Angiotensin converting enzyme 2 level and its significance in COVID‐19 and other diseases patients

Abstract

Background

Angiotensin‐converting enzyme 2 (ACE2) expressions and its modulation are of great interest as being a key receptor for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV2) and the protective arm of the rennin‐angiotensin axis, maintaining cardiovascular homeostasis. However, ACE2 expressions and their modulation in the healthy and disease background are yet to be explored.

Method

We performed a meta‐analysis, extracting the data for ACE2 expression in human subjects with various diseases, including SARS‐CoV2 infection without or with co‐morbidity. The Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines were followed. Out of 203 studies, 39 met the inclusion criteria with SARS‐CoV2 patients without co‐morbidity, SARS‐CoV2 patients with co‐morbidity, cardiovascular (CVD) patients, diabetes patients, kidney disorders patients, pulmonary disease patients, and other viral infections patients.

Results

Angiotensin‐converting enzyme 2 expression was significantly increased in all diseases. There was an elevated level of ACE2, especially membrane‐bound ACE2, in COVID‐19 patients compared to healthy controls. A statistically significant increase in ACE2 expression was observed in CVD patients and patients with other viral diseases compared to healthy subjects. Moreover, subgroup analysis of ACE2 expression as soluble and membrane‐bound ACE2 revealed a remarkable increase in membrane‐bound ACE2 in CVD patients, patients with viral infection compared to soluble ACE2 and pooled standard mean difference (SMD) with the random‐effects model was 0.37 and 2.23 respectively.

Conclusion

It was observed that utilizing the ACE2 by SARS‐CoV2 for its entry and its consequence leads to several complications. So there is a need to investigate the underlying mechanism along with novel therapeutic strategies.

Our review and meta‐analysis substantially add novel knowledge to our understanding of ACE2 expression pattern, its dynamic as soluble and membrane‐bound form. An elevated level of ACE2 in patients with all diseases was observed. Moreover, membrane‐bound ACE2 was remarkably high in patients with SARS‐CoV2 infected patients having co‐morbidities, cardiovascular diseases, and other viral diseases compared to healthy population.

Abbreviations

ACE2

Angiotensin‐converting enzyme 2

CVD

Cardiovascular

EC number

Enzyme Commission number

HPA

Human Protein Atlas

PCT

Proximal convoluted tubule

PRISMA

Reporting Items for Systematic Reviews and Meta‐Analyses

RAS

Renin‐angiotensin system

RBD

Receptor‐binding domain

SARS‐CoV2

Severe acute respiratory syndrome coronavirus 2

SMD

Standard Mean Difference

TMPRSS2

Transmembrane serine protease 2

1. INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV2) is the seventh coronavirus spread all over the globe since December 2019, and the disease is called COVID‐19. Earlier found coronaviruses were SARS‐CoV, MERS‐CoV, SARS‐CoV‐2 HKU1, NL63, OC43, 229 E and were reported to cause mild to severe symptoms. However, SARS‐CoV2 led to unusual pathophysiology pressing the global economy, health at the hardest. ¹ , ²

Severe acute respiratory syndrome coronavirus 2 has a high affinity for angiotensin‐converting enzyme 2 (ACE2; EC number – 3.4.17.23), and ACE2 is another enzyme involved in the renin‐angiotensin system (RAS). The active end product of the RAS cascade is Angiotensin II (AngII), derived from angiotensin I (Ang I), and angiotensin‐converting enzyme (ACE; EC number ‐ 3.4.15.1), commonly found in vascular endothelial cells and lungs, catalyses the process. It is a classical arm of the RAS system. Ang II acts on angiotensin receptors AT1 and AT2. The activation of the AT1 receptors results in vasoconstriction, inflammatory, prothrombotic and arrhythmogenic effects, whereas activation of AT2 receptors antagonize the action of AT1. ACE2 further degrades Ang II into angiotensin 1–7 (Ang‐ (1–7)), and it acts on the MAS receptor. The activation of MAS receptors causes vasodilation, anti‐inflammatory, thrombotic and anti‐arrhythmogenic effects, and it is called a protective arm of the RAS system (Figure S1).

