Angiogenesis assessed by the aortic ring assay was significantly increased in Usp20 ^-/- mice and was suppressed by TPCA-1, an inhibitor of NFκB signaling. Angiogenesis was augmented in USP20(S334D) mouse aortic rings but reduced in USP20(S334A) mice. By screening known angiogenesis factors, we identified matrix metalloproteinase 3 (MMP3), a transcriptional target of NFκB, as a gene that is also regulated by USP20 expression and Ser334 phosphorylation. Inhibiting MMP3 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 under pathological circumstances, like cancer and atherosclerosis.