Angiotensin converting enzyme (ACE) 1 expression in leukocytes of adults from 64 to 67 years old
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Abstract
The renin angiotensin system (RAS) is composed of several enzymes and substrates on which angiotensin converting enzyme (ACE) 1 and renin act to produce angiotensin II. ACE1 and its substrates control blood pressure, affect cardiovascular and renal function, hematopoiesis, reproduction, and immunity. The increased expression of ACE1 has been observed in human monocytes during congestive heart failure and abdominal aortic aneurysm. Moreover, T lymphocytes from hypertensive patients presented increased expression of ACE1 gene after in vitro stimulation with Angiotensin-II (AngII) with the highest ACE1 expression observed in hypertensive patients with low-grade inflammation. Our group and others have shown that aging is associated with comorbidities, chronic inflammation and immunosenescence, but there is a lack of data about ACE1 expression on immune cells during the aging process. Therefore, our aim was to evaluate the levels of ACE1 expression in non-lymphoid as compared to lymphoid cells in association with the immunosenescence profile in adults older than 60 years. Cryopreserved peripheral blood mononuclear cells obtained from blood samples were used. Cells were stained with monoclonal antibodies and evaluated by flow cytometry. We found that ACE1 was expressed in 56.9% of non-lymphocytes and in more than 90% of lymphocytes (all phenotypes). All donors exhibited characteristics of immunosenescence, as evaluated by low frequencies of Naive CD4 + and CD8 + T cells, high frequencies of EMRA CD8 + T cells, and double-negative memory B cells. These findings in addition to the increased C-reactive protein levels are intriguing questions for the study of ACE1, inflammaging, immunosenescence and perspectives for drug development or repurposing.
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Peer review report
Title: Angiotensin converting enzyme (ACE) 1 expression in leukocytes of adults from 64 to 67 years old
version: 2
Referee: Calogero Caruso MD
Institution: Professor Emeritus, University of Palermo
email: Calogero.caruso@unipa.it
ORCID iD: 0000-0001-8004-2363
General assessment
The paper is essentially anecdotal because it studies the cells of 6 subjects without any comparison with other age groups. There is also a serious limitation because beyond the age and sex there is no information on the donors (how and why they were recruited, what drugs they took, etc.). To infer that chronological and biological ages do not match is inappropriate in the absence of the above information.
However, the paper is of some interest because there are few studies on the topic.
Essential revisions that are required to verify the …
Peer review report
Title: Angiotensin converting enzyme (ACE) 1 expression in leukocytes of adults from 64 to 67 years old
version: 2
Referee: Calogero Caruso MD
Institution: Professor Emeritus, University of Palermo
email: Calogero.caruso@unipa.it
ORCID iD: 0000-0001-8004-2363
General assessment
The paper is essentially anecdotal because it studies the cells of 6 subjects without any comparison with other age groups. There is also a serious limitation because beyond the age and sex there is no information on the donors (how and why they were recruited, what drugs they took, etc.). To infer that chronological and biological ages do not match is inappropriate in the absence of the above information.
However, the paper is of some interest because there are few studies on the topic.
Essential revisions that are required to verify the manuscript
Although we do not have data on donors, placing an age and gender column in all tables adds a minimum of useful information for the reader.
Inflamm-ageing means low grade of inflammation. The value of CRP 23.1 suggests acute inflammation (also because albumin has high values, while in chronic inflammation its values decrease). Therefore the Ly averages do not have to take this subject into account.
Other suggestions to improve the manuscript
The authors write that their findings suggest that ACE1 could play a role in several processes linked to aging including the generation and activation of autoimmune cells, due to the experimental evidence that inhibitors of ACE suppress the autoimmune process in a number of autoimmune diseases such as EAE, arthritis, autoimmune myocarditis. [49] They do not appear to have these findings in their paper. So, it needs to change the sentence.
Decision
Requires revisions: The manuscript contains objective errors or fundamental flaws that must be addressed and/or major revisions are suggested.
Decision changed:
Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.