Angiotensin‐converting enzyme 2 acts as a receptor of the SARS‐CoV2 protein and facilitates virus entry into host cells, as depicted in Figure S2. The S‐protein of SARS‐CoV2 SPIKE protein binds to ACE2 receptors of host cells and activates viral spike glycoprotein and cleavage of a c‐terminal segment of ACE2 by proteases like transmembrane serine protease 2 (TMPRSS2) and FURIN, which express mainly in lung tissues. Followed by the fusion with the ACE2 receptor, it delivers the viral nucleo‐capsid inside the cell for subsequent replication. The S‐protein is responsible for syncytial generation between infected cells and other receptor‐bearing cells. ³ , ⁴

Angiotensin‐converting enzyme 2 has a major role in the development of diabetes and in diabetic nephropathy (Figure S3). In pancreatic acini and islet, the distribution of ACE2 is similar to that of ACE, while in the kidney, it is present in the proximal convoluted tubule (PCT) and in the glomerulus. ACE2 is the main target to reduce Ang II expression for treating kidney diseases in diabetic conditions because it is evident that there is a decrease in ACE2 expression associated with albuminuria and glomerular lesions disorder. ⁵ Ang II is highly expressed in the pancreas with low levels of Ang (1–7), and the expression is several fold high in the pancreas compared to plasma, denoting their role in pancreatic functioning. Blockade of Ang II or angiotensin receptors had been associated with increase blood flow and insulin secretion. Several preclinical and clinical studies reported the restoration of glucose homeostasis with RAS pathway inhibition against the diabetic disease backdrop and improvement in clinical outcome. Attenuated ACE2 levels had also been associated with diabetes. ⁶ The plausible underlying mechanism would be a reduced ACE2 expression causing an elevation of Ang II proteins, which delays insulin secretion and decreases blood flow to the islets of Langerhans. Thus the use of ACE inhibitors or Angiotensin II antagonists decreases the incidence of type II diabetes mellitus. ⁶ , ⁷ , ⁸

Severe acute respiratory syndrome coronavirus 2 attenuates the ACE2 levels, essential for the conversion of Ang II to Ang (1–7). Thus, Ang II accumulates and binds to the angiotensin type 1 receptor (AT1R) on the immune cell membrane; this process activates the NF‐κB pathway. It, in turn, induces IL‐6, TNF‐α, IL‐1β, and IL‐10 production. Ang II activates AT1R and regulates mitogen‐activated protein kinases (MAPK) (ERK1/2, JNK, p38MPK) to release the cytokines IL‐1, IL‐10, IL‐12, and TNF‐α. ⁹

The present review and meta‐analysis were designed with the following objectives.

1. To determine the expression of ACE2 in various diseases like COVID‐19, COVID‐19 with comorbidity, other viral infections, diabetes, cardiovascular diseases, pulmonary diseases and kidney diseases.

2. To evaluate the ACE2 expression comparing soluble and membrane‐bound ACE2 in all diseases.

3. To analyse the effect of associated co‐morbidity on the ACE2 expression in COVID‐19.

4. To determine the correlation between various diseases and disorders concerning ACE2 expression using various statistical methods.

2. METHODS

2.1. Search strategy and selection criteria

The ACE2 expression in patients with SARS‐CoV‐2, other diseases and disorders (cardiovascular diseases (CVD), diabetes, pulmonary disorders, kidney disorders, other viral infections) retrieved from National Centre for Biotechnology Information (NCBI). In addition, a search was performed using SCOPUS, WEB OF SCIENCE databases with keywords alone or a combination of them (ACE2, its expression in various diseases, Protein expression of ACE2, mRNA expression of ACE2 in patients having SARS‐CoV‐2, diabetes, pulmonary diseases, CVS, kidney diseases, ACE2 expression in viral infection). Further, the reference list of selected articles was screened as a complementary search. The articles published in the English language were selected. In this study, information on the detection of ACE2 levels either as mRNA or protein using various methods is included. Next, the collected data from different articles were categorized into several groups according to the disease and co‐morbidities associated with the disease. The ACE2 expression is segregated into membrane‐bound ACE2 and soluble ACE2 in circulation. Reporting of the study conforms to broad EQUATOR guidelines. ¹⁰ The Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines were followed. ¹¹ The protocol was registered in the registry PROSPERO (CRD42022302513).

2.2. Inclusion criteria

The studies that reported ACE2 expression in patients with different diseases were included. ⁵ , ⁷ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² , ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ Following the removal of duplicate datasets, the unique datasets were collected. All biopsies' studies related to ACE2 expression in various diseases were also included in the study. Articles were initially screened by titles and abstracts, followed by a full article review to identify eligible studies. Original research articles included if they fulfilled the criteria and provided the data of clinical features and outcome of patients having laboratory‐confirmed tests showing the infection, COVID‐19, and other diseases. The studies having healthy and disease subjects were included for analysis. Discordant results were resolved by discussion between the authors of the studies. Studies reporting haematological, biochemistry and serological laboratory tests, as well as COVID‐19 related comorbidities and clinical outcomes, were selected. The study provided usable quantitative data for estimating 95% confidence intervals. Other information about the ACE2 at mRNA and protein levels across human tissues and recorded pathologies were gathered from Human Protein Atlas (HPA) database for reference purpose only.

