Food-Derived Up-Regulators and Activators of Angiotensin Converting Enzyme 2: A Review

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a key enzyme in the renin-angiotensin system (RAS), also serving as an amino acid transporter and a receptor for certain coronaviruses. Its primary role is to protect the cardiovascular system via the ACE2/Ang (1–7)/MasR cascade. Given the critical roles of ACE2 in regulating numerous physiological functions, molecules that can upregulate or activate ACE2 show vast therapeutic value. There are only a few ACE2 activators that have been reported, a wide range of molecules, including food-derived compounds, have been reported as ACE2 up-regulators. Effective doses of bioactive peptides range from 10 to 50 mg/kg body weight (BW)/day when orally administered for 1 to 7 weeks. Protein hydrolysates require higher doses at 1000 mg/kg BW/day for 20 days. Phytochemicals and vitamins are effective at doses typically ranging from 10 to 200 mg/kg BW/day for 3 days to 6 months, while Traditional Chinese Medicine requires doses of 1.25 to 12.96 g/kg BW/day for 4 to 8 weeks. ACE2 activation is linked to its hinge-bending region, while upregulation involves various signaling pathways, transcription factors, and epigenetic modulators. Future studies are expected to explore novel roles of ACE2 activators or up-regulators in disease treatments and translate the discovery to bedside applications.

1. Introduction

Angiotensin converting enzyme 2 (ACE2), best known as the entry receptor of the pandemic’s severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has attracted considerable attention during the outbreak of COVID-19.¹ ACE2 was first discovered in 2000 from a human cardiac left ventricle cDNA library.² The primary role of ACE2, as a carboxypeptidase, is to catalyze the hydrolysis of angiotensin II (Ang II) into angiotensin (1–7) (Ang (1–7)) or angiotensin I (Ang I) into angiotensin (1–9), by cleaving the carboxyl-terminal amino acid residue.³ Nevertheless, Ang II has a greater affinity for ACE2 than Ang I, leading to the ACE2/Ang (1–7) pathway being approximately 400 times more pronounced than the latter.⁴ Ang II is a vasoconstrictor, while Ang (1–7) acts as a vasodilator, thus ACE2 is protective in the cardiovascular system.⁵ In addition, ACE2 also functions as an aider to gut and kidney amino acid transport, partnering with the neutral amino acid transporter known as broad neutral amino acid transporter 1 (B⁰AT1) (SLC6A19) or the sodium-dependent imino transporter 1 (SLC6A20), and facilitating the transportation of neutral amino acids or proline into the bloodstream, respectively.⁶ Knocking down ACE2 expression leads to Hartnup disease, characterized by reduced absorption of neutral amino acids, such as l-tryptophan, from the small intestine and kidney.⁷ Intestinal ACE2’s role in tryptophan transport also contributes to glucose metabolism during metabolic stress.⁸ Mutations in the SLC6A20 gene hinder the reabsorption of imino acids, causing their excessive excretion in urine—a hallmark of iminoglycinuria.⁹ The three functions of ACE2 are depicted (Figure 1).

Figure 1.

Multifunctional roles of ACE2: SARS-CoVs entry, the RAS regulation, and amino acid transport. (A) ACE2 is the entry receptor of SARS-CoV-2, via interaction with the spike protein of the virus. (B) Ang II increases blood pressure via binding with the Ang II type 1 receptor (AT1R). ACE2 catalyzes the degradation of a vasoconstrictor Ang II to generate vasodilator Ang (1–7). The subsequent binding of Ang (1–7) to Mas receptor (MasR) contributes to the reduction of blood pressure. (C) ACE2 collaborates with the broad neutral amino acid transporter 1 (B⁰AT1) to facilitate the transport of neutral amino acids in the gut (created with BioRender).

The human ACE2 gene is located on the X chromosome and encodes a type I membrane-bound glycoprotein consisting of 805 amino acids.¹⁰ The N-terminal catalytic domain resides extracellularly and harbors a hinge-bending region that serves as a key target for most ACE2 regulators. With a 48% homology to collectrin, the C-terminal collectrin-like domain of ACE2 is more complex, incorporating a neck domain that connects to the protease catalytic domain, a single transmembrane helix, and an intracellular tail (Figure 2). ACE2 exists in two forms: one is the membrane-bound glycoprotein, and the soluble form generated by cleaving segments between the neck domain and the transmembrane helix within the collectrin-like domain. This cleavage results in the release of the soluble ACE2, containing the protease domain, into circulation, as it lacks an anchor to the cell membrane.¹¹

Figure 2.

Two heterodimers of ACE2 and B⁰AT1 combine to form dimers through the neck region of ACE2. ACE2 is composed of an N-terminal protease domain and a C-terminal collectrin-like domain. In its protease domain, the binding sites for spike protein interaction, substrate catalysis, and modulation of enzymatic activity are distinctly segregated. Two widely recognized ACE2 activators, diminazene aceturate (DIZE), and xanthenone (XTN), specifically target the hinge-bending domain, thereby increasing the activity of ACE2. The C-terminal domain contains a neck domain, an intracellular tail, and a transmembrane helix, serving as an anchor that enables ACE2 to localize on the cell membrane. The soluble form of ACE2 is produced through the cleavage of a disintegrin and metalloproteinase 17 (ADAM17) between the neck domain and the transmembrane helix (created with BioRender).

The widespread distribution of ACE2 in the human body highlights its diverse functions.¹² For example, the high abundance of ACE2-expressing ciliated cells in the proximal airway of the nose designates nasal surfaces as the initial site for SARS-CoV-2 respiratory tract infection.¹³ In the cardiovascular system, ACE2 assumes diverse roles, such as reducing blood pressure, alleviating vascular inflammation, mitigating vascular oxidative stress, and modulating cardiac remodeling.⁴ In adipocytes, ACE2 actively participates in glucose metabolism and adipocyte function, while enteral ACE2 shows efficiency against diabetic retinopathy in type 1 diabetes.^14,15

Given the vast potential of ACE2 in regulating numerous physiological functions and human health, as depicted in Figure 3, there is a surge of interest to identify compounds that activate or upregulate ACE2,¹⁶ including those from natural sources such as food protein hydrolysates and bioactive peptides.¹⁷⁻¹⁹ To the best of our knowledge, there is a lack of a comprehensive review on ACE2 activators/up-regulators. Therefore, in this article, we review the key physiological functions of ACE2 concerning health and diseases, provide a research update on ACE2 activators and up-regulators, especially those sourced from natural origins, and further appraise the recent literature on the underlying ACE2 regulatory mechanisms.

Figure 3.

The multifaceted impact of ACE2 on various diseases. Decreased ACE2 activity contributes to heightened Ang II production, amplifying inflammation, and triggering excessive oxidative stress. This cascade culminates in dysfunction across vascular, cardiac, pulmonary, hepatic, and renal systems. Meanwhile, Ang II influences adipose tissue and liver, disrupting the normal metabolism of glucose and lipids (created with BioRender).