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Author response
To: Reviewer: Heikki Vapaatalo, MD, PhD, Emeritus professor of Pharmacolog
Dear reviewer Thank you very much for the insightful suggestions, the manuscript improved a lot with the changes performed. Please find the point-by-point answer to the raised questions. In the main text, all changes are highlighted in yellow. I hope that with the changes made the new version is suitable for publication.
Best regards Valquiria Bueno
General assessment
The study is interesting and the title promises for me more than the MS finally contains.
Answer: The manuscript is part of a project aiming to study ACE1 and ACE2 expression in cells from the immune system of aging and young adults. These initial results suggest that ACE1 (and probably ACE2) plays somehow a role in the process of aging.
The background, question and the aim are …
Author response
To: Reviewer: Heikki Vapaatalo, MD, PhD, Emeritus professor of Pharmacolog
Dear reviewer Thank you very much for the insightful suggestions, the manuscript improved a lot with the changes performed. Please find the point-by-point answer to the raised questions. In the main text, all changes are highlighted in yellow. I hope that with the changes made the new version is suitable for publication.
Best regards Valquiria Bueno
General assessment
The study is interesting and the title promises for me more than the MS finally contains.
Answer: The manuscript is part of a project aiming to study ACE1 and ACE2 expression in cells from the immune system of aging and young adults. These initial results suggest that ACE1 (and probably ACE2) plays somehow a role in the process of aging.
The background, question and the aim are relevant as explained in the introduction.
Answer: We included a piece of information in the “Introduction” trying to link ACE1 expression in tissue cells and age-related diseases, as it follows:
ACE1 has been suggested to influence age-related diseases (i.e. Alzheimer’s, sarcopenia, cancer) but the associated mechanisms are still under investigation. ACE1 polymorphisms were correlated with susceptibility to Alzheimer’s disease (AD). [15, 16] In addition, it was shown recently that in normal aging ACE1 expression is increased in brain homogenates and this expression is unchanged in early stages of AD. [17] Regarding sarcopenia, Yoshihara et al. [18] found a weak correlation between ACE polymorphism and physical function. In cancer (gastric or colorectal), patients presented higher expression of ECA1 in tumor when compared with healthy tissues. [19, 20]
The major criticism concerns the small size of the material (subjects, n=6), the small age difference (64-67 years) and the lack of younger controls.
Answer: We agree that the small number of studied subjects is a limitation of this study. In spite of the interesting results suggesting that ACE1 expression could be linked to the health status, it was not possible to perform correlation analysis due to the small sample size. Even though there is a small chronological difference between the subjects, the biological aging is very different among them and reflects the genetics, lifestyle, nutrition and comorbidities. Another limitation is the lack of younger controls to compare with the subjects studied. Our next steps are to include younger controls, to increase the number of studied subjects, and if possible to get samples from older subjects (i.e. 70-80, 80 and more years old)
Minor notes:
1)Title: Angiotensin converting enzyme (ACE) expression in leukocytes of older adults
Answer: We evaluated only ACE1 expression, and thus, title, abstract, and main text were changed to ACE1 instead of ACE. We decided to change to title for: Angiotensin converting enzyme (ACE) 1 expression in leukocytes of adults from 64 to 67 years old
2)Introduction: The last chapter, the Author should explain in more detail, how references 11-14 suggest that “ACE play an important role in the aging process”. Does this mean, that ACE is somehow regulating the aging process or in increasing age ACE -levels are changed?
Answer: References 11-14 shows that age-related diseases occurring in older adults are associated with changes in the immune system. To complete the text we added:
ACE1 has been suggested to influence age-related diseases (i.e. Alzheimer’s, sarcopenia, cancer) but the associated mechanisms are still under investigation. ACE1 polymorphisms were correlated with susceptibility to Alzheimer’s disease (AD). [15, 16] In addition, it was shown recently that in normal aging ACE1 expression is increased in brain homogenates and this expression is unchanged in early stages of AD. [17] Regarding sarcopenia, Yoshihara et al. [18] found a weak correlation between ACE polymorphism and physical function. In cancer (gastric or colorectal), patients presented higher expression of ECA1 in tumor when compared with healthy tissues. [19, 20]
Material and Methods:
The N-value of the subjects should be mentioned here, as well the relation of females/males.
