Journal: bioRxiv

Article Title: Combustible and electronic cigarette exposures increase ACE2 activity and SARS-CoV-2 Spike binding

doi: 10.1101/2021.06.04.447156

Figure Lengend Snippet: Bronchoalveolar lavage fluids from non-smokers, smokers, and vapers (n=10 each) were concentrated and ACE2 activity was measured using 50 μM specific fluorogenic substrates. Cleaved substrate derived fluorescence as arbitrary unit (AU) following one hour of reaction was reported (A). Human bronchial epithelial cells (HBECs) were cultures at air-liquid interface (ALI) and exposed to smoke from one cigarette per day for one (acute) or four days (chronic). Apical surface was washed with PBS to collect the mucosal secretion and 50 μM fluorogenic substrate was added on the apical surface to evaluate apical ACE2 activity. ACE2 activity on apical side of acute and chronic smoke exposed cultures and corresponding apical washes (B) were reported. Activities are presented as accumulated fluorescence in arbitrary units (AU) of the cleaved products following 10 minutes of reaction. Whole cell lysates were collected from chronically cigarette smoke exposed cultures, and ACE2 and GAPDH protein expressions were evaluated by immunoblotting. Representative blots were shown and GAPDH normalized ACE2 expression in air and smoke exposed cultures as fold change (C) was reported. (D) Acute or chronically smoke exposed cultures were apically exposed to spike pseudovirus suspension and the resulting DsRed marker fluorescence was recorded after three days. Data represented as median, lower and upper quartiles; n=8-9 per group; p-values were provided on the graph.

Article Snippet: We measured cleavage of an ACE2-specific fluorogenic peptide (Vivitide, SFQ-3819-PI) by microplate reader to evaluate ACE2 activity as described ( ). sACE2 activity was significantly increased in BALF from both smokers and vapers compared to age-matched non-smokers , indicating heightened vulnerability towards SARS-CoV-2 infection in both groups.

Techniques: Activity Assay, Derivative Assay, Fluorescence, Western Blot, Expressing, Marker