2. ACE2 in Human Health and Diseases

2.1. ACE2 and Heart Diseases

ACE2’s abundance in cardiomyocytes, cardiac fibroblasts, and coronary endothelial cells highlights its essential role in maintaining heart function.²⁰ Indeed, extensive research on its role in cardiovascular diseases has been carried out since its discovery.³ Targeted knockout of ACE2 in mice worsened Ang II-induced cardiac dysfunction, but ablation of ACE, the Ang II producer, completely abolished the cardiac dysfunction phenotype in ACE2 knockout mice.²¹ Prolonged exposure to Ang II induced myocardial fibrosis, which was mitigated by injecting the lentiviral vector encoding mouse ACE2 (lenti-mACE2).²² In addition, myocardial infarction (MI)-induced cardiac fibrosis was associated with the ACE/Ang II/AT1R axis activation, increased phospho-p38 mitogen-activated protein kinase (MAPK) expression, and reduced ACE2 levels in myocardial tissue. Therefore, supplementing with ACE2 activators may alleviate cardiac fibrosis resulting from MI.^23,24

2.2. ACE2 and Lung Diseases

ACE2 is highly expressed in airway and alveolar epithelial cells.²⁵ Its involvement in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) has been demonstrated in ACE2 gene-deficient mice with ALI induced by cecal ligation and perforation (CLP). These ACE2 knockout mice showed a lower survival rate and worsened lung functions compared to wild-type mice, whereas ALI was rescued after supplementation with recombinant human ACE2 (rhACE2) protein.²⁶ The role of ACE2 was also investigated in pulmonary hypertension and fibrosis. Exogenous ACE2 attenuated lung fibrosis by reversing the reduced local ACE2 levels and decreasing Ang II levels in a lung fibrosis mouse model.²⁷

2.3. ACE2 and Hypertension

The potential involvement of ACE2 in essential hypertension was suggested by its candidacy as a gene for the quantitative trait locus on the X chromosome in various hypertensive models. Hypertensive rats, compared to normotensive rats, showed significantly reduced ACE2 expression.^28,29 Human renal specimens also showed imbalanced ratios of ACE and ACE2 associated with hypertension development, with a positive correlation between the ACE/ACE2 ratio and mean blood pressure.^30,31 Conversely, overexpression of ACE2 in the brain could lead to a long-lasting decrease in blood pressure in spontaneously hypertensive rats (SHRs).³² Sriramula et al. (2011) suggested that hypertension induced by Ang II could be alleviated by upregulating ACE2 expression, resulting in a reduction in pro-inflammatory cytokines in the paraventricular nucleus.³³ Hypertension is featured by abnormal vascular function due to vascular inflammation and excessive oxidative stress, and the ACE2/Ang (1–7)/MasR axis plays a crucial role in mitigating both factors.⁴ Specifically, overexpression of ACE2 could inhibit the secretion of monocyte chemoattractant protein-1 (MCP-1) from macrophages stimulated by Ang II, likely through the elevation of Ang (1–7) levels.³⁴ Conversely, ACE2 deficiency facilitated Ang II-induced inflammation in the mouse aorta, with increased MCP-1, IL-6, and IL-1β levels.³⁵ The ACE2/Ang (1–7)/MasR axis exerts its antioxidative effect by inhibiting nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX), which led to decreased production of reactive oxygen species (ROS). Ang (1–7) treatment effectively prevented NOX enzymatic activity and NOX4 expression in the kidneys of SHRs with diabetes.³⁶

2.4. ACE2 and Type 2 Diabetes Mellitus (T2D)

The role of ACE2 in diabetes remains controversial. Several reports indicate a positive correlation between ACE2 expression and blood glucose levels, as supported by a study employing mendelian randomization analysis.^37,38 Recent research reported ACE2 upregulation in kidney organoids and renal cells within a diabetic milieu,³⁹ while contrasting reports highlight ACE2 downregulation in the kidneys of human subjects with diabetic nephropathy and in cases of neuropathic pain caused by diabetes mellitus.^40,41 Emerging evidence highlights the importance of ACE2 in diabetes primarily by mitigating diabetic complications arising from elevated Ang II levels and concurrent inflammation.⁴² Uncontrolled and prolonged hyperglycaemia consequently gives rise to the chronic complications, including renal dysfunction, diabetic cardiomyopathy, vascular issues, neuropathy, and retinopathy.⁴³ Notably, absence of ACE2 accelerated diabetic kidney injury,⁴⁴ while the use of human recombinant ACE2 improved kidney function and structure in diabetic nephropathy.⁴⁵ Likewise, simultaneous treatment with ACE2 activator (DIZE) and neprilysin inhibitor (thiorphan) effectively prevented diabetic cardiomyopathy by inhibiting inflammatory signaling in cardiac fibrosis.⁴⁶ Widely recognized in the pathophysiology of diabetic retinopathy, Ang II contributes to the augmentation of oxidative stress, angiogenesis, and inflammation in the retina, therefore suggesting potential treatment avenues such as Ang II receptor blockers or the oral supplementation of human ACE2.^47,48 In diabetes management, peroxisome proliferator-activated receptor γ (PPAR-γ) regulates glucose and lipid metabolism by reducing hepatic glucose output and circulating triglyceride levels, primarily in adipocytes. In diabetes, PPAR-γ activity decreases; however, exogenous Ang (1–7) treatment not only reduces hyperglycemia but also restores PPAR-γ in the kidney.⁴⁹ Furthermore, activated PPAR-γ triggers adipocytes to release adiponectin, promoting insulin sensitivity and glucose uptake by facilitating glucose transporter type 4 (GLUT4) translocation.⁵⁰ Simultaneously, adiponectin can activate AMP-activated protein kinase (AMPK) directly through AdipoR2, or indirectly via extracellular Ca²⁺ influx, which is necessary for the activation of Ca²⁺/calmodulin-dependent protein kinase β.⁵¹ As a result, AMPK activation affects ACE2 expression by regulating its transcription factors, such as sirtuin 1 (SIRT1), initiating ACE2 transcription.²⁰

2.5. ACE2 and COVID-19

In addition to its crucial roles in cardiovascular diseases, ACE2 also acts as the receptor of the SARS virus, facilitating its entry into host cells.⁵² The outbreak of COVID-19 caused by SARS-CoV-2 re-emphasized the importance of ACE2.⁵³ Recent studies showed that residue 394 in the SARS-CoV-2 receptor-binding domain interacts with human ACE2’s lysine 31 residue.⁵⁴ The higher binding affinity of SARS-CoV-2 to ACE2 than SARS-CoV explains its greater transmissibility and infectivity.⁵⁵ While ACE2 inhibition was initially considered for COVID-19 prevention,⁵⁶ emerging evidence supports that impaired ACE2 function due to SARS-CoV-2 infection is linked to adverse outcomes or chronic metabolic disorders.^57,58 In a cohort of COVID-19 patients, an increased baseline level of soluble ACE2 serves as a disease signature but is not linked to severity or mortality. More prominently, the dysregulation of ACE2 contributes to the progression of COVID-19 and is correlated with unfavorable outcomes.⁵⁹ The protective effects of ACE2 are further evidenced by the sex-dependent COVID-19 outcomes. As the ACE2/Ang (1–7)/MasR axis can be enhanced by estrogens, women are less likely to experience severe COVID-19 outcomes compared to men.⁶⁰ Upon SARS-CoV-2 binding to ACE2, the transmembrane serine protease 2 (TMPRSS2) cleaves the spike glycoprotein, enabling fusion between host and viral membranes. Given the potential risks of ACE2 inhibitors in individuals with cardiovascular diseases, TMPRSS2 inhibitors might be the better option for COVID-19 treatment.^56,61 As new SARS-CoV-2 variants continue to evolve,^62,63 engineered ACE2 decoys with enhanced binding affinities for the virus, such as rhACE2 protein and a plant-made rhACE2, have been developed.⁶⁴⁻⁶⁶

3. ACE2 Regulation vs ACE2 Activation

3.1. ACE2 Regulation

Upregulation refers to an increase in gene transcription and subsequent protein synthesis.⁶⁷ Compounds that can elevate ACE2 gene expression or protein levels are considered as ACE2 up-regulators, while those that decrease its gene expression or protein levels are classified down-regulators. The COVID-19 pandemic has highlighted that the SARS-CoVs is a down-regulator of ACE2.⁶⁸ The spike protein of the virus induces the translocation of ACE2 from the cellular membrane to intracellular compartments through a clathrin- and caveolae-independent endocytic pathway.⁶⁹ This translocation may be facilitated by Ang II when there is an imbalance between ACE and ACE2.⁷⁰ Meanwhile, accumulation of Ang II subsequently activates a disintegrin and metalloproteinase 17 (ADAM17), which in turn promotes proteolysis and extracellular shedding of ACE2.^71,72 As a result, despite an increase in soluble ACE2 in the circulation, the overall levels of ACE2 decrease.⁷³ ACE2 shedding results in the loss of its tissue-protective effects and thus triggers the detrimental effects of viral infections, such as multiple organ injury affecting the lung, heart, kidney, and others.⁷⁴ Several studies have suggested that physical training, particularly aerobic exercise, may contribute to the upregulation of membrane-bound ACE2, thus protecting individuals from SARS-CoV-2 invasion.⁷⁵ However, due to these intricate interactions, gaining a deep understanding of these processes is crucial for combating the threats posed by viral infections and exploring potential therapeutic interventions for SARS-related diseases.

