Cell differentiation and aging accompanied by depletion of the ACE2 protein

Localization of the ACE2 protein in the cells

The ACE2 protein is mainly located in the cytoplasm, but we also observed fluorescence signals inside cell nuclei. 3D analysis of images from confocal microscopy confirmed the localization of the ACE2 protein inside the cell nucleus (Figure 1A). 3D reconstruction of the nuclear arrangement of the ACE2 protein is shown in lung carcinoma A549 cells, also characterized by a high density of ACE2 in the cytoplasm (Figure 1A, 1B). Additionally, we studied the density of the ACE2 protein in distinct regions of embryonic hearts (at stage e15, e.g., 15 days postconception). We analyzed the following areas of embryonic hearts: right atrium (RA), left atrium (LA), aorta (AO) and pulmonary trunk (PT), right ventriculus (RV), and left ventriculus (LV) or intraventricular septum (IVS). We observed a relatively homogeneous immunofluorescence signal for ACE2 studied in the heart sections. However, the ACE2 level in the regions around the aorta and pulmonary trunk should be considered as an exception (blue ellipse in Figure 1C).

ACE2 level in the tissue of young and old mice

We also studied the ACE2 protein and renin in mouse hearts isolated from young and old adult male and female animals (Figure 2A). Western blot analyses were performed with cell lysates isolated from the ventricular regions of the heart, atrium, and in the anatomical regions around the aorta (AO) and pulmonary trunk (aorta-associated vessels). Interestingly, we observed that the level of ACE2 was the highest in young female aorta regions compared with that in old female aortas. In male aortas, the level of ACE2 was identical to that compared with young and old animals (Figure 2A). When we studied the ACE2 level in mouse brains (olfactory bulbs [OB], hippocampus [HIP], cortex [CXT]), interestingly, the highest level of ACE2 was found in olfactory bulbs compared with that in hippocampi and the brain cortex (Figure 2B). These results were, in many cases, identical in both genders as well as in young and old animals (Figure 2B). In adult mouse lungs, we observed a low level of ACE2 compared with that in mouse embryonic stem cells (mESCs), which were used as reference samples (Figure 2C). However, in adult mouse kidneys, the ACE2 level was very high compared with that in mESCs and higher in male mice than in female mice of the same age (Figure 2D). Notably, old females were characterized by the lowest level of ACE2 protein in the kidneys (Figure 2D). Generally, the comparison of young and old female samples (heart AO, brain OB, HIP, CTX, and kidneys of female mice) showed a decreasing trend in the ACE2 level during aging, particularly in females (Figure 2A–2D).

Downregulation of ACE2 in HDAC1 wt and HDAC dn mouse embryonic stem cells undergoing differentiation into cardiomyocytes

In non-differentiated mESCs (D3 wt), we observed a relatively high level of the ACE2 protein. Compared with HDAC1 (histone deacetylase 1)-depleted mES cells, HDAC1 wt cells were characterized by a higher level of ACE2 (Figure 3A, 3B). Experimentally induced differentiation into cardiomyocytes in both wt and HDAC1 dn cells was accompanied by the downregulation of ACE2 but upregulation of renin (Figure 3A, 3B). According to these results, the ACE2 protein and renin likely work antagonistically (Figure 3B). Importantly, HDAC1-depleted cells were characterized by a late onset of renin upregulation that appeared during cardiomyogenesis (on the 15^th (dd15) and 20^th (dd20) days of differentiation). In HDAC1 wt mESCs, renin started to be upregulated on day 10 (dd10) of differentiation (Figure 3A–3C). Together, we summarize that HDAC1 deficiency affects the properties of both ACE2 and its interacting partner renin.

Additionally, in HDAC1 wt and HDAC1 dn cells, we analyzed the distribution pattern of the ACE2 protein in the cytoplasm and cell nucleus (Figure 4A–4D). In non-differentiated wt and HDAC1 dn mESCs, we observed a relatively low level of ACE2 in the cell nucleus but a high density in the nucleoplasm, particularly at the cell surface, as expected. However, cell differentiation into cardiomyocytes significantly changed the ACE2 density in the cell nucleus and ratio of the ACE2 level measured in the nucleus and cytoplasm (Figure 4E, 4F).

The HDAC inhibitor TSA enhances the level of ACE2 in explanted embryonic hearts

We analyzed explanted embryonic hearts treated with the HDAC inhibitors TSA and SAHA and observed pronounced upregulation of the ACE2 protein in TSA-treated hearts. Interestingly, embryonic hearts were characterized by a significantly higher level of the ACE2 protein than in explanted hearts from adult animals (Figure 5A, 5B). Thus, again, higher levels of ACE2 in embryonic hearts, compared with those in adult tissue, imply that ACE2 upregulation is the main characteristic of tissues of young individuals.