2.3. Exclusion criteria

We excluded animal studies, review articles, and consensus documents. The exclusion criteria were as follows:

1. Review articles, abstracts, letters to the editor, clinical trials, animal studies, comments and consensus documents were excluded from the study.

2. The study did not focus on patients with COVID‐19, diabetes, CVD, Pulmonary diseases, other viral infections or kidney disorders, as well as when the diagnosis was unclear.

3. Studies that included animal‐based experiments and duplicates of previous publications.

4. Studies with in vitro cell‐based assays and unpublished studies.

2.4. Data extraction and statistical analysis

Data were extracted from the studies considering key characteristics including author(s), publication year, journal name, country, and type of study, the race of patients, ACE2 expression, either soluble or membrane‐bound, laboratory findings, comorbidities and final clinical outcomes (Table 1 and Table S1).

TABLE 1.

Details of selected studies

Study	Journal Name	Population	Type of population	Method of detection	Soluble or membrane bound ACE2 (sACE2 or mACE2)	Tissue / expression site
Wijnant et al 2020	Diabetes	106	Caucasian	RTPCR (Protein expression)	mACE2	Lung tissue
G.Y. Oudit et al 2009	European Journal of Clinical Investigation	20	Caucasian	RTPCR and Western blot	mACE2	Autopsy of heart tissue
HN Reich et al 2008	Kidney International	21	Caucasian	Immuno‐histochemistry &RTPCR	mACE2	Renal biopsies
Batlle et al 2006	The Journal of Heart and Lung Transplantation	12	Caucasian	RTPCR	mACE2	Left ventricular biopsies
Purushothaman et al 2013	Cardiovascular Pathology	24	Caucasian	Immunohistochemistry	mACE2	Arteries
Sharif‐Askari et al 2020	Molecular Therapy: Methods &Clinical Development	541	Caucasian	Microarray expression (PBMC)	mACE2	Lung airway and peripheral blood mononuclear cells
Xiao et al 2012	PLoS ONE	150	Caucasian	ELISA (solubleACE2)	sACE2	Urine
Yang et al 2017	RENALFAILURE	360	Asian	ELISA (with HD)	sACE2	Venous blood samples
Guo et al. 2020	Cellular Physiology	18	–	Immunofluorescence (H929andcalu3cells)	mACE2	Normal and fibrotic lung tissues, normal and failed heart tissue.
Hongjing Gu et al 206	Scientific Reports	54	Asian	Western blot and histology	sACE2	Plasma*
Smith et al 2020	Developmental Cell		Caucasian	RTPCR (Human airway epithelial cells)	mACE2	Expression studies (Mouse lungs)
Chen et al 2020	European Society of Cardiology	55	Asian	RTPCR	mACE2	Human heart tissue
Goulter et al 2014	BMC Medicine	32	Caucasian	QRTPCR	mACE2	Human ventricular myocardium
Liu H and Zhe zang et al 2020	European Society of Cardiology	20	Caucasian	QRTPCR	mACE2	Human colorectal tumour and colorectal normal tissues
Ramachand et al 2018	PLoS ONE	79	Asian	Fluorescent assay	mACE2	Fasting blood samples
Anguiano et al. 2015	Nephrol Dial Transplant	2572	Caucasian	Fluorescent assay	sACE2	Plasma
Trojanowics et al. 2016	Nephrol Dial Transplant	61	Caucasian	RTPCR&westernblot	mACE2	Monocytes
SE Park et al 2013	NephrolDialTransplant	621	Asian	ELISA	sACE2	Blood
Herman‐Edelstein et al 2021	CardiovascularDiabetology	79	Caucasian	QRTPCR & ELISA	mACE2	Right atrial tissues
MARobert et al 2013	Nephroldialtransplant	159	Caucasian	QUENCHED FLUORESCENCESUBSTRATEBASEDASSAY	sACE2	Blood
Osman et al 2021	Frontier microbiology	44	Caucasian	RTPCR	mACE2	Blood
T E Walters et al 2016	European society of cardiology	45	Caucasian	quenched fluorescence assay	sACE2	Blood
van lier D et al 2021	Europen respiratory society	15	Caucasian	LC–MS	sACE2	Blood
S K Patel et al 2021	Europen respirtaory journal	70	Caucasian	ELISA	sACE2	Blood plasma
Peters et al 2020	American journal of respiratory and critical care medicine	409	Caucasian	RTPCR	mACE2	Sputum cells
G Li et al 2020	Journal of autoimmunity	183	Asian	RTPCR	mACE2	Data from six independent studies (Lung tissue)
L He et al 2006	Journal of pathology	5	Asian	Immunohistochemistry	mACE2	Lung tissue, heart tissue, etc.
Bao et al 2021	Journal of inflammatory research	64	Asian	RTPCR	mACE2	Lung tissue
Tue W Kragstrup et al 2021	European respiratory journal	384	Caucasian	RTPCR	sACE2	Blood
Michal Herman Edelstein et al 2021	Cardiovascular Diabetology	79	Caucasian	RT‐ PCR & immunohistochemistry	mACE2	Atrial biopsies
Elemam et al 2021	International journal of molecular science	100	Caucasian	qRTPCR	sACE2	Serum
Cousele‐Seijas et al 2020	European Journal of Clinical Investigation	43	Caucasian	ELISA	sACE2	Epicardial and subcutaneous fat biopsies

