ABSTRACT

Infection caused by Novel Coronavirus has been declared a pandemic and is spreading in 213 countries and territories globally with severe implications. Due to the high rate of transmission of the virus through the aerosol and the novelty of the infection to humans, the illness has become a global emergency. 

The angiotensin converting enzyme-2(ACE-2) has been recognized as the receptor for the SARS-CoV-2 viral entry. We review the physiological functions of ACE2, presence of ACE2 in different organs of the body and ACE2 relationship with COVID-19 and other diseases. The goal is to provide the reader with an understanding of the complexity and importance of this receptor. Understanding the regulation of ACE2 in the context of COVID-19 could help understand the physiopathology of the illness and lead to the development of preventive and therapeutic drugs that help fight the pandemic illness. As such, it is now getting renewed attention as a possible target for anti-viral therapeutics. 

Furthermore...

Associating the occurrence of the disease in diverse geographical areas having genetically diverse ethnicities, can offer a scientific foundation for future researches to implement protocols on COVID-19 treatment, considering the rapid advent of the coronavirus disease and the current data shortfall on the virus pathophysiology. The paper highlights the fact that since ACE2 gene polymorphisms is population specific, screening of ACE2 polymorphisms in varied population groups across geographies could be helpful in evaluating the severity to SARS-Cov-2 infection

ACE2 is a transmembrane glycoprotein with a single extracellular catalytic domain which plays an important regulatory role in the renin-angiotensin system (RAS). ACE2 has different roles such as catalytic, transporter of amino acids or viral receptor. It has an important role in different systems, from cardiovascular regulation to viral infection. It catalyzes the formation of angiotensin II from angiotensin I and so playing a key role in the control of cardio-renal function and blood pressure control (Medina‑Enriquez et al.,2020). 

ACE2 is part of the Renin Angiotensin System (RAS), the key system accountable for the regulation of systemic arterial pressure. In addition, the RAS also has a local or paracrine function. RAS is an intricate system that is involved in many biological processes. The functions of the system are wide, which include inflammation, angiogenesis, cell proliferation, memory, sodium and water reabsorption, thrombosis, and plaque rupture (Perdomo-Pantoja et al.,2018; Wegman-Ostrowski et al.,2015). 

ACE2 Function

ACE2 is a main element of the RAS system. Initially, angiotensinogen is converted into Angiotensin I (Ang I) through the action of renin, an aspartyl protease which is produced in kidney. Angiotensin I (Ang I) is converted into angiotensin II (AngII) by the action of angiotensin-converting enzyme (ACE) which induces an increased blood pressure promoting vasoconstriction and inflammation. Lastly, ACE2 converts AngII to Ang-(1–7) which is vasodilatory agent. The increase in the activity of ACE2 might reduce the RAS system by inactivating and enhancing the production of Ang-(1–7). Ang (1-7) polypeptide, which has anti-inflammatory functions like protecting cardiomyocytes, relaxing blood vessels, anti-proliferation, and can enhance the activity of bradykinin which is an inflammatory mediator (Simões e Silva et al.,2016).

ACE2 Protein

The human ACE2 protein is a zinc metallopeptidase which is an ectoenzyme (family of dipeptidyl carboxypeptidase). It contains 805 amino acids. This protein is a kind of transmembrane glycoprotein and its expression is abundant with a single extracellular catalytic domain that mainly localizes at the plasma membrane (Santos et al.,2003; Hamming et al.,2007). There are two forms of the ACE2 protein. The first form of ACE2 protein contains a structural transmembrane domain and points its extracellular domain to the plasma membrane. The second form is the soluble form, which circulates in small amounts in the blood (Batlle et al.,2020).

The ACE2 gene length is 39.98 kb of genomic DNA and contains 18 exons (Turner et al.,2002). It maps to chromosome X at position Xp22.8 It encodes a type I cell-surface glycoprotein of about 100 kDa which is composed by 805 amino acids and characterized by a N terminal signal peptide of 17 amino acid residues, a peptidase domain (PD) with its HEXXH zinc binding metalloprotease motif, a C-terminal Collectrin (a regulator of renal amino acid transport and insulin)-like domain (CLD) that includes a ferredoxin-like fold “Neck” domain, that end with an hydrophobic transmembrane hydrophobic helix region of 22 amino acid residues followed by an intracellular segment of 43 amino acid residues (Li et al., 2003; Cerdà-Costa and Xavier Gomis-Rüth, 2014).

ACE2 IN BODY

ACE2 is an enzyme attached to the cell membranes of cells located in the lungs, arteries, heart, kidney, and intestines ( Hamming et al.,2004; Donoghue et al.,2000) . In 2002, Harmer et al. (Harmer et al.,2002) studied the presence of ACE2 and found that the mRNA of ACE2 is expressed in 72 different tissues. ACE2 present in epithelial cells, which line certain tissues and create protective barriers.ACE2 is attached to the cell membrane of mainly lung type II alveolar cells, enterocytes of the small intestine, arterial and venous endothelial cells, and arterial smooth muscle cells in most organs. ACE2 mRNA expression is also found in the cerebral cortex, striatum, hypothalamus, and brainstem( Medina‑Enriquez et al.,2020). ACE2 expression is high on the luminal surface of intestinal epithelial cells, in this context, ACE2 functions as a co-receptor for nutrient uptake (Hashimoto et a.,2012). ACE2 has other essential actions such as a non-catalytic function and the regulation of renal amino acid transport and pancreatic insulin secretion (Batlle et al.,2010).

The renin–angiotensin–aldosterone system (RAAS) is a key regulator of systemic blood pressure and renal function and a key player in renal, lung and cardiovascular disease. Ang II is the main effector substance of the RAAS, with potent vasoconstrictive, pro-inflammatory, and pro-fibrotic properties. Ang II is converted into Ang(1–7) which mediates vasodilation, antiproliferation, and apoptosis, thereby opposing the effects of Ang II by the action of ACE2. Thus, ACE2 plays an important role in the RAAS system (Hamming et al.,2007). 

ACE2 and Kidney Disease

In the kidney, Ang 1-7 formed by ACE2 acts on the G protein-coupled receptor (GPCR) MAS which leads to vasodilation, anti-fibrosis, anti-proliferation, and anti-inflammatory vascular protection. ACE2 is vastly expressed in kidney, mostly in brush border cells of proximal renal tubules, endothelial cells, smooth muscle cells of renal vessels, and podocytes. According to studies, ACE2 gene knockout can lead to an increase in blood pressure, glomerular damage, and renal fibrosis in diabetic mice and Exogenous human recombinant ACE2 can decelerate the development of diabetic nephropathy (DKD) (Anguiano et al.,2017). 

In study it has been found that in damaged renal tubules, the increase of Ang II may be a possible mediator for further renal damage in human renal diseases. Hypertensive nephropathy is a widespread complication of hypertension, mainly due to inflammation associated with Ang II, oxidative stress, and renal fibrosis. 

According to a study conducted in the school of medicine of Jilin University, Ginsenoside Rg3 can alleviate the Ang II-mediated renal injury in rats and mice by upregulating ACE2 in renal tissue (Liu et al.,2019). 

ACE2 and Cardiovascular Disease 

ACE2 is broadly present in cardiomyocytes, cardiac fibroblasts, and coronary artery endothelial cells. ACE2 is a vital regulatory protein in RAS. The RAS system controls the balance of body fluid and blood pressure and sustains the tension of blood vessels. 

The overactivation (increase of vasoconstriction) or reduction (decrease of vasodilation) of RAS will lead to vascular dysfunction, which is the major reason for atherosclerosis and cardiovascular disease (CVD) (Anguiano et al.,2017). 

Oudit et al., have found that ACE2 knockout mice have serious cardiac dysfunction, and ACE2 may have the potential to transform and regulate heart function. Researchers discovered that local overexpression of ACE2 significantly reduced the development of early atherosclerosis. Uri et al., confirmed the link among the activity of ACE2 in serum and the deterioration of heart failure. The reduction of serum ACE2 activity is a selective biomarker of cardiac dysfunction. Thus, ACE2 is the major defensive pathway against heart failure. 

ACE2 and Diabetes

RAS and natriuretic peptide systems (NPS) are the major reasons for the occurrence and development of diabetic cardiomyopathy (DCM). Neutral lysozyme inhibitors can protect the cardiovascular system via increasing NPS levels and the increase of RAS blockers which include angiotensin blockers, ARB, and ACE inhibitors. The stimulation of the ACE/Ang II/AT1 receptor pathway is linked to processes like inflammation, oxidative stress, fibrosis, and insulin resistance (Yong et al.,2013).

Experiments in diabetic mouse models demonstrated that the ACE/Ang II/AT1 receptor pathway was upregulated, while the ACE2/Ang (1-7)/MAS receptor pathway was downregulated, in both the retinae and kidneys. This is also established in the kidneys of type 2 diabetic patients with diabetic nephropathy. These data suggest that the imbalance between the ACE/Ang II/AT1 receptor and ACE2/Ang (1-7)/MAS receptor pathways is a base for the occurrence and development of diabetic complications (Zhang et al., 2015).

ACE2 AND COVID 19  

Studies show that, like the severe acute respiratory syndrome coronavirus (SARS-CoV) that caused SARS, SARS-CoV-2 binds to human angio-tensin-enzyme II (ACE2), using it as a cell entry receptor to invade respiratory and lung epithelium through the spike (S) protein (Zhou et al., 2020a,2020b). Studies show that, like the severe acute respiratory syndrome coronavirus (SARS-CoV) that caused SARS, SARS-CoV-2 binds to human angio- tensin-enzyme II (ACE2), using it as a cell entry receptor to invade respiratory and lung epithelium through the spike (S) protein (Zhou et al., 2020a,2020b).

SARS-CoV-2 utilizes angiotensin receptor (ACE) 2 to enter human cells (Donoghue et al., 2000; Turner et al., 2002; Li et al., 2003). Similar to other CoV, during viral entry into the host cell, the spike proteins (S) on the envelope of SARS-CoV-2 are cleaved into S1 and S2 subunits (Kira chdoerfer et al., 2016). S2 does not interact with the receptor but it harbors the functional elements required for membrane fusion of the virion. The S1 protein/receptor interaction is the pivotal determinant for SARS-CoV-2 to infect a host species. S1 contains the receptor binding domain (RBD) and directly binds to the peptidase domain (PD) of ACE 2 to enter host cells (Turner et al., 2002; Li et al., 2003; Yan et al., 2020).

As shown in figure, (A) in normal state without infection of SARS-CoV-2, there is an equilibrium in ACE and ACE2 receptor activity. ACE controls the Renin Angiotensin Aldosterone system (RAS) and cuts Ang I to produce Ang II. Ang II is a powerful vasoconstrictor and harmful for endothelial and epithelial function through activating AT1 and AT2 receptors. The balance of the RAS/Ang II output is controlled by ACE2 and Mas/G protein coupled receptor activity. ACE2 cuts Ang I and Ang II into Ang-1-9 and Ang1-7, respectively, thus it triggers MAS/G protein coupled receptors that guard cell death. (B) In case of SARS-CoV-2 infection, SARS-CoV-2 binds to ACE2 to enter into epithelial cells of the lungs. Cleavage of spike proteins by a protease such as trypsin/cathepsin G and or ADAM17 on ectodomain and TMPRSS2 of endo domain sites help viral entry into the cells. This process leads to flaking of host ACE2 receptors and damage of its protective function.

This can lead to...

Damage of function of ACE2 activity stops production of Ang 1-9 and Ang1-7. Lack of Ang1-7 reduces the activity of MAS/G receptor which leads to the loss of its defensive functions which includes vasodilatation and cell protection both at the epithelial and endothelial sites. Loss of ACE2 leads to an imbalance and unrestricted effects of Ang II and upregulation of RAS/Ang II pathway. Upregulation of Ang II triggers vasoconstriction, thrombophilia, micro thrombosis, alveolar epithelial injury, and respiratory failure. Hence, preventing the proteolytic function of trypsin/cathepsin and ADAM17 or TMPRSS2 and or direct activation of MAS/G receptor by enhancing Ang-(1-7) can overcome the damage of function ACE2 and are viable targets to avert tissue damage to the host (Samavati and Uhal.,2020).

SARS-CoV-2 has a higher ACE2 binding affinity than other CoV (Li et al.,2003). According to research of Shang et al. [9], the 3-dimensional structure of the SARS-CoV-2 binding site has an extra compact conformation, enhanced binding stability, and potentially improves the ACE2 receptor binding affinity. According to Sequence-based prediction studies, a more effective cleavage site is inserted at the edge of the S1/S2 subunits of the spike S protein (a host proprotein convertase, furin). This polybasic furin-type cleavage site is unique, and can increase the virus capacity to internalize into cells (Liu et al.,2020). Additionally, studies through surface plasmon resonance have confirmed that the ACE2 binds to the ectodomain of the SARS-CoV-2 spike glycoprotein with about 10- to 20-fold higher affinity than the S protein of the SARS-CoV (Liu et al.,2020). These diverse features may clarify the higher SARS-CoV-2 infectivity.

The angiotensin-converting enzyme inhibitors have been proposed as possible helpful treatments for COVID-19(Sun et al.,2020). According to the study of Monteil et al. it was found that human recombinant soluble ACE2 could block SARS-CoV-2 cell attachment (Monteil et al. 2020). 

ACE 2 POLYMORPHISM AND SUSCEPTIBILITY OF COVID 19

Morbidity and mortality due to COVID-19 increase with age and co-existing health conditions which include cancer, cardiovascular diseases, and although most infected individuals recover, even very young and healthy patients may randomly succumb to this disease (Dong et al.,2020). These explanations beg the question of how much of the variation in COVID-19 disease severity may be clarified by genetic susceptibility. Human genetic factors may contribute to the very high transmissibility of SARS-CoV-2 and to the persistently progressive disease observed in a small but noteworthy proportion of infected individuals. 

Growth of new preventive and/or therapeutic approaches for COVID-19 will be significantly facilitated by methodical identification of host genetic pathways and DNA polymorphisms (variants) which modify the risk of infection and severe illness, including the over-exuberant immune response to the virus. 

COVID-19 pandemic had vast health and financial impacts in 188 countries/regions across the world, but the disease has also hit different racial/ethnic subpopulations. Huge genetic studies in people of geographically varied lineage have confirmed considerable genetic variation in protein coding regions, with broadly variable allele frequencies (Lek et al.,2016). SARS-CoV-2 infection depends on the host cell angiotensin-converting enzyme 2 (ACE2) for access into cells for SARS-CoV-2 spike (S) protein (Hoffmann et al.,2020). 

Mortality rates amongst males and females 

According to studies, occurrence and mortality rates are significantly diverse among male and female COVID-19 patients and the disease is related with pre-existing conditions, such as cancer and cardiovascular disorders and individuals with hypertension getting anti-hypertensive medications (Guo et al.,2020). So, a systematic study of the functional polymorphisms in ACE2 among different populations could pave the technique for precision medicine and personalized treatment strategies for COVID-19.

The expression level and expression pattern of human ACE2 in diverse tissues might be critical for the susceptibility, symptoms, and outcome of COVID-19. Scientist Hou and his teams have investigated genetic susceptibility to COVID-19 by examining DNA polymorphisms in ACE2 genes. They collected a total of 437 non-synonymous single nucleotide variants (SNVs) in the protein-coding regions of ACE2 from three databases:(i) Genome Aggregation Database (gnom AD v3: gnomad. broadinstitute.org, covering 9 geographical areas), (ii) Exome Sequencing Project (ESP: evs.gs.washington.edu/EVS/), and (iii) 1000 Genomes Project (1KGP, www.internationalgenome.org) and used ANNOVAR (Wang et al.,2010) to interpret all non-synonymous variants. By applying Polyphen2 and CADD (Combined Annotation Dependent Depletion) scores, they recognized 63 possibly deleterious variants in ACE2.

What did the research found out?

They found that the circulation of deleterious variants in ACE2 differs among 9 populations. Precisely, 39% (24/61) and 54% (33/61) of deleterious variants in ACE2 occur in African/African-American (AFR) and Non-Finnish European (EUR) populations, respectively. Occurrence of deleterious variants among Latino/Admixed American (AMR), East Asian (EAS), Finnish (FIN), and South Asian (SAS) populations is 2–10%, while Amish (AMI) and Ashkenazi Jewish people do carry such variants in ACE2 coding regions. 

AFR populations carry p.Met383Thr and p.Asp427Tyr variants having  allele frequencies of 0.003% and 0.01%, individually. The p.Pro389His only happens in the AMR populations having an allele frequency of 0.015%. The p.Arg514Gly is a small allele frequency (0.003%) variant in AFR populations. This ACE2 variant is positioned in the angiotensinogen (AGT)-ACE2 contact surface, which is estimated to influence RAS function. The RAS is important for regulation of blood pressure, sodium, and fluid balance, and its dysfunction is related with cardiovascular and kidney illnesses (Kuster et al.,2020). 

The EUR population carries the p.Arg708Trp, p.Arg710Cys, p. Arg710His, and p.Arg716Cys variants having allele frequency of 0.01~0.006%. whereas the EAS and the AMR populations carry p.Arg708Trp and p.Arg710His having  allele frequency of 0.04% and 0.01% . Adding to these four variants, p.Leu731Phe has the main allele frequency in the AFR and EUR populations. In total, these comparative genetic analyses recommend that ACE2 genomic variants may play significant roles in susceptibilities to COVID-19 and its related cardiovascular conditions by changing AGT-ACE2 pathway. In addition to variance polymorphisms which may explain susceptibility and even consequence in different ethnic populations.

What does other research tell us?

Scientist Hatami et al., discovered a probable association between ACE I/D allele frequency and the COVID-19 recovery rate. Also, this study disclosed that the ACE I/D allele ratio is very varied universally. East Asian countries such as China and Japan had a ratio of more than 1 which showed a higher rate of I allele frequency. In East Asia, there seemed to be more I-alleles than D in the people's genome. Though, South Korea has an allele ratio of 0.87 which is higher than in European countries. Like this study scientist Saab et al also studied the average ACE II genotype frequency in different populations of different nations (Saab et al.,2007). They revealed that ACE I allele and genotype frequencies display an association with longitude. 

As per study of Saab and group, a strong association among II genotype frequency and longitude, shown by a decrease in the II frequency from Europe to the Middle East. Summarily, the normal frequency of the II genotype in the north of Europe and Denmark was 23%, whereas the UK and Spain recorded 20% and 15%, while northern and southern Italy registered 14% and 12%, in addition China and Japan averaging 35% and 45% respectively (Saab et al.,2007).

Likewise, this result was established in our study, as Denmark had an ACE I/D ratio of 1.01, which was higher than in other European countries. The result of Saab et al.’s study about the lowest II genotypes of Europe in Italy, makes us ponder about the likely role of genetic factors and the disaster of the COVID-19 outbreak in Italy with a high case-fatality rate (Livingston and Bucher.,2020). 

European countries like Italy, Spain, and the UK have been extensively affected by COVID-19. It is well recognized that ACE I allele frequency in Europe is lesser than in East Asia as seen in our result of this study, which showed the average ACE I/D allele ratio in Europe as 0.55, whereas it was 0.93 in Asia. This discovery approves the results of Wang et al. 's study as they projected the average ACE frequency in East Asia to be 0.63 and 0.43 in Europe (Wang et al.,2013).

The proposition

Khayat and his team propose a biological aspect, the genetic variation at the viral S protein receptor in human cells, ACE2 (angiotensin I-converting enzyme 2), which may have the worst consequence in males and in some regions globally. They accomplished exomics analysis in native and mixed South American populations, also conducted in silico genomics database research in residents from other continents. ten polymorphisms in coding, noncoding and regulatory sites were found that can propose a reasonable biological clarification for these epidemiological differences. In conclusion, there are ACE2 polymorphisms that could affect epidemiological differences detected among lineage and, also between sexes (Khayat et al.,2020).

The racial change of ACE gene polymorphism is well recognized.  In the United States, African Americans are identified to have the highest frequency of the D allele (89%) when compared with Indians (69%) and whites (69%) (Mathew et al.,2001).In Europe, people in Italy, Spain, and France have a high frequency of D allele up to 82% to 87% (Lee et al.,2002) while in Asian populations, such as Chinese, Korean, Taiwanese, and Japanese, have a high frequency of ACE gene II allele, which is higher than the European populations (33% to 51% versus 13% to 27%) (Saab et al.,2007).

It seems like...

The racial variance of ACE I/D genotype appears to coincide with the changes of consequences where the populations with high frequency of D alleles appear to experience higher fatality. African Americans seem to have an excessively high fatality rate in the United States (Yancy 2020; Dyer 2020). Likewise, patients from Italy, Spain, and France also experience high fatality in Europe. 

The low frequency of ACE D/D and high frequency of II genotype seen in Asian populations appear to be related with relatively low fatality of COVID-19 in those nations (https://www.worldometers.info/coronavirus/#countries, last accessed April 1, 2021). 

Though socioeconomic and environmental circumstances may play a role, they do not completely clarify the severity of acute lung injury in COVID-19. According to a Scottish study of influenza, socioeconomic factors do not completely clarify ethnic differences in hospitalization for lower respiratory tract infections (Simpson et al.,2015).

 

Thus, The ACE gene polymorphism, which accounts for the variances of the ACE level in general people, may be accountable for the susceptibility to severe lung injury inCOVID-19 patients. The absence of ACE D/D genotype in patients withCOVID-19 may be defensive against developing severe lung injury.

Conclusion

The role of aerosol exposure to the transmission of SARS-CoV-2 has been proven in many studies. Scientists have underlined that infected individuals represent emission sources of aerosol produced by routine behaviors - such as breathing, speaking, singing, coughing, sneezing, all of which might be capable of transmission of   disease thus it is tough to control it by just mask and social distancing.

SARS-CoV-2 uses ACE2 as the receptor for entry into host cells. Since ACE2 is highly expressed in various organs and tissues, SARS-CoV-2 not only enters into the lungs but also attacks other organs with high ACE2 expression. The pathogenesis of COVID-19 is extremely complex, with numerous factors involved. In addition to the direct viral effects and inflammatory and immune factors, the downregulation of ACE2 and imbalance among the RAS and ACE2/angiotensin-(1–7)/MAS axis may also contribute to the multiple organ injuries in COVID-19. These two characteristics of COVID-19 make it highly riskier for humanity. 

The possibility of a second wave of SARS-CoV-2 infections is very strong so preventive actions are important. It is important to note that vaccines against respiratory syncytial virus (RSV), rhinoviruses, SARS-CoV-1, and MERS-CoV have not yet been successful. So, for SARS-CoV-2, the medical and scientific groups must strengthen their studies in the areas of drug development for discovery of effective therapies and in preventive measures. The spike glycoprotein of SARS-CoV-2 is a possible target for the development of specific drugs, antibodies, and vaccines. Reinstating the balance between the RAS and ACE2/angiotensin-(1–7)/MAS may aid attenuate organ injuries in COVID-19.

Prevention would consist of avoidance of viral contamination and possible identification of genetically susceptible groups within the human population. Differences in COVID-19 severity can be classified as (a) asymptomatic, (b) symptomatic but no hospitalization required, and (c) severely symptomatic with hospitalization required. Explanation of alleles of relevant genes related with these three levels of severity to viral response can help clinicians in dealing with probable future waves of pandemic. Clarification of genomics and genetic pathways related to susceptibility of SARS-Cov-2 infection can become important in combating a future wave.

MVS Pharma is working on aerosol transmission of Viral diseases and this article is written by Dr Disha Trivedi who is expert in the field of biotechnology and has published several research and review articles in international journals.