Apelin, a member of the adipokines family is an endogenous peptide hormone and a ligand of G protein-coupled apelin receptor (APJ).⁷⁶ Like Ang II, apelin is likewise hydrolyzed by ACE2 to remove its phenylalanine residue at the C-terminus.⁵ To improve its resistance to ACE2-mediated degradation, apelin analogues were designed with a chemically modified C-terminal phenylalanine residue showing an improved hypotensive effect in animals.⁷⁷ Meanwhile, apelin is an up-regulator of ACE2. One apelin isomer, apelin-13 peptides, boosted ACE2 promoter activity through the activation of APJ and elevated ACE2 expression in the failing hearts of C57BL/6J mice.⁷⁸

ACE2 is always regulated at the transcriptional level. One such factor is the nuclear transcription factor farnesoid X receptor (FXR), also known as the bile acid receptor, which interacts with the FXR responsive element (FXRE) located in the ACE2 promoter region. FXR is susceptible to modulation by various compounds, each exerting distinct effects on ACE2 expression. For example, an FXR agonist, chenodeoxycholic acid, increased ACE2 expression in cholangiocytes; however, this elevation could subsequently be reduced by ursodeoxycholic acid through the inhibition of FXR signaling.⁷⁹ Similarly, another transcription factor, signal transducer, and activator of transcription 3 (STAT3), binds to the ACE2 promoter region following its nuclear translocation. Sesquiterpene lactone such as 6-O-angeloylplenolin (6-OAP), found in a Chinese medicinal herb, was discovered to suppress the binding affinity between STAT3 and the ACE2 promoter, whereas treatment with interleukin 6 enhanced this interaction.⁸⁰ Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor for genes encoding of antioxidant molecules, which have cytoprotective effects by eliminating cytotoxic electrophiles and reactive oxygen species.⁸¹ It is yet another candidate involved in ACE2 gene expression. Transfection with Nrf2 siRNA prevented the reduction in ACE2 mRNA expression.⁸² Further insight into Nrf2 modulation revealed that Nrf2 inhibitors such as alkaloids stimulated ACE2 mRNA expression, while the Nrf2 activator oltipraz, an organosulfur compound, abrogated the increase.⁸³ SIRT1 modulates ACE2 transcription via AMPK signaling, with 5-amino-4-imidazolecarboxamide riboside (AICAR), an adenosine monophosphate (AMP) mimic, enhancing SIRT1 binding to the ACE2 promoter. The SIRT1 inhibitor EX-527 opposes this interaction, emphasizing SIRT1’s role in ACE2 expression.⁸⁴ The association with AMPK signaling establishes SIRT1 as a metabolic sensor; specifically, SIRT1 regulates liver glucose and lipid metabolism, as well as systemic insulin sensitivity.⁸⁵ In the blood vessels subjected to increased mechanical stretch, higher phosphorylation of p38 MAPK is associated with the activation of the activating transcription factor 3 (ATF3). Once translocated into the nucleus, ATF3 directly binds to the ACE2 promoter, consequently inhibiting ACE2 expression.⁸⁶

Epigenetics includes all mechanisms that control gene expression irrespective of the DNA sequence.⁸⁷ Various epigenetic changes such as histone methylation, histone acetylation, DNA methylation, and microRNAs (miR) affect ACE2 expression. For example, Homo sapiens (hsa)-miR-125a-5p effectively lowered ACE2 transcript levels in lung, kidney, and esophagus tissues.⁸⁸ MiR-421 and members of the miR-200 family also suppressed ACE2 expression.^89,90 Notably, lysine demethylase 5B (KDM5B) indirectly upregulates ACE2 transcript levels by inhibiting miR-125 and miR-200.⁹¹ In addition, KDM5B could also demethylate lysine 4 on histone H3 (H3K4me3). These modifications positively correlate with ACE2 expression in human lung tissue.⁹² Conversely, histone H3 acetylation on lysine 27 (H3K27ac) drives ACE2 overexpression, while methylation at the same site (histone H3 on lysine 27) silences ACE2 expression.⁹³ Corley and Ndhlovu (2020) unveiled variations in DNA methylation levels among different tissue types, genders, and disease states, resulting in varying ACE2 expression levels.⁹⁴Figure 4 depicts the proposed regulatory pathways of ACE2.⁹⁴

Figure 4.

Proposed mechanisms of ACE2 regulation. (A) SARS-CoV-2 enters cells by binding to the ACE2 receptor. The virus-mediated ACE2 translocation downregulates ACE2 levels on the cellular membrane. (B) Accumulation of Ang II, resulting from the loss of ACE2, activates ADAM17. This activation, in turn, mediates the proteolytic cleavage of ACE2, leading to the production of soluble ACE2 (sACE2) in the circulation. (C) ACE2 inactivates apelin by removing phenylalanine residue, while simultaneously, apelin can promote ACE2 expression. (D) Epigenetic changes, such as histone methylation and acetylation, affect ACE2 gene expression, while (E) miRNAs negatively regulate ACE2 gene expression by cleaving ACE2 mRNA or inhibiting its translation. (F) Various transcription factors participate in ACE2 transcription. Certain transcription factors, like FXR and Nrf2, bind to their respective responsive elements in the ACE2 promoter region, regulating ACE2 expression. Others directly interact with the ACE2 promoter region upon stimulation by upstream molecules (created with BioRender).

3.2. ACE2 Activation

The catalytic domain of ACE2 consists of two subdomains (I and II) connected by a hinge-bending region, which forms a deep cleft housing the Zn-active catalytic site.⁹⁵ In the resting state, ACE2 is in an open conformation with the two subdomains separated. Upon substrate binding, ACE2 undergoes a conformational change, transitioning into a closed structure that restricts the access of exogenous molecules to its active catalytic site. ACE2 activators can prevent this full closure by inducing a clamshell-like opening motion that moves the two subdomains away from each other. This motion facilitates substrate entry, accelerates the enzymatic rate, and improves enzyme efficiency (V_max/K_m).^95,96 DIZE is among the most extensively studied ACE2 activator. Originally developed as an anti-infective drug, it was subsequently recognized and repurposed as an ACE2 activator potential antihypertensive medication.⁹⁷ DIZE targets the hinge-bending region at the back of ACE2 and exerts a pronounced effect on ACE2 activation. It leads to a 4-fold increase in V_max and a slight decrease in K_m.⁹⁵

4. ACE2 Up-Regulators or Activators

Currently recognized synthetic ACE2 up-regulators or activators are mainly derived from clinically used drugs, particularly those employed in the treatment of hypertension and diabetes.¹⁶ Other compounds were identified using high-throughput analyses to assess their potential to increase ACE2 expression or activity, with the aim of repurposing them for antihypertensive use. Examples include DIZE and XTN.^97,98 Natural ACE2 up-regulators or activators are sourced from four main categories: vitamins, phytochemicals, Traditional Chinese Medicine, and peptides/protein hydrolysates. These are summarized in Figure 5 and further discussed below.

Figure 5.

Natural and synthetic sources of ACE2 activators and up-regulators. Effective doses and treatment durations of each compound used in in vivo studies are included. All compounds are administered orally, except where specified otherwise (created with BioRender).