Answer: Text was correct as suggested Blood was collected from adults (n=6, four females and two males) aged 64-67 years old in 2015.
Do the Authors really regard 64-67 “older age” nowadays?
Answer: Nowadays the most common term used for individuals older than 65 years is “older adults”.
Why first many years later the assays have been done in comparison to the collection of the blood? Are the samples still useable, not destroyed?
Answer: Samples are part of UNIFESP Biobank and have been kept in adequate conditions. We wanted to test cells from a period anterior to COVID-19 and those samples were the only ones that attended our purpose. We compared samples used in this study with fresh blood samples (cell viability and percentage of CD4+, CD8+ and CD19+) and the results showed good preservation of the cells.
Did the subjects have some diseases and/or drugs because the possibly were from hospital sample bank?
Answer: Samples are part of UNIFESP Biobank, but unfortunately we do not have information about diseases and medicaments.
Express the company details similarly than Amersham, cities and countries.
Answer: Changes were done as required ACE CD143 FITC (R&D Systems, Inc, Minneapolis, USA)
Results:
“Table 1 shows that older adults…..” The comparison between the present data and historical studies belongs to the Discussion.
Answer: Changes were done as required
Give also individual ages and gender of the subjects in the table 1.
Answer: The manuscript version sent to medrxiv@medrxiv.org had age and gender on tables, but due to their request, any possible variable that could identify the studied individual had to be removed. That is why in the present version these variables are not shown.
What means p-values here? Compared with which or interindividual differences in the particular variable? Should be explained
Answer: We used p-value for interindividual differences in each variable since individuals age differently (biological aging) and thus, physiological parameters could be affected by genetics, lifestyle, nutrition, and comorbidities. It is now explained in materials and methods
The numbering of tables and the text seems to me confusing. Only three tables, but in the text mentioned four. Number 4 does not exist.
Answer: For some reason table 2 is missing in the main text, please find the new version with Table 2 included
It would be good to have a list of abbreviations used in the description of the cell types for an unfamiliar reader.
Answer: In each figure and table we are now providing a description of cells evaluated.
Discussion:
A major part of the discussion deals with previous publications and not meaning or clinical significance of the present findings and comparison between the present and earlier studies.
Answer: The discussion was changed as suggested:
Our results show that for the studied population, chronological aging and biological aging don´t go at the same pace. Even individuals having a small chronological difference (64 to 67 years old), they are heterogeneous for physiological parameters such as glucose, urea, glycated hemoglobin (Hbglic), and C-reactive protein (CRP). Changes in the same functional parameters have been reported by Carlsson et al. [22] and Helmerson-Karlqvist [23] in healthy older adults. Carlsson’s study [22] found that CRP value was 2.6 with a coefficient variation of 1.4% whereas in our study, it was observed higher values of CRP in 5 out of 6 individuals. Increased CRP levels has been associated with inflammaging and our findings show that the studied population has changes in functional parameters which are likely associated with an inflammatory profile. [24] The link between RAS and inflammation has been suggested but its role is not completely clear under physiological and pathological conditions. [25, 26] In addition, the association between ACE1 altered expression in tissues (brain, muscle, heart and vessels) and the development and progression of age-related conditions such as Alzheimer’s, sarcopenia, and cardiovascular disease has been suggested but results are controversial. [17, 27, 28, 29, 30] There are few studies showing the association between ACE1 expression in cells from the immune system (monocytes, T cells) and the progression of kidney and cardiovascular disease. [9, 8, 31, 32]. Therefore, considering the lack of information on this issue, we questioned whether ACE1 (CD143) was highly expressed in cells from the immune system during the aging process. We found that ACE1 was expressed in almost 100% of T (CD4+, CD8+) and B lymphocytes and in all phenotypes of these cells. In non-lymphoid cells, ACE1 mean expression was 56,9%. In agreement with our findings, independent studies showed that T cells from healthy donors and monocytes from patients with congestive