Levels of the ACE2 protein in distinct cell types

By western blotting, we studied the ACE2 protein and its relationship to renin, a prominent ACE2-cooperating partner in distinct cell types (Figure 6). The levels of both ACE2 and renin were analyzed in the following cell lines representing an example of tissue preferentially affected by the SARS-CoV-2 virus: human lung adenocarcinoma A549 cells, human intestinal adenocarcinoma HT29 cells (nontreated and differentiated by the HDAC inhibitor sodium butyrate (NaBt), human embryonal kidney cells HEK293, and mouse embryonic stem cells (mESCs; line D3). In mESCs, we used the differentiation potential of these cells that can be differentiated into cardiomyocytes. We confirmed data from Figure 3A–3C that experimentally induced cardiomyogenesis is characterized by the depletion of ACE2, but the renin level was increased (Figure 6). Surprisingly, intestinal HT29 cells were characterized by the appearance of additional ACE2 fragments, whose levels were increased when the density of the main ACE2 fragment was slightly decreased by NaBt treatment initiating the differentiation of these cells into mature enterocytes (Figure 6; see differentiation protocol in [13]). Interestingly, the highest level of the renin protein occurred in intestinal cells and lung carcinoma cells but not in embryonic kidney cells HEK293 (Figure 6). This surprising fact can be explained by the observation of Shaw et al. (2002) [14], showing that widely used HEK293 cells are characterized by unexpected features of neurons; thus, these cells are not typical kidney epithelial cells.

Lung carcinoma cells affected with vitamin D2 and other compounds promising for COVID-19 treatment.

Experiments were performed in A549 lung cancer cells treated with vitamin D2. For our analysis, we used a dose of 100 nM vitamin D2 (Figure 7A). Additionally, we also studied the effect of the PARP inhibitor olaparib, HDAC inhibitor SAHA, and γ-irradiation. After such treatment of lung carcinoma cells A549, the levels of the ACE2 protein and renin were analyzed by western blotting (Figure 7A). We found only subtle changes caused by the selected drug treatment. In particular, treatment with vitamin D2 caused a slight increase in the level of the ACE2 protein, accompanied by renin downregulation. Irradiation by γ-rays and exposure to the PARP and HDAC inhibitors did not affect the ACE2 level in A549 cells (Figure 7A, 7B). Only vitamin D2 enhanced the ACE2 level, which seems to be the benefit of this vitamin, likely reducing the severity of SARS-CoV-2 infection (Figure 7A and 7B; [15]).

We also analyzed the effect of dexamethasone (DEX). Interestingly, at the highest concentration, we observed ACE2 downregulation in A549 cells; at the low concentration, the level of ACE2 was maintained at a similar level to that in nontreated cells (Figure 7C). In this case, the renin protein was upregulated (Figure 7C, orange asterisk).

The ACE2 level is not changed in the olfactory bulbs of laboratory animals exposed to nicotine, but changes appeared in the lungs of these animals.

In the experimental smoking hood, 2- or 4-month-old mice of both genders were exposed to cigarette smoke (Figure 8A). After 14 days of experiments, by western blotting, we compared the level of ACE2 in nonsmoking and smoking animals. We studied the given protein in olfactory bulbs of the mouse brain. Surprisingly, we observed no effect of smoking on the ACE2 level in olfactory bulbs. The results were identical in both 2- or 4-month-old mice (Figure 8B). In experimental animals exposed to cigarette smoke, we also studied the level of ACE2 in the lungs. Analysis of the main 97-kDa ACE2 fragment showed no statistically significant changes. However, analysis of the double band (a secondary ACE2 fragment appearing in mouse lungs; see also Figure 2C) showed the downregulation of ACE2 in male animals. In smoking female mice, there was an increased level of the ACE2 protein in the lungs (Figure 8C).

Cell culture and treatment

Human lung cancer A549 cells and human adenocarcinoma HT29 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium; Sigma Aldrich, Czech Republic) supplemented with 10% fetal calf serum (FCS) (Merck, Germany). A549 lung cancer cells were treated with the PARP inhibitor olaparib (10 μM for 24 hours; #S1060, Selleckchem, Germany) and the inhibitor of histone deacetylase SAHA (5 μM for 24 hours; #10009929; Cayman Chemical Company, USA). Additionally, A549 cells were treated vitamin D2 (100 nM for 24 hours; #S4035; Selleckchem, Germany) or dexamethasone (#D4902; Sigma Aldrich, Czech Republic; 0.1 mM, 1 mM, and 5 mM (treatment was adopted from [24]).

The human intestine adenocarcinoma (HT29) cells were treated with the histone deacetylase (HDAC) inhibitor sodium butyrate (NaBt; 5 mM; #B5887; Sigma Aldrich, Czech Republic) to induce enterocytic differentiation for 48 hours (see [13]). In these experiments, we studied the effect of histone hyperacetylation and cell differentiation on the level of the ACE2 protein.

