Cigarette Smoke Regulates Endothelial ACE2 Expression in the Lung

Abstract

Rationale: Angiotensin-converting enzyme 2 (ACE2) is a vasoactive enzyme involved in regulation of vascular tone and blood pressure by reducing angiotensin II and increasing ang(1-7). It is also implicated in the pathogenesis of coronaviruses including SARS-CoV-2. Epidemiological reports differ in implicating cigarette smoking as a risk factor for SARS-CoV-2 infection (COVID-19). Previous studies have been conflicting regarding the implications of cigarette smoke exposure on ACE2 signaling. We hypothesized that cigarette smoke exposure will increase ACE2 expression and impair endothelial cell function. Methods: Female 8-week-old A/J mice were randomly assigned to either air exposure or 48 minutes per day, 5 days per week of cigarette smoke exposure. Mainstream whole-body cigarette smoke exposure was delivered by the SCIREQ “InExpose” smoking system with standard 3R4F research cigarettes. Mouse were sacrificed at 1 and 12 weeks of smoke exposure, and lungs were homogenized and subjected to ACE2 ELISA (Abcam). To investigate the effect of smoking on ACE2 expression and endothelial barrier function, serum starved human pulmonary microvascular endothelial cells (PMVECs) were exposed to cigarette smoke extract (CSE). CSE was prepared at a concentration of 1 cigarette/5 ml in serumfree DMEM and quiescent PMVECs were treated with 1% CSE, 3% CSE or vehicle. Cells were processed for real-time RT-PCR and ELISA 4 hours later, assessment of apoptosis, or underwent TEER to assess endothelial cell barrier function. Results: Lung tissue ACE2 levels were significantly elevated following 1-week of cigarette smoke-exposure. This increase was accompanied by increased macrophage count in bronchoalveolar lavage. Interestingly, at 12-weeks of cigarette smoke-exposure, lung ACE2 was reduced by 15% response. Chronic cigarette smoke-exposure was accompanied by increased right ventricular systolic pressure and Fulton index. In PMVEC models, CSE dose-dependently increased ACE2 mRNA and protein expression. This was accompanied by altered EC barrier function and EC apoptosis. Conclusions: The dose and duration of cigarette smoke exposure affects ACE2 signaling, leading to altered apoptosis and endothelial cell barrier function. These findings have implications for SARS-CoV-2 pathogenesis as well as for furthering our understanding of the effects of smoking on vascular health.