heart failure expressed ACE1, but there was no investigation on cell phenotype. [25, 26]. Our study is the first to show that either inexperienced (naive) or fully activated (memory) cells expresses ACE1. Our findings suggest a that the expression of ACE1 in lymphoid and non-lymphoid cells reflects the health status since our studied population presented changes in physiological parameters and high levels of ACE1 expression on immune cells. Previous independent studies showed that patients with unstable angina [32] or acute myocardial infarction [33] presented higher expression of ACE1 in T cells and dendritic cells than controls subjects. In addition, markers of the cell (lymphoid and non-lymphoid) functional status such as inflammatory or growth factors production could be modulated by ACE inhibitors (ACEi). Accordingly, mononuclear leukocytes from healthy subjects incubated with endotoxin exhibited high levels of tissue factor activity which was reduced in the presence of captopril in a dose-dependent pattern. This result could be related to the antithrombotic effect of ACEi. [34]. In patients with congestive heart failure, immune cells cultured with LPS secreted high levels of the pro-inflammatory TNF-alpha and these levels were significantly reduced in the presence of captopril. [35]
In those previous studies, also ACE2 has been reported, why not studied here?
Answer: Our next studies will be focused on ACE1 and ACE2 expression in cells from the immune system in both younger and older adults.
In the limitations, the Authors fairly mention the real problem: The small sample size, and I would like to say lack of younger subjects.
Answer: we agree with the limitations pointed and the text was changed as required:
This study have limitations such as the small sample size and the lack of young adults for comparison. As an example, the subject with the highest CRP and albumin also exhibited a high percentage of ACE1 expression on T (CD4+, CD8+), B and non-lymphoid cells in addition to the lowest percentage of CD4+ naive cells, and the highest percentage of CD8+ terminally differentiated (EMRA) and DN B cells. However, due to the small sample size it was not possible to associate the high expression of ACE1 on immune cells with inflammaging and immunosenescence. It would bring important information to correlate physiological parameters/health status with ACE1 expression and to find out whether age and associated chronic diseases could lead to increased ACE1 expression.
The COVID-19 point even tempting today, is too far from this study and unnecessary. Answer: Our point was to emphasize the negative impact of chronic diseases for the outcome of aging population during a viral infection and how ACE1/ACE2 expression could bring information to diagnosis and treatment. Therefore, we would like to maintain this piece of information.
Linguistic checking would improve the MS. Answer: We checked for possible linguistic mistakes
Reviewer, Heikki Vapaatalo:
I read with pleasure the very detailed answers to my comments.
I very warmly recommend acceptance of this MS for publications without any further notes.
Decision changed:
Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.
To: Reviewer: Calogero Caruso
Dear Prof.Caruso Thank you very much for the revision of this manuscript. It is a privilege to have a manuscript reviewed by a research with high expertise on the field of ageing. Please find the answers to your questions and in the main text the changes in bold.
Sincerely yours,
Valquiria Bueno
The paper is essentially anecdotal because it studies the cells of 6 subjects without any comparison with other age groups. There is also a serious limitation because beyond the age and sex there is no information on the donors (how and why they were recruited, what drugs they took, etc.).
It is really a limitation to have only 6 individuals for the study, but they were the only ones fitting in the proposal of the manuscript. The samples were from a central bank of cells at UNIFESP and participants were considered “healthy” but there was not further information in addition to what we displayed on the tables of the manuscript. They were not living on homecares or hospitalized.
Our aim was to evaluate samples from individuals aged 60-69 years previously to COVID-19 and/or vaccination. In addition, there were no samples in the same conditions (PBMCs, -80oC) of young individuals and using fresh blood could bring a result that could not be compared mainly regarding to myeloid cells and B cells as is follows in the below reference. Braudeau C, Salabert-Le Guen N, Chevreuil J, Rimbert M, Martin JC, Josien R. An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses. Cytometry B Clin Cytom 2021;100(6):652-665. doi: 10.1002/cyto.b.21994.