We have also studied the ACE2 level in HEK293 human embryonic kidney cells cultured in 293SFM II growth medium (#11686029, Thermo Fisher Scientific, Czech Republic). This is a serum-free, protein-free medium for the growth and transfection of the suspension-adapted human embryonic kidney (HEK293) cells. For culture, the medium was supplemented with 10% FCS (Merck, Germany).

For irradiation using cobalt-60, the cells were cultured on Petri dishes, irradiated with 5 Gy of γ-rays delivered by the Chisostat irradiation device (Chirana, Czech Republic). Next, the cells were either fixed by 4% formaldehyde for immunostaining experiments or harvested for western blotting 2 hours after γ-irradiation.

Culture of mouse ESCs and differentiation into cardiomyocytes

The following mESC lines were studied: wild-type mESCs, D3 line (mESCs wt, wild-type), and mESCs characterized by histone deacetylase 1 (HDAC 1) deficiency (HDAC1 dn mESCs) [25]. The mouse ESCs were cultured on 0.2% gelatin-coated Petri dishes (valid for wt cells) or Matrigel-coated plastic dishes (#354277; Corning Inc., USA; valid for HDAC1 dncells). The mouse ESCs were grown in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, Czech Republic) supplemented with penicillin and streptomycin, 0.1 mM nonessential amino acids, 1 ng/ml of mouse leukemia inhibitory factor (LIF), 100 μM mono-thioglycerol, and 15% fetal bovine serum (FBS). Differentiation into cardiomyocytes via the formation of embryonic bodies (EBs) was performed following [26, 27]. Cells were cultured at 37° C in a humidified atmosphere containing 5% CO₂. Differentiation into cardiomyocytes was activated by cell seeding into ES culture media without leukemia inhibitory factor (LIF). In this step, we used the "hanging drop" method. On day 3 of culture, the EBs were placed on new plastic dishes. On day 6, the EBs were transferred to gelatin-coated culture dishes with DMEM/F12 (1:1) (#11320-033; Sigma Aldrich, Czech Republic) supplemented with insulin, transferrin, and selenium (ITS-100x; #41400-045; ThermoFisher Scientific, Czech Republic). The differentiation medium (DMEM/F12-ITS) was changed every two days. The duration of the differentiation into cardiomyocytes was up to day 20 (dd20).

Tissue isolated from experimental animals

Explanted hearts from the mouse strain C57Bl6 were used to investigate the distribution pattern of the ACE2 protein and its interacting partner renin. The mice were kept in a pathogen-free (SPF) animal facility (see [26]). For experiments, we obtained approval from the Ethics Commission of the Ministry of Agriculture of the Czech Republic (protocol No. 48/2016). After breading, the embryos were explanted from female animals 15 days postconception (e15), and embryonic hearts were treated with HDAC inhibitors (200 nM TSA and, 16 μM SAHA) (for more details, see [28]). Except for embryonic and young (2 months), old (27 months) female and male hearts, we additionally isolated adult mouse lungs, brains, and kidneys. Using western blotting, we compared the level of the ACE2 protein in selected tissues.

Cell culture immunostaining

Immunofluorescence was performed following [29]. Briefly, the cells were fixed in 4% formaldehyde (PFA) for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 (#194854; MP Biomedicals, USA) for 10 min and 0.1% saponin (#S7900; Sigma Aldrich, Czech Republic) for 12 min. After that, the dishes were washed twice in phosphate-buffered saline for 15 min. We used 1% bovine serum albumin (BSA; #A2153-506; Sigma Aldrich, Czech Republic) dissolved in 1× PBS as a blocking solution. Next, the samples were incubated in blocking solution for one hour at room temperature and then washed in 1× PBS for 15 min. For immunofluorescence analysis, the following antibodies were used: anti-ACE2 (#ab15348; Abcam, UK) and α-actinin (#A7811; Sigma Aldrich, Czech Republic). As secondary antibodies, we used the following: Alexa Fluor 594-conjugated goat anti-rabbit (#A11037; ThermoFisher Scientific, Czech Republic), Alexa Fluor 594-conjugated goat anti-mouse (#A11032; ThermoFisher Scientific, Czech Republic), Alexa Fluor 488-conjugated goat anti-rabbit (#ab150077; Abcam UK) antibodies. The negative control was considered samples incubated without primary antibodies. Cell nuclei (GC-rich sequences of DNA) were stained with 4′,6-diamidino-2-phenylindole (DAPI; Merck, Czech Republic). As a mounting medium, we used Vectashield (#H-1000, Vector Laboratories, USA).

