Fig 2: Quantified area of Sudan IV positive plaque area in the arch of the aorta (a). Data shows median ± 5–95% CI. Box shows the interquartile interval (25–75% percentiles); * p < 0.05 vs. vehicle/vector treated pair; # p < 0.05 vs. diabetes. Micrograph of the aortic arch for each group (b): (A)—Control + Vehicle; (B)—Ace2/ApoE DKO + Vehicle; (C)—ACE2/ApoE DKO + rmACE2; (D)—Diabetes + Vehicle; (E)—Diabetes + rmACE2; (F)—Control + Vector; (G)—Control + ACE2 minicircle; (H)—Diabetes + Vector; (I)—Diabetes + ACE2 minicircle, n = 6–10 mice per group.

Fig 3: The relationship between ACE2 expression in AMs and susceptibility to SARS-CoV-2 infection. (A) BAL-derived cells of non-smokers were treated with 6 µL/well of CoV-2 PsV for 72 h. The infected cells showed green fluorescence. ACE2, IL-1ß, IL-10, IFN-? and CoV-2 PsV of AMs were measured using flow cytometry. (B) The comparison of ACE2 of between CoV-2 PsV infected and non-infected AMs. (C) The comparison of IL-1ß, IL-10, and IFN-? between AMs before and 72 h after CoV-2 PsV inoculation. (D) Comparison of IL-1ß, IL-10, and IFN-? between CoV2-PsV-infected AMs and uninfected AMs. (E) BAL-derived cells of non-smokers were incubated with 5 µg/ml of CoV-2 Sp for 24 h, and the ACE2, IL-1ß, IL-6, and IFN-? MFI of AMs were measured using flow cytometry. The columns and error bars represent the means and SEMs of experiments. The statistically significant differences between groups are indicated with *, ** and *** (*P < 0.05, **P < 0.01, ***P < 0.005, paired t test).

Fig 4: The effect of ROS on ACE2 expression in AMs. (A) To estimate ROS production of AMs, CD11c+Siglec-F+ cells were isolated from lung homogenates of wild-type and Cybb–/– mice using MASC. The ROS production in AMs (CD11c+CD45+Siglec-F+) was then measured using flow cytometry. To estimate ACE2 expression, the second MASC was performed to isolate AMs (CD11c+CD45+Siglec-F+ cells) from CD11c+Siglec-F+ cells, and ELISA was used to measure ACE2 expression in AMs. (B,C) The comparison of intracellular ROS (n = 4) (B) and ACE2 expression (n = 3) (C) between CSE-treated wild type and Cybb–/– AMs. The AMs were treated with 1% CSE for 30 min before measuring ROS production and 24 h before measuring ACE2 expression. Pooled AMs from 5 mice were used for each independent experiment. (D) The comparison of intracellular ROS and ACE2 expression between wild-type and Cybb–/– AMs after treatment of 1 mM H2O2 for 15 min or 10 mM CoCl2 for 60 min. Pooled AMs of 5 mice were used for each independent experiment. (E) The comparison of intracellular ROS and ACE2 expression of AMs between H2O2-treated and control group. The dose and duration of H2O2 treatment were the same as in Fig. 5D. The columns and error bars represent the means and SEMs of experiments. The statistically significant differences between groups are indicated with * and ** (*P < 0.05, **P < 0.01, paired t test).

Fig 5: Correlation between ACE2 expression in AMs and clinical factors. (A) Smoking pack-year (n = 36), (B) Age (n = 56), (C) BMI (n = 56), (D) FEV1 (n = 27), (E) measured FEV1/predicted FEV1 (n = 27) and (F) FEV1/FVC (n = 27). ACE2 MFI was log2 transformed. Correlation was determined using Spearman’s correlation test.