MDM2 inhibitors counteract inflammation by stabilization of ACE2 in vitro

Angiotensin-converting enzyme 2 (ACE2) is a cell surface receptor that converts angiotensin II to angiotensin-(1-7), which has been identified for its anti-inflammatory activity. In agreement, ACE2 has been reported to be downregulated under inflammatory conditions associated with cytokines storm, such as during Covid-19 disease and sepsis. Since inflammation is known to induce endothelial cell dysfunctions associated with vascular complications in patients, the aim of our work was to investigate an efficient treatment to restore ACE2 levels and ameliorate endothelial functions. In a model of alveolar epithelium (A549-hACE2), we preliminarily observed, by westing blotting and immunofluorescence analysis, an efficient upregulation of ACE2 protein after treatment with the MDM2-inhibitor Nutlin-3a, contrasting MDM2-depending ubiquitination of ACE2 protein. In a model of endothelium (HUVEC), we furthermore tested Nutlin-3a both in presence and in absence of inflammatory stimuli induced by TNFα, high glucose or LPS to mimic acute or chronic pathologies. To this end, cells were treated with several concentrations of Nutlin-3a alone or in presence of inflammatory stimuli. After 24 and 48 hours of treatments, cells were collected for analysis of ACE2 levels by western blotting and biological effects mediated by Nutlin-3a-dependent p53 induction (cell viability, cell cycle, apoptosis and protein levels of p53-gene targets). In parallel supernatants were collected for secreted cytokines analysis. Results showed a concentration-dependent upregulation of ACE2, p53 and MDM2 proteins in Nutlin-3a treated cells together with induction of a cell cycle block, low levels of apoptosis, both in physiological and inflammatory environments. Moreover, Nutlin-3a mitigates inflammation by the reduction of IL-6 release respected to cells exposed to inflammatory stimuli. These results indicate an efficient ACE2 stabilization and control of inflammation associated with MDM2 inhibitors, suggesting an efficient tool with therapeutic potential for vascular disorders associated with inflammation.