We calculated Standard Mean Difference (SMD) for continuous data with a 95% confidence interval (CIs). Statistical heterogeneity was assessed using inconsistency index (I ²) statistics. When I ² > 50%, a random‐effects model was selected. The p‐value <.05 and I ² ≥ 50% were considered to have significant heterogeneity. The risk bias analysis was done and presented as a matrix and summary graph. The forest plot was used to assess the overall effect of the included studies. All data were analysed using RevMan software version 5.4.1, The Cochrane collaboration 2020. ⁴⁴

As ACE2 expression was archived from studies having different methods of ACE2 measurement, the data was subjected to normalization. The ACE2 expression in disease patients was calculated as a fold change compared to healthy subjects, as depicted in Tables S2–S8.

We performed statistical analysis of the transformed and pooled data using GraphPad Prism (GraphPad Prism Version 9.2) software. The data were analysed using the nonparametric Kruskal–Wallis test for multiple variable analysis and comparison. We used a two‐tailed p‐value with a statistical significance threshold of .05. Correlation coefficients were calculated using the Spearman correlation coefficient method for subgroup analysis.

2.5. Assessment of methodological quality

Please see Appendix S1.

3. RESULTS

3.1. Search strategy and study design

Total 203 studies were identified according to the search criteria. After removing duplicated records, records noncompliant with inclusion criteria, and reviewing abstracts and titles, 55 articles were selected for the study. However, 15 articles did not report the number of healthy or disease subjects, hence those articles were removed. Finally, 39 articles (n = 39) were selected for meta‐analysis, as shown in a flow diagram (Figure 1 and Tables S1 and S2). These studies include 4686 subjects with diseases (COVID‐19, Diabetes, Cardiovascular diseases (CVD), Pulmonary infections, Viral infections, Kidney disorders) and 3040 healthy subjects. [(1–15)].

FIGURE 1.

Studies included in the review and meta‐analysis is depicted using a PRISMA flow chart.

The expression of ACE2 for different diseases and healthy subjects was summarized in Table 1 and Table S1. The populations of the included studies are of Caucasian and Asian origin. The expression of ACE2 was detected with various techniques like RT‐PCR, ELISA, Western blot, and Fluorescent assay, as depicted in Table 1. Of these 39 articles, five articles compared ACE2 expression in COVID‐19 patients, nine articles compared ACE2 expression in cardiovascular disease (CVD), eight articles compared in diabetic patients, four articles compared in pulmonary diseases, three articles compared in kidney disease, five articles in other viral infection and six articles compared in COVID19 patients with either DM or CVD.

The expression of ACE2 data was selected for meta‐analysis, depicted in Table S1. The ACE2 expression for nonparametric statistical analysis was normalized, and fold changes were calculated, and soluble ACE2 is highlighted (Tables S2–S8).

3.2. Meta‐analysis of ACE2 expression in all diseases vs. healthy subjects

The meta‐analysis of ACE2 expression in all diseases compared with healthy subjects is depicted in Figure 2A. The level of ACE2 was significantly higher in subjects with diseases as compared to healthy subjects, and pooled standard mean difference (SMD) with the random‐effects model was .13, 95% CI: −.02, .29, z = 1.67, p = .10, as shown in Figure 2A. The included studies contributed to high heterogeneity I ² = 84% with τ² = .16, Chi² = 234.11, df = 38, p < .00001.

FIGURE 2.

Forest plot of studies, which measured (A) Total angiotensin‐converting enzyme 2 (ACE2) (B) soluble and membrane bound ACE2 in diseases compared to healthy subjects. The overall effect size was presented as the standard mean difference with 95% CI and inverse variance (IV) method. The square (blue colour) represents the sample size and corresponding weightage.

