IG is an employee of Olink Proteomics. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors declare no other potential conflicts of interest.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) enabling entrance of the virus into cells and causing the infection termed coronavirus disease of 2019 (COVID-19). Here, we investigate associations between plasma ACE2 and outcome of COVID-19.
This analysis used data from a large longitudinal study of 306 COVID-19 positive patients and 78 COVID-19 negative patients (MGH Emergency Department COVID-19 Cohort). Comprehensive clinical data were collected on this cohort, including 28-day outcomes. The samples were run on the Olink® Explore 1536 platform which includes measurement of the ACE2 protein. High admission plasma ACE2 in COVID-19 patients was associated with increased maximal illness severity within 28 days with OR = 1.8, 95%-CI: 1.4–2.3 (
This study suggests that measuring plasma ACE2 is potentially valuable in predicting COVID-19 outcomes. Further, ACE2 could be a link between COVID-19 illness severity and its established risk factors hypertension, pre-existing heart disease and pre-existing kidney disease.
Since December 2019, a previously undiscovered virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a devastating global pandemic. The disease caused by SARS-CoV-2 infection has been termed coronavirus disease of 2019 (COVID-19) with clinical manifestations ranging from asymptomatic and subclinical infection to severe hyperinflammatory syndrome and death [
SARS-CoV-2 binds to the angiotensin converting enzyme 2 (ACE2) receptor enabling entrance into cells through membrane fusion and endocytosis [
ACE2 is part of the renin-angiotensin-aldosterone-system (RAAS). Renin cleaves angiotensinogen leading to formation of angiotensin I (Ang I). Ang I is then converted to the vasoconstricting angiotensin II (Ang II) through cleavage by angiotensin-converting-enzyme (ACE), which is found in the vascular endothelium and more plentifully in the pulmonary endothelium [
ACE2 is a tissue enzyme and thus circulating levels are low; the significance of measuring circulating ACE2 in pathologic conditions remains uncertain [
This study is the first description of the association between circulating ACE2 and disease outcomes in patients with COVID-19 disease. Data were obtained from a large longitudinal COVID-19 study [
Data were downloaded from
Link
COVID-19-negative subjects enrolled were older than COVID-19-positive patients, less Hispanic, and with greater baseline burden of chronic illnesses. Of the 78 COVID-19-negative subjects, 37 (47%) were diagnosed with non-COVID-19 pneumonia or acute lung injury (e.g., aspiration), 12 (15%) with congestive heart failure exacerbation, 6 (7.7%) with COPD exacerbation, 3 (3.8%) with acute pulmonary embolus, 11 (14%) with non-pulmonary sepsis or infection, and 8 (10%) with other illnesses. COVID-19-negative patients were significantly less inflamed than COVID-19-positive patients, median CRP 22 [IQR 9–67] versus 105 [IQR 48–161], p-value < 0.05, but illness acuity and outcomes were very similar between the two groups [
Sample collection and analysis was approved by Partners Human Research Committee (PHRC). The need for informed consent was waived by this committee.
Detailed description is available online (
Group comparisons were made using non-parametric tests. The diagnostic value of plasma ACE2 at baseline was further tested with a receiver operating characteristic (ROC) curve for severe (A1-A2) vs non-severe (A3-A5). The comparison of outcome group differences between COVID-19 positive and negative patients was done using a two-way ANOVA. The association between clinical outcome (response variable) and plasma ACE2 (explanatory variable) was also investigated using ordered logistic regression and results are presented as odds ratios (OR) for worse outcome scores per unit increase in plasma ACE2. The specific statistical method tests used for each analysis is described in the table and figure legends. For all tests, the level of significance was a two-sided
First, we investigated the association between circulating ACE2 at day 0 and maximal acuity outcome group during the 28-day study period (Acuity max, A1-A5). Elevated baseline plasma ACE2 from COVID-19 patients was significantly associated with Acuity max with OR = 1.8, 95%-CI: 1.4–2.3 (
Model | n | OR | 95%-CI | p |
---|---|---|---|---|
1 | 285 | 1.8 | 1.4–2.3 | |
2 | 285 | 1.9 | 1.4–2.4 | |
3 | 285 | 1.9 | 1.4–2.4 | |
4 | 265 | 1.4 | 1.1–1.9 | 0.007 |
The outcome is the maximum acuity score during the 28-day period with death (A1) being the highest acuity possible, and discharge without requiring admission within 28 days (A5) the least acuity possible.
