Decrease in Angiotensin-Converting Enzyme activity but not concentration in plasma/lungs in COVID-19 patients offers clues for diagnosis/treatment

Abstract

Although several therapeutics are used to treat coronavirus disease 2019 (COVID-19) patients, there is still no definitive metabolic marker to evaluate disease severity and recovery or a quantitative test to end quarantine. Because severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects human cells via the angiotensin-converting-enzyme 2 (ACE2) receptor and COVID-19 is associated with renin-angiotensin system dysregulation, we evaluated soluble ACE2 (sACE2) activity in the plasma/saliva of 80 hospitalized COVID-19 patients and 27 non-COVID-19 volunteers, and levels of ACE2/Ang (1-7) in plasma or membrane (mACE2) in lung autopsy samples. sACE2 activity was markedly reduced (p < 0.0001) in COVID-19 plasma (n = 59) compared with controls (n = 27). Nadir sACE2 activity in early hospitalization was restored during disease recovery, irrespective of patient age, demographic variations, or comorbidity; in convalescent plasma-administered patients (n = 45), restoration was statistically higher than matched controls (n = 22, p = 0.0021). ACE2 activity was also substantially reduced in the saliva of COVID-19 patients compared with controls (p = 0.0065). There is a strong inverse correlation between sACE2 concentration and sACE2 activity and Ang (1-7) levels in participant plasmas. However, there were no difference in membrane ACE2 levels in lungs of autopsy tissues of COVID-19 (n = 800) versus other conditions (n = 300). These clinical observations suggest sACE2 activity as a potential biomarker and therapeutic target for COVID-19.

Keywords: COVID-19; SARS-CoV-2 diagnosis/treatment, soluble Angiotensin-Converting Enzyme 2, biomarker in plasma/saliva, Corona virus, COVID-19 disease, ACE2 activity/concentration, RAS pathway; angiotensin-converting-enzyme; biomarker; coronavirus disease; plasma marker; saliva marker.

Graphical abstract

Figure 1

Kinetic reading of plasma ACE2 enzyme activity ACE2 activity determined by cleavage of fluorogenic Mca-APK (Dnp) substrate. ACE2 enzyme activity units (mU/mg) calculated in control and COVID-19 patients. (A) In EDTA-treated samples, 17 control (blue) and 16 COVID-19 (red). (B) In Non-EDTA-treated samples, 10 control (blue) and 43 COVID-19 (red). (C) Both EDTA and non-EDTA samples combined data. Data were analyzed using the Mann-Whitney U test; ∗∗∗∗p < 0.0001.

Figure 2

Kinetic reading of saliva ACE2 enzyme activity (A and B) ACE2 activity determined by cleavage of fluorogenic Mca-APK (Dnp) substrate in 20 control and 21 COVID-19 (red) samples. ΔRFU was calculated by subtracting data of time point 0 min from data of time point 90 min. Data were analyzed using the Mann-Whitney U test; ∗∗p < 0.05 (0.0065). (C) ACE2 enzyme activity units (mU/mg) calculated in control and COVID-19 patients. Data were analyzed using the Mann-Whitney U test; ∗∗p < 0.05 (0.0099).

Figure 3

ACE2 enzyme activity in patients treated with convalescent plasma (A and C) ACE2 activity measured at all timepoints: days 1, 3, 8, 15, 29, and 60 in the convalescent plasma group of 10 patients (n = 45). The plasma ACE2 activity was significantly higher on day 60 compared with day 1 in convalescent plasma-treated patients (∗∗∗p = 0.0005), Mann-Whitney U test). Unavailable samples #001 (days 15 and 29), #007 (day 29), #013 (days 8 and 15), #018 (days 15 and 29), #020 (days 3 and 8), #028 (days 3 and 8), #034 (days 3 and 8), #037 (day 8), and #055 (day 8). (B) ACE2 activity measured on days 1 and 60 in 11 non-CCP-treated patients (n = 22). No significant difference was detected (p = 0.6857).

Figure 4

Measurement of ACE2 and Ang (1–7) concentration in plasma and correlation of ACE2 enzyme activity with Ang (1–7) levels (A) ACE2 concentration determined by ELISA in a subset of seven COVID-19 and 16 control plasma samples. The level of ACE2 is significantly higher in COVID than control (∗∗p < 0.01, Mann-Whitney U test). (B) Ang (1–7) concentration level was quantified in 17 control (blue) and 16 COVID (red) prepared samples by ELISA (∗∗∗p < 0.001, Mann-Whitney U test). (C) Ang (1–7) concentration (pg/mL) in all 17 control (blue) and 16 COVID (red) plasma samples positively correlated with ACE2 activity (ΔRFU). Each dot represents a sample with both ACE2 activity and Ang (1–7) concentration data. The Pearson correlation was performed for correlation analysis. A linear regression line was formulated, and its R square value indicates the coefficient of determination (p = 0.0073).

Figure 5

IHC autopsy lung tissue sections stained for ACE2 (red) Representative image of deconvoluted DAB-stained (IHC Plugin; ImageJ software) control (A) or COVID-19-infected lung tissue (#6, B). (C) Quantitation of ACE2 staining. No statistical significance was observed (p = 0.088; Mann-Whitney test). A total of 800 images from COVID-19 lung ACE-stained 200-μm sections covered 80%–90% of the area. For controls #1 to #4, 100 images were analyzed, and for #5 to #8, 200 images were analyzed.