ACE2 up-regulation by MDM2 inhibition reduces inflammation and monocytes adhesion in HUVEC

Endothelial cells play a pivotal role in vascular homeostasis and innate immunity, modulating inflammation by adapting their activation status and recruiting immune cells. Reduced expression of the cell surface receptor Angiotensin-Converting Enzyme 2 (ACE2) has been identified under inflammatory conditions; therefore, our work aimed to explore a potential treatment to restore ACE2 levels and rescue cells from inflammation. Recent studies have suggested that Murine Double Minute 2 (MDM2), a well-known p53 inhibitor, can control ACE2 ubiquitination. To pursue our goal, we tested nutlin-3a, a known MDM2 inhibitor, to avoid ACE2 degradation and repristinate homeostasis in a model of endothelial inflammation based on Human Umbilical Vein Endothelial Cells (HUVEC) treated with exogenous inflammatory stimuli. Nutlin-3a at different concentrations was used alone or in the presence of 1ng/mL TNFα or 5ng/mL LPS, and biological events on cell behavior were analyzed at 24 and 48 hours’ time points after treatment, with respect to controls. ACE2 protein level was investigated by western blotting and immunofluorescence. In parallel, the impact on cellular proliferation and migration was assessed by xCELLigence real-time assays; cell cycle and apoptosis were evaluated by cytofluorimetric analysis. Protein levels of p53 and MDM2 were assessed by western blotting. For the evaluation of inflammation status, pro-inflammatory cytokines release and monocytes (THP-1) adhesion to cultures were investigated. Nutlin-3a efficiently upregulated ACE2 protein and induced p53-dependent pathway activation, leading to reduced cell proliferation and cell cycle block without significant apoptosis. These effects were also associated with the inhibition of HUVEC cell migration. Interestingly, nutlin-3a decreased IL-6 release and impaired monocyte adhesion to endothelium. These findings indicate that MDM2 is an efficient target for anti-inflammatory therapies and nutlin-3a, used as proof-of-concept MDM2 inhibitor molecule, can restore a physiological condition in inflamed endothelium upregulating ACE2 protein level.