Models (predictors): 1) Plasma ACE2. 2) Plasma ACE2, age, body mass index (BMI). 3) Plasma ACE2, age, body mass index (BMI), pre-existing hypertension, pre-existing heart disease, pre-existing lung disease, pre-existing kidney disease, pre-existing diabetes, pre-existing immunosuppressive condition. 4) Plasma ACE2, C-reactive protein (CRP), absolute neutrophile count, and D-dimer.
n = number of patients included in statistical analysis. OR = Odds ratio for a higher clinical outcome category per unit increase in plasma ACE2, 95%-CI = 95% confidence interval, p = p-value.
Next, we tested whether circulating ACE2 at day 0, day 3, and day 7 was associated with clinical status at the time of blood sampling. Hospitalized patients were grouped according to outcome categories A2 (intubated at the time of sample collection) or A3-A4 (not intubated at the time of sample collection). Elevated plasma ACE2 in COVID-19 patients was significantly associated with higher acuity category at the time of blood sampling at day 0, day 3, and day 7 (
We then analyzed the relationship between ACE2 and comorbidities. Circulating ACE2 in COVID-19-positive patients with hypertension was significantly elevated compared with patients without hypertension (
We further tested associations of circulating ACE2 with age and body mass index (BMI). Elevated plasma ACE2 in COVID-19-positive patients was significantly associated with increasing age (
Next, we analyzed whether circulating ACE2 differed between COVID-19 patients and non-COVID-19 patients with respiratory symptoms. Plasma ACE2 showed a clear overlap between the two groups. There was no significant difference between plasma ACE2 in COVID-19-positive versus negative patients (
Outcome group A1-2 vs. A3-5 | p | 95%-CI | |
---|---|---|---|
COVID-19-negative | 0.33 | 0.15 | -0.12–0.77 |
COVID-19-positive | 0.49 | <0.0001 | 0.27–0.71 |
Difference | 0.16 | 0.52 | -0.34–0.66 |
Maximal acuity category at 28 days is the maximum score within first 28 days with death (A1) being the highest acuity possible, and discharge without requiring admission within 28 days (A5) the least acuity possible.
p = p-value, 95%-CI = 95% confidence interval.
SARS-CoV-2 uses ACE2 as a functional receptor for entry into cells [
Elevated baseline plasma ACE2 in COVID-19 patients was significantly associated with increased disease severity during the 28-day study period. This indicates that abundant ACE2 production could be involved in increased viral spread and disease burden, as previously shown in experimental models of SARS-CoV infection [
It is desirable to predict disease outcomes in order to specialize treatment, since the drugs used in the treatment of COVID-19 can have deleterious side effects [
Circulating ACE2 in COVID-19-positive patients with hypertension was significantly increased compared with plasma from patients without hypertension. Patients with hypertension are often treated with ACE-inhibitors and AT1R-blockers. During this pandemic, it has been highly debated whether or not the use of these antihypertensive medications should be discontinued in patients with COVID-19. A study performed on rats showed that the use of ACE inhibitors and/or use of AT1R-blocker led to an increased expression of ACE2 in cardiac tissue [
In contrast, differences in plasma ACE2 does not seem to explain the risk of severe COVID-19 disease associated with pre-existing lung disease, diabetes, or immunosuppression. Plasma ACE2 was associated with age, which is in line with recent observations showing higher serum levels of ACE2 in adults compared to a pediatric cohort [
Baseline plasma ACE2 was not significantly different between COVID-19-positive and negative patients, which is in line with the results of a recent study with a much smaller sample size [
There are some overall limitations of this study. First, plasma ACE2 was measured as relative protein concentrations using NPX (Normalized Protein eXpression) values. Therefore, it is not possible to determine a plasma ACE2 cut-off value to predict severe outcome or to compare findings in this study with results in studies measuring protein concentration or enzymatic activity. Second, several continuous variables were categorized in the publicly available data set. This decreases the statistical power of some of the analysis. Third, some potentially important variables such as gender and treatment were not available. Fourth, severity driven recruitment criteria (respiratory distress at emergency department) might have introduced a bias. Thus, patients with more severe COVID-19 at admission have both higher ACE2 at time of inclusion and a poorer prognosis. However, our study reflects clinical practice where laboratory tests are performed when patients are admitted to the hospital. Finally, causality of associations between plasma ACE2 and severity of COVID-19 disease cannot be drawn from this study.