The ACE2 is present in the human body as free circulating ACE2, termed as soluble ACE2, as well as membrane‐bound ACE2. Therefore, we segregated the data into ACE2 soluble and membrane‐bound ACE2 in disease patients and subjected them for analysis to evaluate the predominance of any form in the backdrop of disease, as shown in Figure 2B. The subgroup analysis of ACE2 expression as membrane‐bound ACE2 (mACE2) and soluble ACE2 (sACE2) showed higher membrane‐bound ACE2 in all diseases compared to a soluble form. The pooled standard mean difference (SMD) with the random‐effects model for membrane bound ACE2 was .21, 95% CI: −.02, .43, z = 1.76, p = .008, as shown in Figure 2B. The 75% heterogeneity was observed with included studies.

3.3. ACE2 expression in COVID‐19 without comorbidities and COVID‐19 with co‐morbidities

The membrane‐bound and soluble ACE2 expression in COVID‐19 patients without comorbidities were analysed and compared with healthy subjects. The soluble ACE2 expression was higher in subjects with COVID‐19 as compared to healthy subjects, and pooled standard mean difference (SMD) with the random‐effects model was .44, 95% CI: −.08, .96; z = 1.65 (p = .10). The included studies showed high heterogeneity 71% (τ² = .21, Chi² = 14.67, df = 4, p = .005, I ² = 73%) (Figure 3A). The membrane‐bound ACE2 was lower in COVID‐19 patients without any comorbidities (Figure 3A), and pooled standard mean difference (SMD) with the random‐effects model was −.28, 95% CI: −.19, .79; z = .87 (p = .38).

FIGURE 3.

Forest plot of studies, which measured soluble and membrane‐bound angiotensin‐converting enzyme 2 in (A) COVID19 patients, (B) COVID19 patients with comorbidities, (C) cardiovascular (CVD) patients, (D) diabetes patients, (E) kidney disorders patients, (F) pulmonary disease patients and (G) patients with other viral infection compared to healthy subjects. The overall effect size was presented as the standard mean difference with 95% CI and inverse variance (IV) method. The square (blue colour) represents the sample size and corresponding weightage.

The membrane–bound and soluble ACE2 levels in COVID‐19 patients with comorbidities (Table S1) were found to be high compared to healthy subjects, as shown in Figure 3B. The pooled standard mean difference for membrane‐bound ACE2 was .41, and 95% CI: −.24, 1.05; z = 1.23 (p = .22). The included studies showed heterogeneity of 90% (τ² = .2, Chi² = 9.6, df = 1, p = .02, I ² = 90%) (Figure 3B). The SMD for soluble ACE2 was .10, 95% CI: −.03, .23; z = 1.43 (p = .15). The included studies showed heterogeneity of 0% (τ² = .00, Chi² = 1.99, df = 3, p = .58, I ² = 0%).

3.4. ACE2 expression in CVD

As shown in Figure 3C, the membrane‐bound ACE2 expression was significantly higher in subjects with CVD as compared to healthy subjects, and pooled standard mean difference (SMD) with the random‐effects model was .37, 95% CI: .13, .62, z = 2.97 (p = .003). Heterogeneity was found to be 0% (τ² = .00, Chi² = 5.76, df = 6, p = .45, I ² = 0%). The soluble ACE2 expression was in favour of healthy than CVD patients and pooled SMD was −.50, 95% CI: −2.12, 1.11, z = .61 (p = .54) with significant heterogeneity and it was 96% (τ² = 1.30, Chi² = 25.44, df = 1, p < .00001, I ² = 96%).

3.5. ACE2 expression in diabetes

The membrane‐bound and soluble ACE2 expression in diabetes patients did not differ significantly compared to healthy subjects (Figure 3D). The pooled standard mean difference (SMD) with the random‐effects model for membrane bound ACE2 was 1.31, 95% CI: −.49, 3.11, z = 1.43, p = .64. Heterogeneity was found to be 93%, with τ² = 2.63, Chi² = 41.02, df = 3, p < .00001, as depicted in Figure 3D. The soluble ACE2 SMD was 0, 06, 95% CI: −.11, .23, z = .67, p = .50. Heterogeneity was found to be 0%, with τ² = 0, Chi² = .04, df = 3, p < .00001.

3.6. ACE2 expression in kidney diseases

As shown in Figure 3E, the soluble and membrane‐bound ACE2 expression did not change in patients with kidney diseases in comparison to healthy subjects. However, the soluble ACE2 was higher in kidney patients compared to membrane‐bound. The pooled standard mean difference (SMD) with the random‐effects model for soluble ACE2 was .04, 95% CI: −.14, .22; z = .42, p = .67. Heterogeneity was found to be I ² = 38%, with τ² = .01, Chi² = 1.61, df = 1, p = .20. For membrane‐bound ACE2, SMD was −.33, 95% CI: −.85, .18; z = 1.27, p = .21.