4.1. Synthetic Compounds

Synthetic ACE2 up-regulators primarily derive from pharmaceutical drugs used to treat hypertension, diabetes, lipidemia, and inflammation.¹⁶ The major categories of antihypertensive drugs include angiotensin converting enzyme inhibitors (ACEIs), Ang II receptor blockers (ARBs), calcium channel blockers (CCBs), diuretics, and mineralocorticoid receptor (MR) antagonists.^99,100 Even though their mechanisms of lowering blood pressure differ, many can upregulate ACE2. In addition, as previously highlighted, AMPK activation plays a crucial role in the regulation of ACE2 transcription. Therefore, certain antihyperglycemic medications which can stimulate AMPK also act as ACE2 up-regulators, with metformin being one example.¹⁰¹ Lipid-lowering drugs can be mainly categorized into two classes: statins and fibrates. Only statins, but not fibrates, have been reported as ACE2 up-regulators. Specifically, statins prevent the key rate-limiting step controlled by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in cholesterol synthesis, as the HMG-CoA analogues.¹⁰² Statins not only act as lipid modulators but also protect against diabetic cardiovascular complications, such as intimal thickening, through ACE2 upregulation.¹⁰³ Moreover, nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, also possess ACE2 up-regulatory activity, enhancing ACE2 expression in human alveolar type II pneumocytes.¹⁰⁴ In addition to the prescribed medications mentioned above, DIZE and XTN are two critical ACE2 up-regulators/activators. They not only act as ACE2 activators by directly binding to the hinge-bending region of ACE2, but also upregulate ACE2 expression, enabling them to protect against various ACE2-centered conditions.^105−109

4.2. Natural Compounds

4.2.1. Vitamins

Calcitriol, the active form of vitamin D3 (1,25-(OH)2D3), was initially used to treat vitamin D-resistant rickets.¹¹⁰ More recently, its ACE2 up-regulatory activity has renewed interest in its clinical applications, particularly for conditions like acute lung injury and diabetic kidney disease.^111,112 For example, the ACE2 up-regulatory activity of calcitriol has been implicated in mitigating lipopolysaccharide (LPS)-induced lung injury in rats by suppressing Ang II production. Its protective effects were also validated in pulmonary microvascular endothelial cells stimulated with LPS, indicating its therapeutic potential in lung-related pathologies.¹¹¹ Another compound with ACE2 up-regulatory activity is all-trans retinoic acid (RA), the active metabolite of vitamin A from the retinoid family. RA upregulated ACE2 in various disease models, including cardiac remodeling,¹¹³ essential hypertension,¹¹⁴ and renal interstitial fibrosis.¹¹⁵ Similarly, Ang II acts as a stimulus of left ventricular remodeling through its actions on AT1R. Therefore, the suppression of Ang II production achieved by increasing cardiac ACE2 expression and subsequent binding with AT1R through RA treatment led to the reversal of pressure overload-induced left ventricular dysfunction.¹¹³ Unlike vitamins A and D, vitamin C is a known ACE2 inhibitor. It suppressed ACE2 expression in various cell types, including HEK293T (kidney), Caco-2 (colon), 2fTGH (fibroblast), and A549 (lung), in a dose-dependent manner. Its inhibitory effect on ACE2 expression was not evident at the gene level but was observed at the protein level, where it promotes lysine 48-linked polyubiquitination and lysosome-dependent degradation.¹¹⁶

4.2.2. Phytochemicals

Long-term oral administration of resveratrol, a well-known anti-aging compound, enhanced aortic ACE2 expression in aging mice.¹¹⁷ Baicalin, a flavonoid derived from Scutellaria baicalensis, confers a protective effect in Ang II-induced human umbilical vein endothelial cells (HUVECs) by activating the ACE2/Ang (1–7)/MasR axis.¹¹⁸ Moreover, curcumin, the main active component of turmeric, and its analogue B6, could reverse ACE2 downregulation in rat models challenged with Ang II or streptozotocin, respectively.^119,120 Furthermore, several other compounds were identified as ACE2 up-regulators, including rosmarinic acid from rosemary,²³ puerarin from Kudzu root,¹²¹ thapsigargin from thapsia,¹²² osthole from Cnidium monnieri Cusson,¹²³ and naringenin from citrus fruits.¹²⁴

Several saponins derived from ginseng, such as ginsenoside Rg3 and ginsenoside Rc, enhanced ACE2 expression in various tissue and cell types. For example, ginsenoside Rg3 resulted in a higher ACE2 expression in renal tissue of SHRs, leading to reduced Ang II levels and attenuated Ang II-induced inflammation, oxidation, and fibrosis.¹²⁵ Meanwhile, ACE2 activating activity renders ginsenoside Rc anti-inflammatory and antioxidant properties in HUVECs. These effects, however, could be abolished by the addition of ACE2 inhibitor MLN-4760.¹²⁶ Similarly, Panax notoginseng saponins activated ACE2 and exerted protective effects in post-MI ventricular remodeling in rats.¹²⁷ In addition, licorice root-derived saponin glycyrrhizic acid blunted LPS-induced acute lung injury in mice by restoring pulmonary ACE2 expression. In accordance with in vivo study, glycyrrhizic acid markedly enhanced ACE2 expression in HUVECs after LPS stimulation.¹²⁸ Furthermore, harpagoside, an iridoid glycoside, was emerged as a promising ACE2 activator with even higher potency than DIZE. Harpagoside has a higher binding affinity to ACE2, with an equilibrium dissociation constant (K_d value) of 86.1 μM, compared to the K_d value of 4.9 μM for DIZE. Notably, harpagoside also tends to promote ACE2 transcription as ACE2 mRNA expression increased in a dose-dependent manner in HUVECs treated with harpagoside. Taken together, harpagoside not only functions as an ACE2 enzymatic activator but also serves as an ACE2 transcriptional up-regulator.⁵⁸

4.2.3. Traditional Chinese Medicines (TCMs)

While most TCMs were reported for their ACE2 inhibitory activity,¹²⁹ some TCMs were found to activate ACE2. Sini decoction, which contains aconite, liquorice and ginger rhizome, markedly activated the ACE2/Ang (1–7)/MasR axis in mouse lung tissues and plasma, resulting in attenuated acute lung injury in a mouse model.¹³⁰ This result was later confirmed by Chen et al. (2019), who also demonstrated the ACE2 activating activity of Sini decoction in HUVECs under inflammatory conditions.¹³¹ Recently, the network-based degree distribution analysis was used to identify 144 active ingredients within a complex formula decoction known as Wuwei Jiangya decoction (WJD). These active ingredients could regulate key parameters related to hypertension. Subsequently, the antihypertensive activity of WJD was observed in the SHRs with improved left ventricular mass index and ameliorated cardiac hypertrophy and vascular injury. This positive outcome was achieved through the activation of the ACE2/Ang (1–7)/MasR axis in both the thoracic aorta and left ventricular myocardium.¹³²

In addition, a Tibetan remedy called Tsantan Sumtang, known for its ability to increase ACE2 expression in the right ventricle, could attenuate hypoxia-induced cardiac remodeling by suppressing Ang II production in pulmonary arterial hypertensive rats.¹³³ Another TCM, Fugan Wan, alleviated hepatic fibrosis by activating the ACE2/Ang (1–7)/MasR axis while inhibiting the ACE/Ang II/AT1R axis, suggesting the significant role of the balanced RAS in preventing chronic liver diseases.¹³⁴ However, considering the complex composition of TCMs, the identification and isolation of active compounds with ACE2 activating ability could deepen our understanding of their mechanisms of action, thereby improving their therapeutic efficacy.