Overall, this study suggests a potential utility of measuring ACE2 in COVID-19 to predict disease outcome. Further, circulating ACE2 could be a link between severe COVID-19 disease and its risk factors, namely hypertension, pre-existing heart disease and pre-existing kidney disease. The design of the data analysis using the Olink platform does not allow assessment of quantitative differences. However, previous studies have described a positive correlation between plasma ACE2 and ACE1 activity. This is interesting because ACE1 (serum ACE) analysis is a standardized test in most hospital laboratories. Therefore, our study encourages quantitative investigations of both plasma ACE 1 and 2 in COVID-19.
(DOCX)
Data provided by the MGH Emergency Department COVID-19 Cohort (Filbin, Goldberg, Hacohen) with Olink Proteomics. We want to thank Aparna Udupi, Department of Biostatistics, Aarhus University for technical assistance running the statistical analyses.
Angiotensin converting enzyme
Angiotensin I
Angiotensin II
Analysis of variance
Type 1 angiotensin II receptor
Area under the curve
Body mass index
Coronavirus
Corona virus disease 2019
C-reactive protein
Interleukin
Normalized Protein eXpression
Proximity extension assay
Renin-angiotensin-aldosterone-system
receiver operating characteristic
Severe acute respiratory syndrome coronavirus 2
Tumor necrosis factor
PONE-D-21-06434
Plasma ACE2 levels predict outcome of COVID-19 in hospitalized patients
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Reviewer #1: In the current paper, the authors investigate the link of ACE2 signals obtained from a proximal extension assay multiplex panel in a cohort of 306 COVID-19 patients and 78 SARS-CoV-2 negative controls. The authors suggest a link between baseline signals for ACE2 obtained from a commercially available proximal extension assay and the outcome of COVID-19 disease. The findings are interesting and in-line with previous observations regarding ACE2 regulation during COVID-19. The authors should also check for more recent literature on the topic, describing a link between soluble ACE2 activity and COVID-19 severity.
The aspect of a prognostic value of baseline ACE2 for COVID-19 outcome appears to be interesting at the first glance, but considering the design of the clinical readout, it might be introduced by the fact that "Day 0" in the current study was actually the admission to the emergency department respiratory distress, which could happen at very variable time points related to disease onset. Knowing that COVID-19 severity is linked to plasma ACE2 activity with a peak in concentrations between 1 and 2 weeks after disease onset, severity driven recruitment criteria (respiratory distress at emergency department) might have introduces a certain bias, as more severe cases have higher ACE2 at time of inclusion, with obviously having poorer outcomes.