3.7. ACE2 expression in pulmonary diseases

The membrane‐bound ACE2 expression did not differ between pulmonary diseases patients and healthy subjects, and pooled standard mean difference (SMD) with the random‐effects model was .01, 95% CI: −.14, .16; z = .15, p = .88. Heterogeneity was found to be 0% (τ² = .00, Chi² = .07, df = 3, p = .99, I ² = 0%) (Figure 3F).

3.8. ACE2 expression in other viral infections

The analysis of membrane‐bound ACE2 levels indicated a difference in patients with a viral infection and healthy subjects, and pooled standard mean difference (SMD) with the random‐effects model was 2.23, 95% CI: −1.01, 3.44, z = 3.59, p = .0003 (Figure 3F). Heterogeneity was found to be I ² = 0%, with τ² = .00, Chi² = .04, df = 1, p = .84. The soluble ACE2did does not show any difference in healthy and disease subjects, as depicted in Figure 3F.

3.9. Multivariate analysis of ACE2 expression in all diseases vs. healthy subjects

The fold change in total ACE2 expression significantly increased in patients with viral infections, diabetes, cardiovascular diseases, pulmonary diseases, and SARS‐CoV2 infection, except for kidney disorders, as shown in Figure 4A. The total ACE2 expression in patients with CVD and viral infections showed statistically significant elevated levels compared to healthy subjects, and these results are in line with meta‐analysis data.

FIGURE 4.

The violin rank plot of angiotensin‐converting enzyme 2 (ACE2) expression (fold change) showed an elevated levels in all diseases except kidney disorders. The numbers in parenthesis indicate the total subjects. The solid (−) and dashed (‐‐) lines indicate the median value and interquartile range, respectively. *p < .05 indicates statistical significance vs healthy subjects and # p > .05 statistically nonsignificant vs healthy subjects. (B, C) The heat map of ACE2 expression in various diseases and their correlation were analysed with the Spearman correlation coefficient method.

Spearman correlation of total ACE expression among different diseases showed a significant positive association. ACE2 expression in COVID‐19 and CVD showed a significant positive association with a correlation coefficient (r) of .97 (Figure 4B). Similarly, ACE2 expression in diabetes, kidney disorders and other viral infections showed a positive association with a correlation coefficient (r) of .94 and .88, respectively. COVID‐199 patients with co‐morbidities and patients with pulmonary diseases showed a positive association for ACE2 expression with a correlation coefficient (r) of .87, as demonstrated in Figure 4A,B. The soluble and membrane‐bound ACE2 were analysed for Spearman correlation and found to have an inverse association with a correlation coefficient (r) of −.02 as shown in Figure 4C.

4. DISCUSSION

4.1. ACE2 levels and its significance

Angiotensin‐converting enzyme 2 is a crucial access point for SARS‐CoV2 in host cells, and the expression of ACE2 and their dynamic as soluble and membrane‐bound ACE2 are of utmost importance, and deliberation on it remains unknown to date. ⁴⁵ , ⁴⁶ The computational approach depicted that electrostatic features drives the binding affinity of SARS‐CoV2 towards ACE2. Several changes in the SARS‐CoV‐2 receptor binding domain residue increase the binding affinity of SARS‐CoV‐2 to ACE2. ⁴⁷ Additionally, the ACE2‐binding ridge in the SARS‐CoV‐2 receptor binding domain has a more compact conformation. The binding of ACE2 to coronavirus includes a series of events that results into the fusion of viral and host cell membrane for entry. It further leads to the dissociation of S1 and S2 proteins of the virus, which prompts the transition of S2 from a metastable pre‐fusion to a more stable post‐fusion state required for membrane fusion. ³ , ⁴⁸ , ⁴⁹

Severe acute respiratory syndrome coronavirus 2 virus identifies ACE2 as its receptor via receptor‐binding domain (RBD), which is ever‐shifting among standing‐up position for receptor binding and a lying‐down position for immune evasion. ⁵⁰ Additionally, the spike also required activation by proteolysis at the S1/S2 boundary, so that S1 dissociates and S2 undergoes a dramatic structural change. The cell surface and lysosomal proteases, TMPRSS2 and cathepsin, are necessary for protease activity which will facilitate immediate spread in the host. Upon fusion with the host membrane with a virus, intact ACE2 or its transmembrane domain is internalized with the virus. Spike glycoprotein does not block the catalytical active site of ACE2, thus, the overall process of fusion is independent of the peptidase activity of ACE2. ³ Some other transmembrane proteases are also involved in the process of binding and membrane fusion are disintegrin and metallopeptidase domain 17 [ADAM17], transmembrane protease serine 2 [TMPRSS2], TNF‐converting enzyme. ⁶ The ADAM17 is a TNF convertase that facilitates the cleavage of ACE2 for ectodomain shedding, while TMPRSS2 cleaves ACE2 to facilitate viral uptake. ³