Our goal from now on is to expand this study with young and old adults samples since it is important to understand whether ageing is associated with an increase in ACE expression on immune cells.
-To infer that chronological and biological ages do not match is inappropriate in the absence of the above information.
This piece of information regarding chronological and biological age was required by another reviewer. I agree that the concept does not match without more information on the donors. However, the information is now referenced and should be considered when older adults are studied. Vasto S, Scapagnini G, Bulati M, Candore G, Castiglia L, Colonna-Romano G, Lio D, Nuzzo D, Pellicano M, Rizzo C, Ferrara N, Caruso C. Biomarkes of aging. Front Biosci (Schol Ed) 2010;2(2):392-402. doi: 10.2741/s72. PMID: 20036955.
-However, the paper is of some interest because there are few studies on the topic.
Thanks for this positive comment. Few studies on the topic was the reason why we decided to send the manuscript for publication even though there were some important information on the donors missing and limited number of individuals.
Essential revisions that are required to verify the manuscript
- Although we do not have data on donors, placing an age and gender column in all tables adds a minimum of useful information for the reader.
The first table submitted with age, but for requirement of MedRxiv, gender and age could no be linked to the metabolic results to preserve the anonymity of the donors.
- Inflamm-ageing means low grade of inflammation. The value of CRP 23.1 suggests acute inflammation (also because albumin has high values, while in chronic inflammation its values decrease). Therefore the Ly averages do not have to take this subject into account.
Thank you for this comment. In a review of literature it was found an article (below) with CRP variation from 0.1 to 19.8 (Heumann Z, Youssim I, Kizony R, Friedlander Y, Shochat T, Weiss R, Hochner H, Agmon M. The Relationships of Fibrinogen and C-Reactive Protein With Gait Performance: A 20-Year Longitudinal Study. Front Aging Neurosci 2022;14:761948. doi: 10.3389/fnagi.2022.761948). There is also an article from your group showing CRPs <5g/dL and >5g/dL (Cancemi P, Aiello A, Accardi G, Caldarella R, Candore G, Caruso C, Ciaccio M, Cristaldi L, Di Gaudio F, Siino V, Vasto S. The Role of Matrix Metalloproteinases (MMP-2 and MMP-9) in Ageing and Longevity: Focus on Sicilian Long-Living Individuals (LLIs). Mediators Inflamm 2020;2020:8635158. doi: 10.1155/2020/8635158) that will be used to discuss how ageing impacts CRP levels. Considering the already small number of donors, data were maintained and statistics (mean + SD) with and without 23.1 mg/dL are now shown.
This will be the new version (discussion) about CRP Carlsson’s study [22] found that CRP value was 2.6 with a coefficient variation of 1.4% whereas in our study, it was observed higher values of CRP in 5 out of 6 individuals. In addition, it was shown by Cancemi et al. in an evaluation of individuals from 40 years to older than 95 years (long-living) that CRP increases in an age-dependent manner. Increased CRP levels has been associated **with low grade of chronic inflammation (inflammaging) ** and our findings show that the studied population has changes in functional parameters which are likely associated with an inflammatory profile. [24] However, an individual presented CPR 23.1 mg/dL suggesting acute inflammation instead, but as all donors were not hospitalized or living on homecares, this sample was considered as part of the study. Another study evaluating gait speed found CRPs varying from 0.1 to 19.8mg/dL (Front Aging Neurosci 2022;14:761948.). Our study has an important limitation that is the lack of data on donors such as the use of continuous medicaments or sarcopenia, hypertension, cognition, among others, and thus it was not possible to correlate CRP with age-related conditions.