In other words, patients being included with a more severe disease manifestation ion day 0 (e.g. higher ACE2), may just be at another time-point in the course of COVID-19 disease, with an already pre-determined worse outcome. It would add a lot of valuer to the study, if the measurement time points would be related to the time of symptom onset, first positive test result or any other earlier time point that may would assure the direct comparability of disease severity groups. The authors should further describe the control group of 78 Covid-negative patients described more clearly. Why did these patients at emergency units with respiratory distress?
Finally, the use of terminology within the whole paper is misleading, as the authors keep using the terms "ACE2 levels" and "ACE2 concentrations". Of note, the result of the proximal extension assay the authors used as a basis for their interpretation is given in the form of an artificial unit (NPX), that has been calculated by the manufacturer by arithmetically linking a series of Ct value based correction algorithms. Even the manufacturers point out on their webpage that the given readout cannot be compared to actual protein levels, which disables comparability to other studies.
From a technical perspective, the big open question is why no calibration of the readout is performed to be able to provide actual protein levels. With a highly reproducible and standardized method as described by the manufacturers on their webpage, it should be easy to retrospectively include a valid calibration allowing for providing actual ACE2 concentrations instead of manufacturer invented units that prevent comparability with other studies. Moreover, a certain analytical validation vor ACE2 in the used PEA panel should be shown that compares the used "NPX" values with real concentration units or standard activity units in a defined set of clinical samples. It should also be noted that reading the method section needs some revision as it currently reads like an advertisement for the used (commercially available) technology rather than an objective method description to be published in a research paper.
Reviewer #2: .This study investigated the association between plasma ACE2 levels and outcomes of COVID-19 patients, using clinical data and plasma samples from 306 COVID-19 positive and 76 COVID-19 negative patients. High baseline plasma ACE2 levels are reported to be associated with worse COVID-19 outcomes and patients with hypertension, pre-existing heart conditions or kidney disease had higher plasma ACE2 levels than those without.
Even as a marker I doubt very much that it will be useful for COVID-19 because the changes found in figure 2 are so small.
Main criticisms and suggestions for improvement:
- Overall, the paper is an excellent contribution but the authors should acknowledge the limitations of measurements of ACE2 in plasma where the levels are usually very low and even when mildly elevated in pathological conditions the significance remains uncertain. Specifically, it must be stated that ACE2 is a tissue enzyme and that the levels in the circulation are low in all species studied, including humans. Appropriate references should be given.
Please acknowledge that the changes in COVID-19 are so small that they are not likely to be of any significance regarding the metabolism of the substrates of ACE2
- In the introduction the part on ACE2 receptor distribution must be modified to include the kidney as a main site of ACE2 . The authors do not seem aware of a critical important observation, namely that the expression of ACE2 in the lung is very low as shown by Serfozo et al using western blot and confirmed by others
See
These references must be cited as well as others showing that the kidney is next to the intestine the organ that has the highest abundance of ACE2.
- Perhaps the authors are not aware that ACE2 RNA levels do not necessarily imply protein levels. Actually, ACE2 can be post translationally regulated and be increased when the levels of mRNA are not. The authors may consult and cite a review on this by Lores et al.
- In the discussion it is stated that “ACE2 as a decoy receptor… is now being explored in COVID-19 disease.” Appropriate references should be given to be faithful to the literature. Perhaps they are not aware of these papers because they are recent but they should be cited. For instance,
- “A study performed on rats showed that the use of ACE inhibitors and/or use of AT1R-blocker led to an increased expression of ACE2”. It should be specified that in ref 34 ACE2 was only examined in cardiac tissue and not kidney or lung. Others found a decrease of kidney ACE2 after ACEi and ARBs and no effect on lungs. For instance, see
- “The population sampled on day 3 and day 7 therefore consists of patients with more severe disease compared with day 0.” Maybe ACE2 for day 3 and day 7 should be shown in a way that they are only compared to their matching samples of day 0?
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Response letter
Dear editor and reviewers,
We are very pleased with a constructive review of our paper. We readily acknowledge that reviewers have in depth knowledge on ACE2 physiology and the role of this protein in COVID-19 disease. We are therefore very happy to accommodate all suggestions made by the reviewers.