4.2.4. Food-Derived Bioactive Peptides

Food-derived bioactive peptides possess various physiological functions and can be obtained through enzymatic digestion or fermentation of parent proteins. Bioactive peptides possess various health benefits, including antihypertensive activity.⁴ Most antihypertensive peptides are identified as inhibitors of ACE, while the blood pressure lowering activity of peptide IRW is via upregulating ACE2 transcription and then activating the ACE2/Ang (1–7)/MasR cascade.¹⁷ IRW, derived from ovotransferrin, is the first food-derived ACE2 up-regulatory peptide. Pea protein-derived peptide LRW, an isomer of IRW, was previously identified as ACE inhibitor.¹³⁵ In vascular smooth muscle cells (VSMCs), this peptide shows similar activity in upregulating ACE2 gene and protein levels as that of IRW;¹³⁶ in SHRs, LRW failed to reduce blood pressure and upregulate ACE2 in SHRs due to its susceptibility to degradation by the gastrointestinal enzymes.¹³⁷ AKSLSDRFSY, also derived from pea, and its two GI-digested peptides, LSDRFS and SDRFSY, were shown to upregulate ACE2 in VSMCs.¹³⁸ However, it is not known if these peptides also possess in vivo activity. Several ACE2 up-regulatory/activating peptides were also identified from other food sources. IQP and VEP, isolated from Spirulina platensis, were reported to activate the ACE2/Ang (1–7)/MasR axis in the myocardium of SHRs.¹³⁹ Peptides VKW, VHPKESF, VVHPKESF, and VVHPK, released from chicken muscle proteins via thermoase digestion, were able to increase ACE2 protein levels in VSMCs.¹⁴⁰ Among these peptides, VVHPKESF enhanced cellular ACE2 enzymatic activity and upregulated ACE2 protein levels in a dose-dependent manner from 50 to 200 μM.¹⁴¹ Further, its ACE2 up-regulatory activity was confirmed in the aortas of SHRs, suggesting its potential as an antihypertensive agent in this rat model.¹⁴² Moreover, rapeseed-derived peptides LY and GHS both positively affected ACE2 gene and protein levels in myocardium tissue of SHRs. Peptide RALP failed to increase ACE2 mRNA expression but significantly improved ACE2 protein levels in rats’ myocardium.¹⁴³ Garlic protein-derived peptides, MGR and HDCF, were chosen based on their high ACE inhibitory activities determined through simulated hydrolysis and in silico screening. Further, both peptides showed antihypertensive effects in SHRs even at a low dosage of 10 mg/kg body weight. This was associated with enhanced renal function and endothelial function, as well as the restoration of a balanced RAS in the kidney, with reversal of ACE overexpression and ACE2 inhibition.¹⁸ Given the role of a high-sodium diet in hypertension development, a low-sodium pork sausage was specifically formulated to counteract this effect. The identification of two peptides, LIVGFPAYGH and IVGFPAYGH, exhibiting both ACE inhibitory and ACE2 up-regulatory activities, positions this sausage as a potential antihypertensive product.¹⁴⁴ Similarly, two novel peptides, SNHANQLDFHP and PVQVLASAYR, identified from pumpkin seed meal (PSM), showed both ACE inhibitory and ACE2 up-regulatory activities, providing new sustainable solutions for PSM after oil extraction.¹⁴⁵ Moreover, ACE inhibitory peptides RIY derived from rapeseed and IKW from chicken muscle could upregulate ACE2 in both plasma and aorta of SHRs, thus contributing to their antihypertensive activities. Although most ACE2 activating/up-regulatory peptides possess ACE inhibitory activity, ACE2 activating or up-regulatory activity is not a common feature of ACE inhibitory peptides.¹⁹ By up-regulating ACE2, soybean protein isolate hydrolysate ameliorated endothelial dysfunction in HUVECs, which was reserved by MLN-4760, an ACE2 inhibitor. Two novel peptides, IVPQ and IAVPT, derived from this isolate, activated ACE2 enzymatic activity and enhanced ACE2 expression in HUVECs.¹⁴⁶Figure 6 summarizes the food-derived peptides exhibiting ACE2 activating/up-regulatory activities.

Figure 6.

Bioactive peptides with ACE2 activating/up-regulatory activities (created with BioRender).

Significantly, bioactive peptides often have low bioavailability and may undergo degradation and metabolism after oral administration.¹⁴⁷ Therefore, we cannot dismiss the possible contribution of peptide fragments or their metabolites to the physiological effects in vivo, an aspect that could be validated through time-course metabolomics. By employing this method, kynurenine, a metabolite of the peptide IRW, was identified as the molecule partially responsible for the antihypertensive effects of IRW after its degradation and metabolism in SHRs.¹⁴⁸ In addition, although several ACE2 activating peptides have been identified, further exploration of these peptides is warranted to establish a comprehensive database for future structural and activity studies. This will facilitate the determinization of the specific structural requirements responsible for ACE2 activation and the development of a peptide with the highest ACE2 activity.¹⁴⁹

4.2.5. Others

In addition to the aforementioned categories of natural compounds, other natural substances, such as palmitic acid,¹²² 17β-estradiol,¹⁵⁰ nicotine,¹⁵¹ and walnut supplementation (a rich source of polyunsaturated omega-3 fatty acids),¹⁵² all demonstrated the potential to upregulate ACE2 expression. A novel gaseous mediator, hydrogen sulfide (H₂S), which is endogenously produced during cysteine metabolism, could attenuate atherosclerosis through ACE2 activation.¹⁵³ Lipoxin A4, an eicosanoid derived from arachidonic acid through transcellular metabolic pathways, could also act as ACE2 activator.¹⁵⁴ The detailed study information on natural compounds with ACE2 activating activity is summarized in Table 1, and information regarding natural compounds with ACE2 up-regulatory activity is included in Table 2.

Table 1. Natural Compounds with ACE2 Activating Activity In Vivo or In Vitro.

compounds	aim(s)	treatments	results	ref.
Phytochemicals
Harpagoside	Screen ACE2 activators within pharmacological compounds using a combination of in silico virtual docking screening methods and in vitro measurements	Treatment of HUVEC lysates with 0.5 × 10–7 M and 1.5 × 10–6 M harpagoside for 30 min	↑ ACE2 enzymatic activity in HUVEC lysates in a dose-dependent manner	(58)
Food-derived peptides/protein hydrolysates
Ovotransferrin-derived peptide IRW	Evaluate the ACE2 activating activity of IRW in VSMCs	Addition of 10–1 to 10–13 M IRW in cell-free ACE2 activity assay	↑ ACE2 activity in a dose-dependent manner from 10–7 to 10–3 M	(168)
	Investigate the mechanism underlying the activation of ACE2 by IRW through specific cell-bound receptors	Treatment of VSMCs with 50 and 25 μM of IRW for 24 h	IRW facilitates the exchange of GDP for GTP, leading to the activation of G protein-coupled receptor 30 (GPR30) This activation subsequently results in ACE2 transcriptional activation	(169)
Soybean protein isolate hydrolysate (SPIH) and soybean-derived peptides IVPQ and IAVPT	Identify ACE2 activating peptides from soybean protein hydrolysate and investigate their roles in Ang II-induced endothelial dysfunction	(1) Treatment of HUVECs with 0.5 or 1.0 mg/mL of SPIH for 4 h before 24 h-Ang II (1 μM) stimulation	(1) ↑ ACE2 activity with SPIH at 1.0, but not 0.5 mg/mL	(146)
		(2) Treatment of HUVECs with peptides at 10 and 50 μM for 24 h	(2) ↑ ACE2 activity with both peptides at only 50 μM
Others
Bile acid derivatives (BARs)	Select BARs as ACE2 activators through virtual screening and molecular dynamics	MD simulations were conducted for apo ACE2 and ACE2 in complex with BAR708 and BAR107	↑ ACE2 activity by inhibiting the spontaneous closure of Sub I to Sub II	(170)
Lipoxin A4	Investigate the effects of lipoxin A4 in LPS-induced acute lung injury	Injection of lipoxin A4 (100 μg/kg) to adult male Kun Ming mice (4–6 weeks old) 30 min before LPS challenge (10 mg/kg) with A779 pretreatment (10 μg/kg)	50% ↑ renal ACE2 activity after lipoxin A4 treatment, which 36% ↓ by A779 administration	(154)

Table 2. Natural Compounds with ACE2 Up-Regulatory Activity In Vivo or In Vitro^a.

