Cell differentiation and aging accompanied by depletion of the ACE2 protein

Figure 1. ACE2 in lung A549 cells and mouse embryonic heart sections. (A) 3D-projection of images obtained by confocal microscopy. The ACE2 protein (red fluorescence) was visualized by immunofluorescence. (B) ACE2 protein (green fluorescence) expression in A549 cells. (C) ACE2 expression in mouse heart section (red fluorescence) [see right atrium (RA), left atrium (LA), aorta (AO) and pulmonary trunk (PT), right ventriculus (RV), and left ventriculus (LV) or intraventricular septum (IVS)]. The blue ellipse shows the anatomical part associated with the aorta.

Figure 2. ACE2 and renin levels in mouse hearts, brains, lungs, and kidneys isolated from young and old animals. Adult mouse hearts were sectioned following the following anatomy: ventricular parts (V), atrium (A), and vessels associated with the aorta (AO). The levels of selected proteins were studied by western blotting, and the data were normalized to the total protein levels. The level of the ACE2 protein was examined in (A) mouse hearts, (B) brains, (C) lungs, and (D) kidneys isolated from young and old male and female mice. Quantification was performed using ImageJ software. Red asterisks indicate a decrease in the protein level; blue asterisks show a high ACE2 protein level compared with other samples analyzed in panel B. The nonparametric Mann–Whitney test was used for statistical analysis. Asterisks (*) indicate α=0.05.

Figure 3. Downregulation of ACE2 in mESCs undergoing differentiation into cardiomyocytes. (A) western blot analyses were performed in HDAC1 wt (D3 wt) and HDAC1-depleted (dn) mES cells, non-differentiated and differentiated into cardiomyocytes. The data show the levels of the ACE2 protein and renin normalized to the total protein levels. (B) Quantification of the protein levels, as assessed by western blotting, was performed using ImageJ software; the bar chart shows the comparison of protein levels in differentiated wt mESCs and differentiated HDAC1 dn mESCs. (C) The graphical illustration shows the comparison of ACE2 and renin (separately) in differentiated wt mESCs and differentiated HDAC1 dn mESCs.

Figure 4. Distribution of ACE2 (green) in wt and HDAC1-depleted mES cells, non-differentiated and differentiated into cardiomyocytes. Immunofluorescence analyses were performed in (A) HDAC1 wt mESCs, (B) HDAC1 dn mESCs, (C) α-actinin (red)-positive cardiomyocytes generated from HDAC1 wt mESCs, and (D) α-actinin (red)-positive cardiomyocytes generated from HDAC1 dn mESCs. DAPI (blue) was used as a counterstain. (E) The nucleo/cytoplasmic ratio of the level of ACE2 is shown for HDAC1 wt mESCs. (F) The nucleo/cytoplasmic ratio of the level of ACE2 is shown for HDAC1 dn mESCs.

Figure 5. Level of the ACE2 protein studied in explanted embryonic hearts treated by HDAC inhibitors. (A) In nontreated, TSA (trichostatin A), and SAHA (suberoylanilide hydroxamic acid)-treated explanted mouse hearts at stage e15, the ACE2 protein level was analyzed. (B) Quantification of the protein levels, as assessed by western blotting, was performed using ImageJ software.

Figure 6. Levels of the ACE2 and renin in distinct cell types. The analysis was performed in mouse embryonic stem cells (mESCs) differentiated into cardiomyocytes, human embryonic kidney cells HEK293, human intestinal adenocarcinoma HT29 cells (nontreated and differentiated using the HDAC inhibitor sodium butyrate [NaBt]), and human lung adenocarcinoma A549 cells. The asterisks show a statistically significant change in the protein levels. The nonparametric Mann–Whitney test (STATISTICA software) was applied for the analysis; data with an asterisk show statistically significant differences between the tested samples (α=0.05).

Figure 7. ACE2 and renin levels after distinct treatments of A549 cells. (A, B) The effect of the following drugs was analyzed: γ-irradiation, PARP inhibitor (olaparib), HDAC inhibitor (SAHA), and vitamin D2. © Level of ACE2 in dexamethasone-treated A549 cells. The data were normalized to the total protein levels, as shown in the bar chart. Quantification of the protein levels, as assessed by western blotting, was performed using ImageJ software. The blue asterisk indicates a slight increase in the ACE2 level, and orange asterisk shows the renin level decrease, in cells treated with vitamin D2 (panel B). The orange asterisk in panel C indicates renin upregulation caused by dexamethasone treatment. The data were analyzed by the nonparametric Mann–Whitney test.

Figure 8. The level of ACE2 is unchanged in the olfactory bulbs of smoking animals. (A) The experiments were performed in the experimental chamber-simulated closed room with regulated ventilation. (B) The level of ACE2 was studied by western blotting in 2- or 4-month-old mice of both genders (smoking and nonsmoking groups). The data from 4-month-old mice were quantified by ImageJ software. The level of ACE2 was studied in explanted olfactory bulbs. (C) The level of ACE2 was analyzed by western blotting in the explanted lungs of mice exposed to cigarette smoke. The nonparametric Mann–Whitney test was used for statistical analysis (* indicates α=0.05).