Table 1. Updated
Other suggestions to improve the manuscript The authors write that their findings suggest that ACE1 could play a role in several processes linked to aging including the generation and activation of autoimmune cells, due to the experimental evidence that inhibitors of ACE suppress the autoimmune process in a number of autoimmune diseases such as EAE, arthritis, autoimmune myocarditis. [49] They do not appear to have these findings in their paper. So, it needs to change the sentence.
Sentence changed to: According to experimental evidence, ACE inhibitors suppress the autoimmune process in a number of autoimmune diseases such as EAE, arthritis, autoimmune myocarditis. [49] Extrapolating these findings to our results, it is possible to suggest that ACE1 play a role in several processes linked to aging including the generation and activation of autoimmune cells.
Rviewer: Calogero Caruso
Decision changed:
Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.
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Peer review report
Title: Angiotensin converting enzyme (ACE) expression in leukocytes of older adults
version: 1
Reviewer: Heikki Vapaatalo, MD, PhD, Emeritus professor of Pharmacology
Institution: Department of Pharmacology, Medical Faculty,University of Helsinki, Finland
email: heikki.vapaatalo@helsinki.fi
General assessment
The study is interesting and the title promises for me more than the MS finally contains.
The background, question and the aim are relevant as explained in the introduction.
The major criticism concerns the small size of the material (subjects, n=6), the small age difference (64-67 years) and the lack of younger controls.
In the following minor notes:
Title: ACE > better ACE1, or does the sophistic, elegant method include both ACE:s? The same should be explained and taken into consideration throughout the text.
I…
Peer review report
Title: Angiotensin converting enzyme (ACE) expression in leukocytes of older adults
version: 1
Reviewer: Heikki Vapaatalo, MD, PhD, Emeritus professor of Pharmacology
Institution: Department of Pharmacology, Medical Faculty,University of Helsinki, Finland
email: heikki.vapaatalo@helsinki.fi
General assessment
The study is interesting and the title promises for me more than the MS finally contains.
The background, question and the aim are relevant as explained in the introduction.
The major criticism concerns the small size of the material (subjects, n=6), the small age difference (64-67 years) and the lack of younger controls.
In the following minor notes:
Title: ACE > better ACE1, or does the sophistic, elegant method include both ACE:s? The same should be explained and taken into consideration throughout the text.
Introduction: The last chapter, the Author should explain in more detail, how references 11-14 suggest that “ACE play an important role in the aging process”. ACE plays. Does this mean, that ACE is somehow regulating the aging process or in increasing age ACE -levels are changed?
Material and Methods: The N-value of the subjects should be mentioned here, as well the relation of females/males. Do the Authors really regard 64-67 “older age” nowadays? Lack of younger controls! Why first many years later the assays have been done in comparison to the collection of the blood? Are the samples still useable, not destroyed? Did the subjects have some diseases and/or drugs because the possibly were from hospital sample bank? Express the company details similarly than Amersham, cities and countries.
Results: “Table 1 shows that older adults…..” The comparison between the present data and historical studies belongs to the Discussion. Give also individual ages and gender of the subjects in the table 1. What means p-values here? Compared with which or interindividual differences in the particular variable? Should be explained The numbering of tables and the text seems to me confusing. Only three tables, but in the text mentioned four. Number 4 does not exist. It would be good to have a list of abbreviations used in the description of the cell types for an unfamiliar reader.
Discussion: A major part of the discussion deals with previous publications and not meaning or clinical significance of the present findings and comparison between the present and earlier studies. In those previous studies, also ACE2 has been reported, why not studied here? In the limitations, the Authors fairly mention the real problem: The small sample size, and I would like to say lack of younger subjects. The COVID-19 point even tempting to-day, is too far from this study and unnecessary. Linguistic checking would improve the MS.
As a summary: I recommend the acceptance of the MS for publication after the Authors careful rethinking of the message of the results and correction of the minor comments. I hope that in the future the possible age -related correlations to old age up to >80 years would be possible.
Decision
Verified with reservations: The content is scientifically sound but has shortcomings that could be improved by further studies and/or minor revisions.
Decision changed:
Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.
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