Three native speaking authors have carefully proof read the manuscript. This included a few changes worth mentioning.
1) The Odds ratios have been inverted to report odds ratios for a smaller category instead of a larger category. Seems like it would make more intuitive sense to have higher severity as the primary outcome in analyses. The text as written supports this approach.
2) Fig 2B has been changed to better align with the other figures (no colors).
We have made two general corrections throughout the manuscript based on reviewer suggestions and co-author comments after seeing the review. These changes are not highlighted in the manuscript because the corrections are found abundantly throughout.
1) As suggested by reviewer #1 we have replaced all mentions of ”levels” and ”concentrations” with ”plasma ACE2” or ”circulating ACE2”.
2) We have also systematically changed the terminology of the disease groups from “WHO groups” to “clinical outcome groups/Acuity groups”. “WHO groups” was part of the publicly available information downloaded from the source link at the Olink website. However, the scores were not based on the WHO Outcome scale. This is because COVID19 is not treated with non-invasive ventilation or high-flow nasal cannula (therefore, no patients were categorized in previous group 3). The group definitions are the same but have been renamed accordingly. 1 = Death. 2 = Intubated, ventilated, survived. 3 = Hospitalized, supplementary O2 required. 4 = Hospitalized, no supplementary O2 required. 5 = Not hospitalized. These changes are not highlighted in the manuscript.
All other changes have been highlighted in the revised manuscript.
We believe the paper has been greatly improved. Thank you.
On behalf of the authors,
Tue Wenzel Kragstrup
Tue Wenzel Kragstrup
Associate Professor, MD, PhD
E-mail:
Biomedicine, Health
Aarhus University
Wilhelm Meyers Alle 4, 4fl.
DK-8000 Aarhus C
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Reviewer #1: In the current paper, the authors investigate the link of ACE2 signals obtained from a proximal extension assay multiplex panel in a cohort of 306 COVID-19 patients and 78 SARS-CoV-2 negative controls. The authors suggest a link between baseline signals for ACE2 obtained from a commercially available proximal extension assay and the outcome of COVID-19 disease. The findings are interesting and in-line with previous observations regarding ACE2 regulation during COVID-19. The authors should also check for more recent literature on the topic, describing a link between soluble ACE2 activity and COVID-19 severity.
Response: We would like to thank the Reviewer for taking time to carefully read our paper. We agree with all his/her comments. We believe that we have addressed all comments and this is a great improvement to the manucript.
We reviewed the literature again and found the very recent publications below evaluating circulating ACE2 in COVID-19. They have been cited in the manuscript.
Lundström A, Ziegler L, Havervall S, Rudberg A-S, von Meijenfeldt F, Lisman T, et al. Soluble angiotensin-converting enzyme 2 is transiently elevated in COVID-19 and correlates with specific inflammatory and endothelial markers. medRxiv. 2021:2021.03.03.21252841. doi: 10.1101/2021.03.03.21252841.
Nagy B, Jr., Fejes Z, Szentkereszty Z, Suto R, Varkonyi I, Ajzner E, et al. A dramatic rise in serum ACE2 activity in a critically ill COVID-19 patient. Int J Infect Dis. 2021;103:412-4. Epub 2020/11/30. doi: 10.1016/j.ijid.2020.11.184. PubMed PMID: 33249290;
van Lier D, Kox M, Santos K, van der Hoeven H, Pillay J, Pickkers P. Increased blood angiotensin converting enzyme 2 activity in critically ill COVID-19 patients. ERJ Open Res. 2021;7(1). Epub 2021/03/20. doi: 10.1183/23120541.00848-2020.