4.2. Tissue‐specific ACE2 expression

Severe acute respiratory syndrome coronavirus 2 is accepted as an airborne transmitted respiratory virus. Therefore, the virus must initially infect the respiratory tract cells. In situ RNA analysis and single‐cell sequencing of the human respiratory tract revealed that ACE2 is highly expressed in nasal epithelial cells and has lesser expression in bronchial epithelial cells and alveolar type 2 (AT2) cells. In vitro studies were consistent with that finding showing upper respiratory tract cells were more permissive to SARS‐CoV‐2 infection than lower respiratory tract cells. ⁵¹ Recently, Zhao and colleagues ⁵² revealed with single cell RNA profiling study that, ACE2 is expressed only in .64% of pulmonary cells, and the majority of the expression is concentrated on AT2 cells (about 83%). Therefore, SARS‐CoV‐2 seems to efficiently utilize AT2 cells for reproduction and spread. The high AT2 expression of ACE2 may be the reason for severe alveolar injury following the initial infection.

Previous studies demonstrated, ACE2 is widely distributed in multiple other tissues, including the heart, vascular smooth muscle cells, kidney, gastrointestinal tract, testis, adipose tissue and brain tissue. ACE2 is expressed mainly in the epithelial cells of these organs. Xu and colleagues performed RNA‐Seq for 14 organs and showed that ACE2 is highly expressed in (from highest to lowest) the colon, gallbladder, heart muscle, kidney, epididymis, breast, ovary, lung, prostate, oesophagus, tongue, liver, pancreas and cerebellum. ⁵³

The same study revealed high ACE2 expression in oral cavity epithelial cells, consistent with the initiation of infection at the upper respiratory tract. Hikmet and colleagues analysed the protein expression profiles in a variety of human tissues. ACE2 was mainly expressed in renal tubules, intestinal enterocytes, cardiomyocytes, vasculature gallbladder, male reproductive cells, ductal cells, placental trophoblasts and the eye. ⁵⁴

4.3. Membrane‐bound and soluble forms of ACE2, and mechanism of shedding

As a transmembrane glycoprotein enzyme, ACE2 is composed of a short cytoplasmic domain, a transmembrane domain, a catalytic ectodomain and an amino‐terminal signal peptide. Similar to most other transmembrane proteins, ACE2 undergoes proteolytic cleavage from the cell surface to release its catalytic ectodomain part as a soluble form, a process known as ectodomain shedding. Shedding is a key post‐translational modifications mechanism for transmembrane proteins such as ACE2. Therefore, ACE2 can be found as membrane‐bound or as soluble/circulating within the body. ⁵⁵ Because the catalytic site of ACE2 is located in the ectodomain site, the release of the catalytically active part of the enzyme following shedding can influence the enzymatic activities in the cellular microenvironment, but also can influence distal tissue once the soluble enzyme migrates to distal organs via systemic circulation. ⁵⁶

To improve therapies, it is important to understand how ACE2 shedding, as well as membrane‐bound and soluble ACE2 levels, affect SARS‐CoV‐2 infection. Li and colleagues were the first groups showing that the S1 domain of the SARS‐CoV S‐protein binds efficiently to ACE2 isolated from SARS‐CoV‐permissive Vero E6 cells. ⁵⁷

When a major portion of the cytoplasmic domain of ACE2 was deleted, there was no effect on S‐driven infection, suggesting that the cytoplasmic domain is not critical for receptor function for infection. Another important finding was that a soluble form of ACE2 could successfully block the association of the S1 domain with the cells. Viral replication was inhibited when the cells were cultured in the presence of an anti‐ACE2 antibody. Hoffmann et al ⁵⁸ showed that ACE2 expression levels in various cell lines directly correlated with the susceptibility to SARS‐CoV S‐driven infection. They also demonstrated that pre‐incubation of cell cultures with soluble ACE2 ectodomain inhibits SARS‐CoV S‐driven infection in a dose‐dependent manner, further confirming that soluble ACE2 can effectively block the binding of SARS‐CoV S‐protein to membrane‐bound ACE2. These findings directed interest to mechanisms for ACE2 shedding, which would increase soluble ACE2 levels and inhibit viral infection. Lambert and colleagues investigated the involvement of several ADAM family proteins, which largely mediate the shedding of membrane proteins. ⁵⁸ , ⁵⁹