compounds	aim (s)	animal model	treatments	results	ref.
Vitamins
Calcitriol	Investigate the role of calcitriol in LPS-induced ALI and its impact on the activity of components in RAS	Wistar rats (male, 3–4 months old)	(1) Oral administration of 1, 5, or 25 mg/kg calcitriol for 3 days before LPS challenge (5 mg/kg)	(1) In LPS-challenged rats, a ∼ 50% ↑ pulmonary ACE2 mRNA expression only after 25 mg/kg treatment	(111)
			(2) Incubation of 100 μg/mL LPS with calcitriol (5, 20, or 100 nM) in pulmonary microvascular endothelial cells	(2) ↑ ACE and ↓ ACE2 expression by LPS treatment, whereas calcitriol treatment (100 nM) ↓ the effects of LPS in vitro
	Evaluate the effect of calcitriol on ACE and ACE2 in diabetic nephropathy	Wistar rats	Oral administration of 0.03 g/kg of calcitriol to both nondiabetic and STZ-induced diabetic rats for 16 weeks	A 14-fold ↑ tubular ACE/ACE2 ratio in diabetic rats compared to the nondiabetic rats, which 50% ↓ by calcitriol treatment	(112)
Vitamin D2	Analyze ACE2 levels across cell types and treat them with promising compounds for COVID-19 therapy to investigate the role of RAS in COVID-19 pathophysiology		Treatment of A549 lung cancer cells with vitamin D2 (100 nM)	↑ ACE2 and ↓ renin protein levels	(171)
All-trans retinoic acid (atRA)	Investigate the effects of atRA on ACE2 expression and blood pressure	Wistar rats and SHRs (male, 12 weeks old)	Injection of atRA at a dose of 10 mg/kg/day to WKY, or atRA (10 or 20 mg/kg/day) to SHRs for one month	(1) 100% and 500% ↑ cardiac mRNA expression after low- and high-dose atRA treatment, respectively, in SHRs; 200% and 300% ↑ renal mRNA expression after low- and high-dose atRA treatment, respectively, in SHRs	(114)
				(2) 30% ↑ cardiac and renal ACE2 protein levels after atRA treatments
				(3) atRA administration had no effects on cardiac and renal ACE2 gene expression or protein levels
	Investigate the preventive role of atRA in the development of cardiac remodeling	Sprague–Dawley rats (male)	Oral administration of atRA (30 mg/kg/day) for 3 days to rats with aortic constriction	↑ cardiac ACE2 protein levels, resulting in ↓ Ang II level in plasma	(113)
	Study the effects of atRA on RAS components in renal interstitial fibrosis		Treatment of renal tubular epithelial cells with oxidative stress and cell injury with atRA (0.1 μM) for 48 h	↑ ACE2 mRNA and protein levels in cells treated with atRA	(115)
Phytochemicals
Resveratrol	Investigate the effects of resveratrol on RAS components and their role in vascular aging	C57BL/6J mice (male, 8 months old)	Administration of normal chow with 40 mg/kg/day of resveratrol for 6 months	100% ↑ aortic ACE2 protein levels	(117)
Baicalin	Investigate the protective role of baicalin in Ang II-induced endothelial dysfunction		Treatment of HUVECs with baicalin (12.5, 25, and 50 μmol/L) and/or Ang II (1 × 10–6 mol/L)	↑ ACE2 mRNA and protein levels in a concentration-dependent manner	(118)
Curcumin	Explore the role of curcumin in Ang II-induced myocardial fibrosis	Sprague–Dawley rats (male)	Oral administration of 150 mg/kg/day of curcumin during Ang II infusion at a rate of 500 ng/kg/min for 4 weeks	∼30% ↓ transmural tissue ACE2 with the 4-week Ang II infusion, but 80% ↑ ACE2 with curcumin treatment	(120)
Curcumin analogue B6	Investigate the effects of a curcumin derivative in T2D	Wistar rats (male)	Oral administration of 1 (low), 3 (middle), and 9 mg/kg/day (high) B6 to T2D rats for 8 weeks	(1) ∼ 40% ↓ renal interstitial ACE2 protein levels, while 40% and 50% ↑ after low- and middle-dose treatment, respectively	(119)
				(2) 33% ↓ renal interstitial ACE2 gene expression, while ∼20% and 40% ↑ after low- and middle-dose treatment, respectively
Rosmarinic acid (RA)	Investigate the protective effect of RA on cardiac fibrosis in the established MI rat model	Sprague–Dawley rats (male)	Oral administration of 50, 100, and 200 mg/kg/day of RA to MI rats for 4 weeks	40% ↓ myocardial ACE2 protein levels in MI rats, while 50% ↑ after RA treatment (only 200 mg/kg/day)	(23)
Puerarin (Pue)/Felodipine (Felo)	Investigate the protective role of the combination of Pue and Felo in renovascular hypertension	Sprague–Dawley rats (male)	Administration of 25 mg/kg/day of Pue and 0.4 mg/kg/day of Felo for 8 weeks	∼62.5% ↓ renal ACE2 mRNA expression in rats with ischemia, while ∼150% ↑ after Felo and Pue treatment	(121)
Thapsigargin (Tg)	Investigate the effect of ACE2-modulated ER stress on preventing nonalcoholic fatty liver disease		Treatment of HepG2 hepatocytes with 1 μM of Tg for 12 or 24 h	↑ ACE2 mRNA and protein levels	(122)
Osthole	Investigate the protective role of osthole in pulmonary fibrosis	Sprague–Dawley rats (male)	Oral administration of 40 mg/kg/day of osthole to rats with pulmonary fibrosis for 4 weeks	50% ↓ pulmonary ACE2 gene expression and protein levels, while 100% and 180% ↑ protein levels and gene expression, respectively, after osthole treatment	(123)
Naringenin	Investigate the effects of naringenin in renovascular hypertensive renal damage	Sprague–Dawley rats (male)	Oral administration of 200 mg/kg/day naringenin for 10 weeks	(1) 40% ↓ ACE2 protein levels in cortex, while 130% ↑ after treatment	(124)
				(2) 60% ↓ ACE2 protein levels in medulla, while 200% ↑ after treatment
Ginsenoside Rg3 (Rg3)	Evaluate the effects of Rg3 in hypertensive nephropathy	SHRs (male, 16–17 weeks old);	(1) Oral administration of 20 mg/kg/day of Rg3 to SHRs for 42 days	(1) 120% ↑ renal ACE2, 85% ↑ Ang (1–7) (6.5 and 12 pg/mg, respectively, for SHRs with or without receiving Rg3), and 42%↓ Ang II (70 and 40 pg/mg, respectively) in SHRs	(125)
		C57BL/6 mice (male, 10 weeks old)	(2) Oral administration of 20 mg/kg/day of Rg3, with or without concurrent Ang II infusion (1.5 mg/kg/day), to C57BL/6 mice for 14 days	(2) 110% ↑ renal ACE2 protein levels in Ang II-induced mice that received Rg3 treatment
Ginsenoside Rc (Rc)	Investigate the role of ACE2/Ang (1–7)/MasR axis in insulin resistance and endothelial dysfunction	C57BLKS/J mice (male, 8 weeks old)	(1) Oral administration of Rc (20 mg/kg/day) for 4 weeks with or without MLN-4750 (0.5 mg/kg/day, intraperitoneal) to leptin receptor-deficient db/db mice	(1) 30% ↑ aortic ACE2 protein levels after Rc treatment in mice with or without receiving MLN-4750	(126)
			(2) Treatment of HUVECs with glucose (7 mM) plus Rc (50 μM) with or without ACE2 inhibitor MLN-4760 (100 nM) for 24 h before a 30 min insulin incubation	(2) ↑ ACE2 protein levels upon Rc treatment, which was partially abrogated by MLN-4760 in HUVECs
Panax notoginseng saponins (PNS)	Investigate the role of AEC2 and TNFα in post-MI ventricular remodeling	Sprague–Dawley rats (male, 10 weeks old)	Oral administration of PNS (10, 20, and 40 mg/kg/day) for 4 weeks	↑ cardiac ACE2 and ↓ TNFα in all dose groups	(127)
Glycyrrhizic acid (GA) derived from licorice root	Explore the effects of GA in LPS-induced ALI	Balb/c mice (male, 8 weeks old)	(1) Intraperitoneal injection of GA (200 mg/kg) for 1 h with or without 10 mg/kg LPS for 8 h	(1) 12% ↓ pulmonary ACE2 protein levels induced by LPS, whereas 64% ↑ after GA treatment in mice with ALI	(128)
			(2) Treatment of HUVECs with GA (100 μg/mL) for 1 h with 8 h LPS (1 μg/mL)	(2) Similar results were found in HUVECs
Harpagoside	Screen ACE2 activators within pharmacological compounds using a combination of in silico virtual docking screening methods and in vitro measurements		(1) Treatment of HUVECs with harpagoside (4, 20, and 100 μM) for 16 h	(1) ↑ ACE2 mRNA expression in HUVECs in a dose-dependent manner	(58)
			(2) Treatment of AML12 cells with 4 and 100 μM of harpagoside for 16 h	(2) ↑ ACE2 mRNA expression with both treatment doses
Traditional Chinese Medicines (TCMs)
Sini decoction (SND)	Investigate the role of SND in acute lung injury	ICR mice (male)	Oral administration of 5 g/kg of SND twice daily for 1 day, after being treated with LPS (8 mg/kg)	∼69% ↓ pulmonary ACE2 protein levels in mice receiving LPS, whereas 500% ↑ after SND treatment	(131)
Wuwei Jiangya decoction (WJD)	Investigate the antihypertensive effects of WJD in SHRs and screen the active compounds from WJD using network-based degree distribution analysis	SHRs (12 weeks old); Wistar rats (male, 12 weeks old)	Oral administration of 3.24 (low), 6.48 (medium) or 12.96 g/kg/day (high) of WJD to SHRs for 8 weeks	4% and 35% ↑ ACE2 protein levels in thoracic aorta and left ventricular myocardium, respectively, after high-dose treatment	(132)
