ACE2 expression is affected by Angiotensin‐II type I receptor (AT 1 R) and kinin B1 receptor (B 1 R) in primary human aortic endothelial cells

Abstract

Angiotensin Converting Enzyme type 2 (ACE2) acts as a critical player in the renin-angiotensin system (RAS) by converting Angiotensin (Ang)-II to Ang-(1-7) and in the kallikrein-kinin system (KKS) by converting Des-Arg9-Bradykinin (DABK) into inactive peptide. We previously reported in Neuro-2A cells that Ang-II mediates ACE2 internalization through its type 1 receptor (AT1R) via ubiquitination and ultimately degradation in lysosomes. In addition, we recently identified UBR1 and BRCC3, as potential ubiquitinase and deubiquitinase, respectively that may modulate ACE2 ubiquitination and internalization in hypertensive conditions. Here, we hypothesize that both AT1R and the bradykinin B1 receptor (B1R) promote ACE2 ubiquitination and degradation in human aortic endothelial cells. Protein expression for ACE2, UBR1 and BRCC3 was assessed using capillary western in endothelial cells pre-treated with media (control group), an AT1R antagonist (Telmisartan,10 μM) or a B1R antagonist (R-715, 10 μM), one hour prior to stimulation with a B1R agonist DABK (1 μM) or Ang-II (100 nM) for 4 hours. Also, immunocytochemistry against ACE2 along with a plasma marker was performed to determine the localization of ACE2. ACE2 protein expression was significantly decreased with Ang-II (~50 %) or DABK (~40 %) treatments in comparison to untreated cells (n=6; Dunnett's test, p<0.05), while Telmisartan and R-715 fully blocked the effects of Ang-II and DABK. In addition, UBR1 protein level was significantly increased with Ang-II (~25 %) or DABK (~50 %) treatments (n=3; Dunnett's test, p<0.05). Moreover, treatment with DABK and Ang-II produced a synergistic effect resulting in a 4-fold increase in UBR1 expression. On the other hand, both agonists produced a significant reduction of BRCC3 (n=3; Dunnett's test, p<0.05) while simultaneous administration resulted in a paradoxical 3-fold upregulation. Immunocytochemistry showed that Ang-II and DABK treatments resulted in a similar reduction of ACE2 colocalization on the plasma membrane. In summary, stimulation with DABK or Ang-II increases the protein expression of the ubiquitinase UBR1 while decreasing the deubiquitinase BRCC3. These effects were associated with a reduction of ACE2 expression on the plasma membrane, mediated by both AT1R and B1R. These data suggest that hypertension and inflammation may contribute to the loss of ACE2 compensatory activity on the plasma membrane in endothelial cells. Therefore, designing treatments targeting AT1R and B1R as well as intracellular components such as UBR1 and BRCC3, involved in the regulation of ACE2 expression may be effective for the treatment of cardiovascular and pulmonary diseases related to ACE2.