In the present meta‐analysis, we observed an elevated level of ACE2 in patients with all diseases, albeit membrane‐bound ACE2 expression is high compared to soluble form ACE2. Further, subgroup analysis of two forms of ACE2 expression revealed an elevated membrane‐bound ACE2 in patients with diabetes, cardiovascular diseases, viral diseases, and SARS‐CoV2 infection with co‐morbidities, whereas relatively low expression of membrane‐bound ACE2 in patients with SARS‐CoV2 infection without co‐morbidities and kidney diseases. The multivariate analysis depicted a statistically significant increase in total ACE2 levels in patients with cardiovascular diseases, and viral infection.

Numerous studies showed the high expression of ACE2 in patients with chronic obstructive pulmonary disease (COPD), influenza infection and their susceptibility to SARS‐COV2 infection mediated severity. ⁶⁰ , ⁶¹ Similarly, differential levels of expression correlated directly with the signs and symptoms, site of entry, and invasion for SARS‐CoV2 infection. For instance, Chen et al. showed elevated levels of ACE2 in the olfactory neuroepithelium, suggestive of loss of taste and smell. ⁶² The low expression of ACE2 in the nasal epithelium of children compared to adults or low expression of ACE2 in asthma patients compared to COPD protect them from SARS‐CoV2 infection has been demonstrated by several reports. ⁶⁰ , ⁶³

The patients with SARS‐CoV2 infection with or without comorbidity have high levels of soluble ACE2, suggesting shedding of ACE2, however, high expression of membrane‐bound ACE2 in comorbid patients with SARS‐CoV2 infection was observed. It could be a plausible underlying reason corroborating the findings of Li and colleagues' data depicting blocking viral entry with soluble ACE2. Therefore, COVID‐19 patients with co‐morbidity are prone to have an infection due to high membrane‐bound ACE2 despite having high levels of soluble ACE2. The investigation of the underlying mechanism for elevated levels of soluble ACE2 in response to SARS‐CoV2 infection is warranted. Next, the soluble form of ACE2 did not show any regulation in diabetic and kidney disease subjects. However, the membrane‐bound ACE2 levels were reduced in SARS‐CoV2 infected and kidney disease patients.

The differential expression of ACE2 and its dynamic in normal physiology and pathophysiology is crucial to decipher its role at the backdrop of SARS‐CoV2 infection.

4.4. Limitations of the study

The limitations of the present meta‐analysis include diverse methods employed for the estimation of the ACE2 levels in the selected studies. Moreover, ACE2 expressions are analysed at transcriptional and translational levels in the included studies, and samples taken for expression studies were also diverse in origin. Hence, for meta‐analysis and multivariate analysis, data were analysed, comparing ACE2 levels in a healthy population included in the respective study. In multivariate analysis, fold change was calculated with respect to healthy population ACE2 levels, and nonparametric analysis was performed.

5. CONCLUSION

Several reports suggested that the patients with co‐morbidity are prone to have SARS‐CoV‐2 infection than age match healthy subjects. The underlying reason could be associated with elevated ACE2 levels in these patients. The dynamic of soluble and membrane‐bound ACE2 did not investigate so far in detail. The measurement of soluble and membrane‐bound ACE2 could delineate a link between the severity and clinical outcome of the patients with co‐morbidity. Interestingly, in hospitalized patients, measuring ACE is the standard method. Our study encourages ACE2 measurements along with ACE in patients with COVID‐19 infection. Moreover, the ACE2 forms (soluble vs. membrane‐bound), their significance, regulation, the expression pattern in various tissue and corresponding diseases are still unexplored. Further investigation would warrant the strategic design of therapeutic targets for emerging multitargeted complex infectious diseases like COVID‐19.

AUTHOR CONTRIBUTIONS

Vandana S. Nikam contributed to the study conception and design. Material preparation, data collection and analysis were performed by Vandana S. Nikam, Dipanjali Kamthe, Swapnil Sarangakr, Manali Dalvi and Netra Gosavi. The first draft of the manuscript was written by Vandana S. Nikam and Dipanjali Kamthe, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Supporting information

Appendix S1

Figures S1–S4

Tables S1–S10

Kamthe DD, Sarangkar SD, Dalvi MS, Gosavi NA, Nikam VS. Angiotensin converting enzyme 2 level and its significance in COVID‐19 and other diseases patients. Eur J Clin Invest. 2023;53:e13891. doi: 10.1111/eci.13891