Fagyas M, Kertesz A, Siket IM, Banhegyi V, Kracsko B, Szegedi A, et al. Level of the SARS-CoV-2 receptor ACE2 activity is highly elevated in old-aged patients with aortic stenosis: implications for ACE2 as a biomarker for the severity of COVID-19. Geroscience. 2021. Epub 2021/01/21. doi: 10.1007/s11357-020-00300-2.
And per request from reviewer #2 we added these publications better refelcting the current litterature:
Lores E, Wysocki J, Batlle D. ACE2, the kidney and the emergence of COVID-19 two decades after ACE2 discovery. Clin Sci (Lond). 2020;134(21):2791-2805.
Lee IT, Nakayama T, Wu CT, et al. ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs. Nat Commun. 2020;11(1):5453.
Serfozo P, Wysocki J, Gulua G, et al. Ang II (Angiotensin II) Conversion to Angiotensin-(1-7) in the Circulation Is POP (Prolyloligopeptidase)-Dependent and ACE2 (Angiotensin-Converting Enzyme 2)-Independent. Hypertension. 2020;75(1):173-182.
Batlle D, Wysocki J, Satchell K. Soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy? Clin Sci (Lond). 2020;134(5):543-545.
Wysocki J, Ye M, Hassler L, et al. A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids. J Am Soc Nephrol. 2021.
Epelman S, Shrestha K, Troughton RW, et al. Soluble angiotensin-converting enzyme 2 in human heart failure: relation with myocardial function and clinical outcomes. J Card Fail. 2009;15(7):565-571.
Tikellis C, Bialkowski K, Pete J, et al. ACE2 deficiency modifies renoprotection afforded by ACE inhibition in experimental diabetes. Diabetes. 2008;57(4):1018-1025.
Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB, Lindsey SH, Chappell MC. Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes. Am J Physiol Renal Physiol. 2012;302(11):F1374-1384.
Wysocki J, Lores E, Ye M, Soler MJ, Batlle D. Kidney and Lung ACE2 Expression after an ACE Inhibitor or an Ang II Receptor Blocker: Implications for COVID-19. J Am Soc Nephrol. 2020;31(9):1941-1943.
The aspect of a prognostic value of baseline ACE2 for COVID-19 outcome appears to be interesting at the first glance, but considering the design of the clinical readout, it might be introduced by the fact that "Day 0" in the current study was actually the admission to the emergency department respiratory distress, which could happen at very variable time points related to disease onset. Knowing that COVID-19 severity is linked to plasma ACE2 activity with a peak in concentrations between 1 and 2 weeks after disease onset, severity driven recruitment criteria (respiratory distress at emergency department) might have introduces a certain bias, as more severe cases have higher ACE2 at time of inclusion, with obviously having poorer outcomes. In other words, patients being included with a more severe disease manifestation ion day 0 (e.g. higher ACE2), may just be at another time-point in the course of COVID-19 disease, with an already pre-determined worse outcome. It would add a lot of valuer to the study, if the measurement time points would be related to the time of symptom onset, first positive test result or any other earlier time point that may would assure the direct comparability of disease severity groups.
Response: We agree with this comment. However, we are not able to get access to a longitudinal study of samples included at symptom onset or at the time of a positive test result. We have included the comment from the reviewer in the discussion because it is obviously a limitation to the study (page 22 line 16). We also mention that our study likely reflects clinical pratice were prognostic tests will not be carried our with no or mild symptoms. We would like to thank the Reviewer for bringing up this relevant point.
”Fourth, severity driven recruitment criteria (respiratory distress at emergency department) might have introduces a bias. Thus, patients with more severe COVID-19 at admission have both higher ACE2 at time of inclusion and a poorer prognosis. However, our study reflects clinical practice where laboratory tests are performed when patients are admitted to the hospital.”
The authors should further describe the control group of 78 Covid-negative patients described more clearly. Why did these patients at emergency units with respiratory distress?
Response: We agree with this comment. We included a description of the control group as below, while we are also referencing the original study where full patient characteristics are provided (page 7 line 5).