Tsantan Sumtang	Investigate the role of Tsantan Sumtang in right ventricular (RV) structure remodeling and fibrosis	Sprague–Dawley rats (male)	Intragastrical administration of 1.0, 1.25, or 1.5 g/kg/day of Tsantan Sumtang for 28 days	↑ ACE2 protein and ACE2 mRNA levels in RV tissue after Tsantan Sumtang administration at doses of 1.25 and 1.5 g/kg/day (statistical data not provided)	(133)
Fugan Wan (FGW)	Investigate the role of FGW in hepatic fibrosis	Wistar rats (6–8 weeks old)	Oral administration of 6.43 g/kg/day of FGW for 4 weeks	∼140% ↑ hepatic ACE2 mRNA expression after FGW treatment in rats with hepatic fibrosis	(134)
Food-derived peptides/protein hydrolysates
Ovotransferrin-derived peptide IRW	Identify the genes critical for the antihypertensive activity of IRW	SHRs (male)	Oral administration with IRW (15 mg/kg/day) for 18 days	23-fold ↑ ACE2 transcripts, further qPCR showed 17-fold ↑	(172)
	Evaluate its ACE2 up-regulatory activity in VSMCs		Treatment of VSMCs with IRW (10 or 50 μM) for 24 h	↑ ACE2 mRNA and protein levels in VSMCs with 50 μM of IRW treatment	(168)
	Evaluate the ACE2 up-regulatory and activating effects of IRW in SHRs	SHRs (male, 12–14 weeks old)	Oral administration of 15 mg/kg/day of IRW for 18 days	(1) 114% ↑ circulating ACE2 levels	(17)
				(2) 13-fold ↑ aortic ACE2 protein levels
Spirulina platensis derived peptides IQP, VEP and its hydrolysates	Investigate the regulatory effects of IQP, VEP, and the hydrolysates on renal RAS components in SHRs at different ages and compare their efficacy in blood pressure regulation with that of captopril	SHRs (male, 5 weeks old)	Oral administration with IQP, VEP, Spirulina platensis hydrolysates, and captopril (10 mg/kg/day) for 6 weeks	(1) 11-week-old rats: 40%, 50% and 80% ↑ myocardial ACE2 mRNA expression after IQP, hydrolysates and captopril, respectively; 14-week-old rats: 80%, 60%, 90% and 130% ↑ after IQP, VEP, hydrolysates and captopril, respectively; 16-week-old rats: 40% and 50% ↑ after hydrolysates and captopril, respectively	(139)
				(2) similarly, ↑ myocardial ACE2 protein levels after IQP, VEP, hydrolysates and captopril in 11- or 14-week-old rats; in 16-week-old rats, ↑ myocardial ACE2 protein levels only after hydrolysates and captopril treatments
Spent hen protein hydrolysate prepared using thermoase PC10F (SPH-T)	Compare the ACE2 up-regulatory activity of SPHs hydrolyzed by different enzymes		Treatment of VSMCs with 2.5 mg/mL of SPHs for 24 h	↑ ACE2 protein levels	(173)
	Investigate the antihypertensive effects of SPH-T	SHRs (male, 12–14 weeks old)	Oral administration of 250 (low) and 1000 mg/kg/day (high) for 20 days	∼186% and 150% ↑ circulating and aortic ACE2 levels, respectively, after high-dose treatment	(174)
Hen protein-derived peptides VKW, V–F and V–K	Identify both ACE inhibitory and ACE2 up-regulatory peptides from antihypertensive SPH-T		(1) Treatment of VSMCs with SPH-T at doses of 0.25, 0.5, 1.0, 1.5, and 2.5 mg/mL for 24 h	(1) ↑ ACE2 protein levels with treatment of SPH-T at doses of 1.0, 1.5, and 2.5 mg/mL	(140)
			(2) Treatment of VSMCs with peptides (50 μM) for 24 h	(2) ↑ ACE2 protein levels upon peptides treatment (50 μM)
	Investigate the blood pressure-lowering activity of peptide V–F	SHRs (male, 12–14 weeks old)	Oral administration of 15 mg/kg/day of V–F for 18 days	123% and ∼113% ↑ aortic and circulating ACE2 protein levels, respectively	(142)
Pea-derived peptides AKSLSDRFSY, LSDRFS and SDRFSY	Identify ACE2 up-regulatory peptides from pea protein hydrolysate		Treatment of VSMCs with 50 μM of peptides for 24 h	↑ ACE2 protein levels	(138)
Pea-derived peptide LRW	Explore the regulatory effects of LRW on RAS components in Ang II-stimulated cells		Treatment of VSMCs with 10 or 50 μM of LRW for 24 h	↑ ACE2 protein levels upon LRW treatment (50 μM)	(136)
Rapeseed protein-derived peptides LY, RALP, and GHS	Assess the long-term antihypertensive effects of selected ACEi peptides	SHRs (male, 10 weeks old)	Oral administration of 30 mg/kg/day of peptides for 5 weeks	(1) 75% and 175% ↑ ACE2 mRNA expression in the myocardium after LY and GHS treatments, respectively	(143)
				(2) 50%, 80% and 60% ↑ ACE2 protein levels after LY, GHS and RALP treatments, respectively
Garlic protein-derived peptides MGR and HDCF	Screen garlic protein-derived ACEi peptides from established database and investigate their impact on the blood pressure of SHRs	SHRs (male, 12 weeks old)	Oral administration of peptides at doses of 10 (low), 30 (medium), or 50 mg/kg/day (high) for 7 weeks	9.5%, ∼ 29%, ∼ 43% ↑ renal ACE2 protein levels after low-, medium-, and high-dose treatment of MGR, respectively	(18)
				Similarly, 111%, 178%, 267% ↑ after low-, medium-, and high-dose treatment of HDCF, respectively
Pork sausage-derived peptides LIVGFPAYGH and IVGFPAYGH	Identify ACEi peptides in sodium-reduced pork sausage		Treatment of HUVECs with peptides (100 and 300 μM) for 4 h before 12 h Ang II treatment (1 μg/mL)	↑ ACE2 protein levels with peptides at both doses	(144)
Chicken muscle protein-derived peptide IKW and rapeseed-derived peptide RIY	Investigate the ACE2 up-regulatory activity of ACE inhibitory peptides in vitro and in vivo	SHRs (male, 12–14 weeks old)	(1) Oral administration of peptides (15 mg/kg/day) for 7 days	(1) 100% and 116% ↑ aortic ACE2 protein levels after IKW and RIY treatment, respectively	(19)
			(2) Treatment of VSMCs with peptides (50 μM) for 24 h	(2) ↑ ACE2 protein levels with IKW treatment in VSMCs
Soybean protein isolate hydrolysate (SPIH) and soybean protein-derived peptides IVPQ and IAVPT	Identify ACE2 up-regulatory peptides from soybean protein hydrolysate and investigate their ACE2 up-regulatory activities against Ang II-induced endothelial dysfunction		(1) Treatment of HUVECs with 0.5 or 1.0 mg/mL of SPIH or 1 μM Ang II for 24 h	(1) ↑ ACE2 protein levels with SPIH at both doses	(146)
			(2) Treatment of HUVECs with both peptides at 10 and 50 μM for 24 h	(2) ↑ ACE2 protein levels with IVPQ at 50 μM, but not 10 μM
Others
Palmitic acid (PA)	Confirm the role of hepatic ACE2 in endoplasmic reticulum stress		Treatment of hepatoblastoma cell line (HepG2 cells) with 0.4 mM of PA for 12 or 24 h	(1) ↑ ACE2 mRNA expression after PA treatments	(122)
				(2) ↑ ACE2 protein levels in 12 h PA treatment
17β-Estradiol (E2)	Investigate the alleviative effects of E2 on Alzheimer’s disease	Wistar rats (female, 12 weeks old)	Subcutaneous injection of 25 μg/kg/day of E2 and/or oral administration of 3 mg/kg/day of Telmisartan (Tel) for 6 weeks	62.5% ↓ hippocampal ACE2 protein levels in D-gal-injected ovariectomized rats, which 87%, 73%, 133% ↑ after Tel, E2, and Tel+E2 treatment, respectively	(150)
Nicotine	Investigate the mechanism underlying the increased susceptibility to SARS-CoV-2 associated with nicotine usage		(1) Treatment of 0.01, 0.1, and 10 μM of nicotine to A549 type II pulmonary adenocarcinoma cells for 12, 24, or 48 h	(1) ↑ ACE2 mRNA expression by nicotine at concentrations of 0.01 μM and 0.1 μM for 24 h	(151)
			(2) Treatment of 0.01 μM of nicotine for 24 h or 0.1 μM of nicotine for 1, 24, or 48 h	(2) ↑ ACE2 protein levels following nicotine treatment after 24 h, but ↓ at 48 h
Walnut supplementation	Investigate the protective effects of walnut supplementation on chronic inflammation and metabolic syndrome	Wistar rats (male, 3-week-old)	After providing water or a 10% (w/v) fructose solution for 9 weeks, half of the rats in each group were administrated 2.4 g of dietary walnuts for 6 weeks	(1) ∼ 31% ↑ cardiac ACE2 protein levels in rats fed with a fructose solution upon walnut supplementation	(152)
				(2) No significant change in cardiac ACE2 protein levels in rats that consumed water, even supplemented with walnuts
Hydrogen sulfide (H2S)/NaHS	Investigate the alleviative role of H2S in atherosclerosis	C57BL/6J apoE–/– mice (male)	(1) Following the assignment of mice to a partial ligation of the left carotid artery, intraperitoneal injections of 1 mg/kg/day of NaHS for 4 weeks	(1) In carotid arteries, 48% ↑ ACE2 expression after NaHS treatment in mice underwent LCA partial ligation	(153)
			(2) Treatment of HUVECs with 50, 100, and 200 μM of NaHS for 24 h	(2) In HUVECs, ↑ ACE2 mRNA and protein levels in a time- and dose-dependent manner with NaHS treatment
			(3) Treatment of HUVECs with 100 μM of NaHS for 0, 6, 24, or 48 h

