No abstract

Background: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II (Ang II), a potent vasoconstrictor, to Ang 1-7 and is also a membrane protein that enables COVID-19 infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of post-translational modification (PTM) of ACE2 in vascular endothelial cells (ECs) is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the PTM of ACE2 via its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH. Methods: Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH. Results: Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH. Conclusions: Maladapted PTM (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.

Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-b (TGF-b), TGF-b receptor 2 (TGFBR2), b-catenin, connective tissue growth factor (CTGF), interleukin-1b (IL-1b), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-b, TGFBR2, b-catenin, CTGF, IL-1b, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses.