The Angiotensin converting enzyme-2 (ACE2) is a significant regulator of the Renin-Angiotensin-Aldosterone system (RAAS), which plays key roles in the control of cardiovascular (CV) system (1). In the canonical pathway, the RAAS precursor, angiotensinogen, is degraded to the octapeptide angiotensin-II (Ang-II) by consecutive digestions of renin and angiotensin convertase enzyme (ACE). Ang-II could be then converted by aminopeptidase-A to Ang-III, which conserves similar proprieties mostly mediated by two distinct G protein-coupled receptors named AT1R and AT2R (2). Overactivation of the ACE/Ang-II/AT1R pathway has been associated with several CV pathologies, including hypertension, heart failure, vascular inflammation and remodeling, coagulation, and atherosclerosis (3). However, activation of AT2R can trigger anti-inflammation and vasodilation. In this regard, the stimulation of the non-canonical axis of the RAAS may compensate ACE/Ang-II/AT1R actions. The mono-carboxypeptidase ACE2 can convert Ang-II into angiotensin 1–7 [Ang (1-7)], which binds to the Mas receptor (MasR) (1). The ACE2/Ang (1-7)/MasR axis can reduce blood pressure and CV hypertrophy and fibrosis, stimulating vasoactive prostaglandins and lessening redox imbalance (4). Ang (1-7) also protects ACE-degradation of bradykinins and ameliorates inflammatory responses, and vascular permeability (1). Moreover, ACE2 also degrades Ang l into Ang (1–9), decreasing Ang-II levels. Given the opposite effects of Ang (1-7) and Ang-II in the CV homeostasis, it might be imperative to maintain a minimum ACE2 level or activity.

Interestingly, the ACE2 gene is highly conserved in mammals and expressed in testes, renal and CV system, and especially in gastrointestinal tissues (5). It encodes a type I membrane-bounding glycoprotein composed by 805 amino acids divided in three functional domains including a C-terminal transmembrane anchoring region, a N-terminal signal peptide motif, and an HEXXH zinc-binding metalloprotease region (Figure 1). ACE2 maps in chromosome Xp22 and contain 17 introns and 18 exons, where a high degree of genetic polymorphism can be found (6). There are at least 510 valid variants in ACE2 (mostly intronic regions) (7), and some single nucleotide polymorphisms (SNP) may affect ACE2 structure and stability, interfering with its cardioprotective functions. In adults with different gender, ethnic and presence of pre-existing CV disease, the rs4646188, rs2158083, rs233575, rs879922, and rs2074192 SNPs (Figure 1) have been related to hypertension, ventricular hypertrophy, or type-2 diabetes mellitus (T2DM), and increased plasma total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), or reduced high-density lipoprotein-cholesterol (HDL-C) and Ang (1-7) (8–11). In children or adolescent cohorts, where existence of confusing factors (i.e., epigenetic alterations) is lower, boys but not girls showed association of rs2158083, rs233575 and rs2074192 with hypertension (12, 13). Nowadays, regulation of the ACE2 expression and activity has become essential for Coronavirus disease (COVID)-19 subjects, particularly for those with cardiovascular complications (6, 14). Thus, further studies analyzing ACE2-SNPs and related haplotypes in young populations, could suggest predictive markers for CV diseases and molecular targets for CV therapy.

In our study, we examine five ACE2 SNPs in relationship with the presence of overweight and obesity in a representative sample of 12-to 16-year-old children, and we assess the potential association of the polymorphisms with the lipid profile and insulin levels.

In this work we have analyzed five SNPs in the ACE2 gene in a general population of apparently healthy adolescents from Spain. The prevalence of these polymorphisms in females and males was comparable to previous findings (12, 22, 23), but the occurrence of some of them was associated with CV risk factors only in girls.

At these ages, females showed lower rates of overweight and obesity, SBP and glycemia than males. Although girls showed higher TC and LDL-C, they enhanced HDL-C, as compensation. They also exhibited lower TG/HDL ratio, which is a predictor of metabolic syndrome and CV diseases (i.e., coronary artery disease or atherosclerosis), and an estimator of LDL-C particle size (20). Thus, theoretically, young girls may be cardio-protected, as previously described (24, 25). Spite of the differences in hormonal regulation between sexes (26), a potential explanation of this cardio-protection could be based on the expression of ACE2 and subsequent effects on the RAAS activity. Since ACE2 is encoded in chromosome X, and specifically in a region that escape from X-inactivation, ACE2 may be upregulated in girls. However, this escape gene also shows heterogeneous sex bias that may lead to lower expression (and tissue-specific) and its activity can be regulated by hormone release (27). Nevertheless, the ACE2/Ang (1-7)/MasR activation could preserve pancreas, skeletal muscle, adipose tissue or the CV system from oxidation, inflammation, and fibrosis (28). Moreover, the elevation of ACE2 involve a reduction of Ang-II, which enhances the NADH/NADPH oxidase activity and reduces the nitric oxide-dependent vasodilatation at systemic level (29). Also, Ang-II stimulates cholesterol biosynthesis, overexpresses lipid receptors (i.e., LRP1, LOX-1), facilitates the uptake and oxidation of LDL-C, and induces pro-inflammatory matrix proteins and matrix-degrading enzymes (i.e., MCP-1, IL-18, PAI-1) (30, 31). In addition, ACE2 may defend the CV system by direct actions. ACE2 increases mitochondrial ATP production and reduces pro-oxidant factors such as NOX4. It also ameliorates lipid deposition and endoplasmic reticule stress via GRP78/eIF2α/XBP-1/ATF4/CHOP expression (32).

Importantly, a reduction of ACE2 levels and subsequent Ang (1-7)-MasR signaling can promote CV failures (33). ACE2 knockout mice showed increased lipid accumulation, ER stress and mitochondrial dysfunction in skeletal muscle, and overexpression of ACE2 reduced these responses and improved glucose and lipid metabolism through the IKKβ/NFkB/IRS-1 pathway (32, 34). Similarly, MasR knockout exhibited dyslipemia and insulin resistance, along with decreased adiponectin secretion and glucose uptake (35). Furthermore, during hypertension or diabetes, a deficiency of ACE2 and increased ACE/ACE2 ratio have been noted (36), and cardioprotective treatments with ACE inhibitors and Ang-II receptor blockers exert their effects partly by increasing ACE2 levels (37). In this regard, we propose that in those girls who carried specific intergenic ACE2 SNPs, the integrity, activity and/or stability of the ACE2 RNA-messenger and resultant protein could have been affected (38, 39), and Ang-II signaling may overcome those of ACE2/Ang (1-7)/MasR. In fact, the existence of rs879922, rs233575, and rs2158083 SNPs of ACE2 was associated with overweight/obesity and elevated plasma TG in Spanish girls. The rs879922 and rs233575 polymorphisms were also related to upper TG/HDL-C ratio, and the rs2074192 and rs2158083 with higher TC levels. Reinforcing our findings, we observed that the C-G-C haplotype of rs879922, rs233575, and rs2158083 was associated with excessive weight and elevated TG levels. Previous data on ACE2 haplotypes linked higher risk of cardiac hypertrophy with minor alleles of rs2074192 and rs2106809 in females (10) or with those of rs2106809 and rs6632677 in males (40).

Little is known about rs2158083 and rs233575. The former is encoded in ACE2's intron 4 and was previously associated with higher blood pressure (12). Similarly, rs233575 (intron 16) was related to higher blood pressure in adolescents and its interaction with obesity was present only in females (13). It was also linked to increased LDL-C (≥1.8 mmol/L) (8) and T2DM (11) and associated with higher LVMI (41). The rs233575 SNP may alter binding of regulatory factors such as RNA-binding proteins involved in splicing (e.g., ARID5A), which is stimulated under inflammation (42). Moreover, rs879922 (intron 11) can be considered a significant quantitative trait locus for essential hypertension in female Chinese, where it also associated with increased TC (≥5.2 mmol/L) and LDL-C (≥1.8 mmol/L) (8). rs879922 was linked with T2DM (11) and cardiac hypertrophy (41), and importantly, the relative expression of ACE2 was significantly lower in subjects with the CC genotype of rs879922 compared with those carrying the GG/CG genotype (43). Finally, rs2074192 (intron 16) has been associated with CV risk (i.e., LVH, carotid arteriosclerosis stenosis, microalbuminuria), retinopathy in T2DM individuals, hypertension, and cardiac hypertrophy (10–12). rs2074192 was also linked to increased TC (≥5.2 mmol/L) in hypertensive population (8), and neonates with rs2074192 were more likely to be born as small for gestational age babies, which contribute to metabolic syndrome and CV diseases in later life (44). Interestingly, rs2074192 was associated with reduced circulating Ang (1-7) levels (22), and in COVID-19 patients, rs2074192 correlated to more severe outcomes (i.e., bilateral pneumonia, dyspnea, high fever) (13, 45).

This study lays the groundwork for subsequent trials in patients and experimental animals. The ACE2-SNPs, rs879922, rs233575, rs2158083, and rs2074192, could help to classify girls at risk of obesity and dyslipidemia in adulthood. The presence of these SNPs should be confirmed in populations of adult women with obesity, dyslipidemia and/or cardiovascular diseases. Also, this work suggests that variability of the ACE2 gene could predispose patients to certain metabolic or cardiovascular conditions that are key in the evolution of other diseases (i.e., COVID-19). Furthermore, these ACE2-mutations might be generated in rodent models with/without obesity and cardiovascular failures in order to characterize atherogenic responses under ACE2 modulation.

At these ages, girls and boys may produce different sexual hormones and factors, which can influence on the lipid profile. Thus, their puberal stage may add important information to explain differences between sex. Also, the precise biological mechanism and other possible factors underlying the association of the ACE2 gene with overweight/obesity or plasma lipids elevation remain to be clarified. Finally, functional investigations of the association between C-G-C haplotype with overweight/obesity or TG elevation could be required.

The ACE2 gene may become a double-sword factor for CV protection, at least in women. Those female adolescents who carry the wild-type ACE2 gene may be somehow protected from increased adiposity and hyperlipidemia. However, Spanish girls carrying the rare alleles of the ACE2-SNPs rs879922, rs233575, rs2158083, or rs2074192 may be vulnerable to future obesity and CV injuries. The haplotype rs879922-rs233575-rs2158083 may be considered a valid biomarker for both pathologies. Therefore, these ACE2-SNPs might be addressed for therapeutic and prognostic purposes against CV diseases. In this sense, new studies might evaluate their role as predictors of worse evolution in COVID-19 subjects, where CV homeostasis may be seriously damaged.

The data presented in the study are deposited in the dbSNP repository, submission batch ID 1063361 (dbSNP Build ID: B156).

The study protocol was approved by the Ethics Committee of Clinical Investigation of the Fundación Jiménez Díaz (Code number: PIC016-2019 FJD). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

JL-C, CV-V, and IP-N quantified the gene expression of ACE2-SNPs and analyze its associations with clinical variables. IM-F performed the statistic studies. LS-G, OL, and CG designed and wrote the manuscript. All authors participate in the discussion of the work.

This research was funded by SPACE2-CV-COVID-CM grant from REACT-EU, Comunidad de Madrid and European Regional Development Fund, and by the Fondo de Investigación Sanitaria-IS. Carlos III (ref.: PI18/01016 and PI20/00923), Biobank (ref.: FEDERRD09/0076/00101), and Ciberdem (ref.: CB07/08/2007). JL-C was granted by FPI contract from Universidad Autónoma, Madrid.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.