”COVID-19-negative subjects enrolled were older than COVID-19-positive patients, less Hispanic, and with greater baseline burden of chronic illnesses. Of the 78 COVID-19-negative subjects, 37 (47%) were diagnosed with non-COVID-19 pneumonia or acute lung injury (e.g., aspiration), 12 (15%) with congestive heart failure exacerbation, 6 (7.7%) with COPD exacerbation, 3 (3.8%) with acute pulmonary embolus, 11 (14%) with non-pulmonary sepsis or infection, and 8 (10%) with other illnesses. COVID-19-negative patients were significantly less inflamed than COVID-19-positive patients, median CRP 22 [IQR 9-67] versus 105 [IQR 48-161], p-value < 0.05, but illness acuity and outcomes were very similar between the two groups.”
Finally, the use of terminology within the whole paper is misleading, as the authors keep using the terms "ACE2 levels" and "ACE2 concentrations". Of note, the result of the proximal extension assay the authors used as a basis for their interpretation is given in the form of an artificial unit (NPX), that has been calculated by the manufacturer by arithmetically linking a series of Ct value based correction algorithms. Even the manufacturers point out on their webpage that the given readout cannot be compared to actual protein levels, which disables comparability to other studies. From a technical perspective, the big open question is why no calibration of the readout is performed to be able to provide actual protein levels. With a highly reproducible and standardized method as described by the manufacturers on their webpage, it should be easy to retrospectively include a valid calibration allowing for providing actual ACE2 concentrations instead of manufacturer invented units that prevent comparability with other studies. Moreover, a certain analytical validation vor ACE2 in the used PEA panel should be shown that compares the used "NPX" values with real concentration units or standard activity units in a defined set of clinical samples.
Response: We of course agree with these limitations. Unfortunately, many factors hinder us in generating actual protein concentrations. The manufacturer cannot generate protein levels from current data, we are not able to re-analyse the same samples in a different assay, and we are not in a position to collect samples from a validation cohort. We highlighted this in the limitations as described below (page 22 line 10). We have also reworded all mentions of ”levels” and ”concentrations” with ”plasma ACE2” or ”circulating ACE”.
“The plasma ACE2 levels were measured as relative protein concentrations using NPX (Normalized Protein eXpression) values. Therefore, it is not possible to determine a plasma ACE2 cut-off value to predict severe outcome or to compare findings in this study with results in studies measuring protein concentration or enzymatic activity.”
It should also be noted that reading the method section needs some revision as it currently reads like an advertisement for the used (commercially available) technology rather than an objective method description to be published in a research paper.
Response: We specifically referred to the website where the data is publicly available, although we agree that the methods should be tailored for scientists and we have therefore removed the unnecessary text about the technology. Thanks for this comment.
”Detailed description is available online (https://
Reviewer #2: .This study investigated the association between plasma ACE2 levels and outcomes of COVID-19 patients, using clinical data and plasma samples from 306 COVID-19 positive and 76 COVID-19 negative patients. High baseline plasma ACE2 levels are reported to be associated with worse COVID-19 outcomes and patients with hypertension, pre-existing heart conditions or kidney disease had higher plasma ACE2 levels than those without.
Even as a marker I doubt very much that it will be useful for COVID-19 because the changes found in figure 2 are so small.
Response: We would like to thank the Reviewer for taking the time to help improving our manuscript. We really appreciate it. We agree with the comment that changes in ACE2 are small. Our study will require validation by others in assays designed to measure protein concentration or as mentioned further investigation with measuring ACE(1) as mentioned in the paper. We have reviewed our discussion and believe that the conclusions are balanced and in line with the reviewers comments, particularly as we have now included additional limitations of the study.
Main criticisms and suggestions for improvement:
- Overall, the paper is an excellent contribution but the authors should acknowledge the limitations of measurements of ACE2 in plasma where the levels are usually very low and even when mildly elevated in pathological conditions the significance remains uncertain. Specifically, it must be stated that ACE2 is a tissue enzyme and that the levels in the circulation are low in all species studied, including humans. Appropriate references should be given.
