Baseline Characteristics of the Total Study Population
We analyzed lung samples obtained from white subjects, untreated (n=42) and treated with ACEI/ARB (n= 41). Mean systolic and diastolic pressure of those subjects were 120+5.4 and 67.3+2.0 mmHg in the untreated group and 124.4+5 and 73.3+4.4 mmHg in the ACEI/ARB treated group, respectively. The mean age of untreated subjects was 51+17 and of ACEI/ARB-treated subjects was 63+9 years old. The male-to-female ratio was 42/41. Characteristics of the population investigated are presented under table 1.
Lung Ang II levels are Influenced by Age, Sex, Smoking and ACEI/ARB Treatment
ACE2 has high catalytic efficiency for Ang II. In order to investigate ACE2 activity as the ratio between Ang-(1-7) and Ang II, we measured Ang II and Ang-(1-7) levels by radioimmunoassay. Figure 1 shows Ang II levels in pg/g tissue in lung parenchyma of untreated and ACEI/ARB-treated subjects. We found that age, sex, smoking and ACEI/ARB treatment influence on Ang II levels (p<0.05). Within the untreated population, subjects under 60 years old, smokers or never smokers, showed no difference in Ang II levels irrespective of their sex. Conversely, Ang II levels were higher in smoker and former smoker men older than 60 years old compared to smoker and former smoker women of the same age. In addition, Ang II levels were higher in older smokers men compared to younger smoker and never smoker women. Unfortunately, our study lacks samples from never smoker subjects older than 60 years old to make a comparison between older never smoker and smokers. Regarding Ang-(1-7) levels, we observed a trend (p= 0.08) to have reduced Ang-(1-7) levels in untreated older and smoker subjects, female and male compared to younger smoker subjects, that is, age and smoking showed a tendency to induce a decrease in Ang-(1-7) levels in untreated subjects (Figure 2). Altogether, our results showed that untreated older smoker or former smoker men displayed higher Ang II levels compared to women, while Ang-(1-7) levels were not changed by sex at the same age. In contrast, younger smoker women showed slightly higher Ang II levels compared to men.
Age and smoking also influence Ang II levels in those subjects under RAS blockade (p= 0.001). We found that Ang II levels were reduced in those subjects treated with ACEI/ARB, never smoker or smoker, under 60 years old compared to subjects older than 60 years old, independently of their sex (Figure 1). RAS blockade treatment showed a tendency to increase Ang-(1-7) levels in subjects under 60 years old of any sex, smoker or never smoker compared to older subjects (p= 0.08). Instead, antihypertensive treatment did not change Ang-(1-7) levels in subjects over 60 years old (Figure 2).
Because ACE2 catalyzes the conversion of Ang II into Ang-(1-7), we then evaluated Ang-(1-7)/Ang II ratio as an indirect measurement of ACE2 activity. We observed no difference in Ang-(1-7)/Ang II ratio between the investigated groups, suggesting no change in ACE2 activity (Figure 3S).
ACE2-expressing type II pneumocytes are Influenced by Age, Smoking and ACEI/ARB Treatment
Because type II pneumocytes are key cells to maintain lung homeostasis8 and that ACE2 is mainly expressed in these cells9,10,12,32, we measured the percentage of ACE2-expressing type II pneumocytes by immunohistochemistry in lung parenchymal from untreated and subjects under ACEI/ARB treatment. If we consider only untreated and ACEI/ARB-treated subjects, we found no difference in the percentage of type II pneumocytes that expresses ACE2 (Figure 3A), being ACE2 present in 61.8 ± 7.5 % of type II pneumocytes from untreated subjects and in 62.4 ± 8.6 % of type II pneumocytes from ACEI/ARB-treated subjects. We also did not find significant differences if we only compare never smokers, former smokers or smokers (Figure 3B) or male and female (Figure 3C). However, we observed that subjects over 60 years old exhibited lower percentage of ACE2-expressing type II pneumocytes (p= 0.012) (Figure 3D). When we compared all the variables at the same time, that is, smoking, sex, age and antihypertensive treatment, we found a significant interaction between ACEI/ARB treatment and smoking on the percentage of ACE2-expressing type II pneumocytes (p= 0.026). This means that the percentage of ACE2-expressing type II pneumocytes depends on whether the subject is a smoker, former smoker or never smoker, under or not RAS blockade treatment. Regarding smokers subjects, those under RAS blocking treatment exhibited higher percentage of ACE2-expressing type II pneumocytes than untreated ones. Within the group of subjects under ACEI/ARB treatment, the percentage of ACE2-expressing type II pneumocytes was higher in smokers compared to never smokers (Figure 4). Altogether, these data suggest that smoking and RAS blocking treatment enhances the percentage of ACE2-expressing type II pneumocytes.
ACE2 Protein Levels are Influenced by Age and Smoking
We evaluated ACE2 immunostaining intensity, indicative of ACE2 protein levels. We found a significant association between ACE2 immunostaining intensity and smoking on subjects equal or older than 60 years old (p= 0.05). The greater percentage of subjects that exhibited higher ACE2 protein levels was that of older smokers and former smokers (Figure 5B).
We next evaluated whether RAS blockade and age are associated with ACE2 protein immunostaining intensity. Although the p value of that association was lower than 0.1, this fact shows a tendency to older subjects treated with ACEI/ARB to display higher ACE2 protein expression levels (Figure 5C). Thus, age and RAS blockade trends to enhance ACE2 protein expression levels.
TMPRSS2-expressing type II pneumocytes are Influenced by Age and Sex
Since TMPRSS2 is needed for SARS-CoV2 entry7, we next determined the percentage of TMPRSS2-expressing type II pneumocytes. We found a significant difference by age (p= 0.04) and sex (p= 0.026) but not by smoking in the percentage of TMPRSS2-expressing type II pneumocytes (Figure 6). Although we did not observe a significant difference between TMPRSS2-expressing cells in untreated vs ACEI/ARB-treated subjects, the p value of 0.06 indicated a trend of hypertensive treated subjects to have higher percentage of TMPRSS2-expressing cells compared to those untreated (Figure 6).
TMPRSS2 Protein Levels are Influenced by Age, Smoking and RAS blockade
We then evaluated the association between TMPRSS2 immunostaining intensity (indicative of protein levels) with age, sex, smoking and ACEI/ARB treatment. We found a significant association with age and smoking (p= 0.001) being the higher TMPRSS2 protein levels in older smoker and former subjects (Figure 7A). Regarding ACEI/ARB treatment, we observed a significant association in TMPRSS2 protein levels with age (p= 0.001) (Figure 7B) and smoking (p= 0.039) (Figure 7C). Thus, the greater percentage of ACEI/ARB-treated subjects that exhibited higher TMPRSS2 protein levels was that of olders and smokers.