Heart tissue cryosectioning

Fixed mouse embryonic hearts (immersed in 30% sucrose) were frozen in embedding medium (OCT embedding matrix; Leica Microsystems, Mannheim, Germany) at −80° C. The mouse hearts were sectioned using a Leica Cryo-microtome (Leica CM 1800; Leica, Germany). The cryosections were washed in PBS, and the thickness of the sections was 13-14 μm. For immunofluorescence, we used the protocol published in [28]. The primary antibody was anti-ACE2 (#ab15348; Abcam, UK). As the secondary antibody, we used Alexa Fluor 594-conjugated goat anti-rabbit antibody (#A11037 ThermoFisher Scientific, Czech Republic). DNA was counterstained using 4′,6-diamidino-2-phenylindole (DAPI; Merck, Czech Republic), and the mounting medium was Vectashield (#H-1000, Vector Laboratories, USA).

Exposure of laboratory animals to nicotine

In a ventilated hood, the mice were exposed to 3 cigarettes/day for 14 days. For experiments, we used both female and male animals exposed to the fumes from cigarettes P&S black (Tobacco Group PLC, 121 Winterstoke Road, Bristol BS3 2LL; 10 mg/cigarette; nicotine 0.8 mg/cigarette; carbon monoxide 10 mg/cigarette). The mice were maintained in the smoking box and exposed to nicotine and related air-pollution for 90 min each day. The experiments were performed in a closed chamber with closed airflow. Twelve 2- or 4-month-old C57BL6 mice (6 males and 6 females, in duplicated experiments) were tested. Animals of both genders were randomly divided into two experimental groups: a control group and a smoking group. Water and food were provided ad libitum, and no animal died during the experiments. The animals were sacrificed according to protocol No. 48/2016, and olfactory bulbs were explanted and used for analysis by western blotting.

Tile scanning

Cryosections of whole embryonic hearts (at embryonic stage e15) were stained using appropriate antibodies, as mentioned above. For analysis, we used the "tile-scanning" mode, a tool of the Leica SP-5 confocal microscope (Leica, Germany). To acquire images, we used the HCX PL APO lambda blue 20.0× 0.7 IMM UV objective (Leica Microsystems, Germany) (see [30]).

Laser scanning confocal microscopy

We used a Leica TCS SP8-X SMD confocal microscope (Leica Microsystems, Germany), equipped with a 63× oil objective (HCX PL APO; lambda blue) with a numerical aperture (NA) = 1.4. Image acquisition was performed using a white light laser (WLL; wavelengths of 470-670 nm in 1-nm increments) with the following parameters: 1024 × 1024-pixel resolution, 400 Hz, bidirectional mode, and zoom 8-12 using the Leica Application Suite (LAS X) software.

Western blotting

Western blot analysis was performed as described previously by [31]. Cell culture or tissue samples were washed with PBS and lysed in sodium dodecyl sulfate (SDS) lysis buffer (50 × 10³ mol/l Tris-HCl, pH 7.5; 1% SDS; 10% glycerol). The total protein concentration was determined using the DC protein assay kit (#5000111; Bio-Rad, Czech Republic) and ELISA Reader μQuant (BioTek, USA). The proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 2% low-fat milk or 2% gelatin for one hour and then immunoblotted overnight at 4° C using the following primary antibodies against the ACE2 protein (#ab15348; Abcam, UK) and anti-renin (#PA5-102432; ThermoFisher Scientific, Czech Republic). Primary antibodies were diluted at 1:1000. As a secondary antibody, we used goat anti-rabbit IgG (#AP307P; Merck, Czech Republic; 1:2000). The western blot data were normalized to the amount of the total proteins.

Statistical analyses and quantification of fluorescence intensity

For densitometric analysis of the western blot bands and fluorescence intensity analysis, we used ImageJ (NIH freeware) and ImageQuant TL software, respectively.

For statistical analysis, we used the Mann-Whitney U test (STATISTICA software), which is a nonparametric test of the null hypothesis that is applied for X and Y values, randomly selected from two experimental units [32]. The primary step of this analysis contains the U statistic involving the use of a symmetric real-valued function h(x,y). The following formula describes the approach:

$U_n=\frac{1}{\left(\frac{n}{2}\right)}{\displaystyle \sum }_{1\le s<i}{\displaystyle \sum }_{len}h\left(u_s,u_t\right)$

• (n/2) is the binomial coefficient,

• (u_t) are independent and identically distributed variables,

• Σ = summation notation

The following two formulas are applicable for the Mann-Whitney U Test. R is the sum of ranks in the sample, and n is the number of items in the sample.

$\begin{array}{c}{U}_1=R_1-\frac{n_1(n_1+1)}{2}\\ or\\ U_2=R_2-\frac{n_2(n_2+1)}{2}\end{array}$

We ran the test at the 5% level of significance (i.e., * means α=0.05). The experiments were repeated three times; however, but in the case of smoking, we performed only two repetitions to minimize the loss of animal life.