Response: Thank you for the comment. We completely agree that citing previous studies on measuring circulating ACE2 levels is a valuable adition. We have now added the following referneces and included a comment to highlight the limitation that ACE2 levels in circulations are low (page 4 line 22).
Epelman S, Shrestha K, Troughton RW, et al. Soluble angiotensin-converting enzyme 2 in human heart failure: relation with myocardial function and clinical outcomes. J Card Fail. 2009;15(7):565-571.
Tikellis C, Bialkowski K, Pete J, et al. ACE2 deficiency modifies renoprotection afforded by ACE inhibition in experimental diabetes. Diabetes. 2008;57(4):1018-1025.
Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB, Lindsey SH, Chappell MC. Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes. Am J Physiol Renal Physiol. 2012;302(11):F1374-1384.
”ACE2 is a tissue enzyme and circulation levels are low and the significance of measuring circulating ACE2 in pathologic conditions remains uncertain.”
Please acknowledge that the changes in COVID-19 are so small that they are not likely to be of any significance regarding the metabolism of the substrates of ACE2
Response: Thank you for bringing this to our attention. We did not consider this previously. It is an important perspective. We have mentioned this in the Discussion with the below wording (page 21 line 19):
”Importantly, changes in ACE2 levels in COVID-19 disease observed here are small and are likely not to be of any significance regarding the metabolism of the substrates of ACE2.”
- In the introduction the part on ACE2 receptor distribution must be modified to include the kidney as a main site of ACE2 . The authors do not seem aware of a critical important observation, namely that the expression of ACE2 in the lung is very low as shown by Serfozo et al using western blot and confirmed by others
See
These references must be cited as well as others showing that the kidney is next to the intestine the organ that has the highest abundance of ACE2.
Response: We agree and have added the below sentence including the mentioned references (page 4 line 12).
- Perhaps the authors are not aware that ACE2 RNA levels do not necessarily imply protein levels. Actually, ACE2 can be post translationally regulated and be increased when the levels of mRNA are not. The authors may consult and cite a review on this by Lores et al.
Response: We would like to thank the reviewer for underlining this, we have now included this important reference. However, the Olink assay measures protein, albeit with all the limitations already discussed above, therefore we did not measure RNA levels of ACE2.
- In the discussion it is stated that “ACE2 as a decoy receptor… is now being explored in COVID-19 disease.” Appropriate references should be given to be faithful to the literature. Perhaps they are not aware of these papers because they are recent but they should be cited. For instance,
Response: Thank you very much for bringing these references to our attention. Indeed, we were not aware of this progress. We have included both citations.
- “A study performed on rats showed that the use of ACE inhibitors and/or use of AT1R-blocker led to an increased expression of ACE2”. It should be specified that in ref 34 ACE2 was only examined in cardiac tissue and not kidney or lung. Others found a decrease of kidney ACE2 after ACEi and ARBs and no effect on lungs. For instance, see
Response: Again we can only thank the reviewer for updating us and improving the manuscript. We have included this in the paper.
”Others found a decrease of kidney ACE2 expression and no effect on lung ACE2 expression with ACE-inhibitors and AT1R-blockers.”
- “The population sampled on day 3 and day 7 therefore consists of patients with more severe disease compared with day 0.” Maybe ACE2 for day 3 and day 7 should be shown in a way that they are only compared to their matching samples of day 0?
Response: Thank you for this suggestion. We agree that this gives a nice representation of the data. We have included this in the Results section and shown data as Supplementary figure S1. The results are the same. We have therefore removed the above mentioned sentance in the Discussion.
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Plasma ACE2 predicts outcome of COVID-19 in hospitalized patients
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Plasma ACE2 predicts outcome of COVID-19 in hospitalized patients
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