^aQuantitative dose–effect data and information on experimental models are added to the in vivo studies

5. Discussion and Future Perspectives

ACE2 has multifaceted functions: its extracellular spike protein binding domain designates it as the entry receptor for coronaviruses, its monocarboxypeptidase activity maintains physiological homeostasis and safeguards various organ systems, and its collectrin-like domain functions as a neutral amino acid transporter. While recent scientific research refutes the initial notion of using ACE2 inhibitors to prevent the binding between the coronavirus and ACE2, further insights suggest that upregulating and/or activating ACE2 can prevent the cytokine storm induced by COVID-19.¹⁵⁵ In this review, we noted that the complexity of the mechanisms governing ACE2 upregulation, with only a few identified ACE2 transcription factors and epigenetic modulators. In contrast, ACE2 activation results from a singular mechanism that targets its hinge-bending region. Despite the distinction between upregulation and activation, molecules that can upregulate or activate ACE2 both show vast therapeutical value, as ACE2 within the renin-angiotensin system plays protective roles in various diseases. Consequently, novel ACE2 modulators have been identified from natural sources, particularly food protein hydrolysates and peptides, as part of a dietary strategy to improve human health. Future research on ACE2 is foreseen to consider the following aspects:

5.1. Continue to Explore the Functions of ACE2 in Health and Disease Control

Despite debates about ACE2’s roles as an entry receptor of some coronaviruses and a monocarboxypeptidase in generating the vasodilator Ang (1–7), it is crucial to note the transient nature of COVID-19, in contrast to the persistent presence of cardiovascular diseases and diabetes. Acknowledging ACE2’s protective roles in these enduring conditions, numerous up-regulators and activators have been identified.¹⁶ Novel roles of ACE2 in other diseases emerge. In cancer, ACE2, through Ang (1–7) and MasR, inhibits reactive oxygen species production, suppressing cell proliferation, invasion, and angiogenesis by inhibiting vascular endothelial growth factor.¹⁵⁶ Similarly, ACE2 is implicated in neurodegenerative diseases such as Parkinson’s, stroke, and Alzheimer.¹⁵⁷ In addition, ACE2 is an emerging key regulator in reproductive health, influencing steroidogenesis, sperm cell function, and epididymal contractility through Ang (1–7).^158,159

5.2. Understanding the Mechanisms Underlying ACE2 Upregulation

When summarizing ACE2 up-regulators, there is a notable gap in exploring the underlying mechanisms of ACE2 regulation; many highlight the therapeutic effects of ACE2 up-regulators and their modulation of the RAS components. ACE2 transcription can be influenced by various transcription factors and epigenetic modulators, suggesting that ACE2 transcription is the cumulative outcome of these diverse regulatory mechanisms. Therefore, understanding the regulatory pathway, such as identifying ACE2 transcription factors, the upstream signaling targeting these transcription factors, and epigenetic modulators, not only facilitates the development of novel molecules targeting these modulators involved in ACE2 transcription, but also eases repurposing drugs for other diseases linked to similar transcription factors or pathways.⁹⁸

5.3. Identifying ACE2 Activators Using Novel Techniques

In contrast to the diverse pathways influencing ACE2 upregulation, ACE2 activation results from molecules binding to its hinge-bending region, with XTN and DIZE being two well-studied ACE2 activators. The field of drug discovery has witnessed a growing application of artificial intelligence (AI) tools.¹⁶⁰ For example, researchers have established a protein language model to predict ACE inhibitory peptides.¹⁶¹ Similarly, there is an expectation to construct a corresponding model for predicting potential ACE2 activators targeting ACE2’s hinge-bending region.

5.4. Conducting Clinical Studies for Prospective Real-World Applications

While numerous novel ACE2 modulators have been identified, the real-world application of ACE2 activators or up-regulators is currently lacking. In contrast to these modulators, rhACE2 emerges as a promising candidate for ACE2-centered disease treatments. Nevertheless, it is important to recognize that every medication introduced to the market undergoes rigorous clinical studies.¹⁶² The discovery of ACE2 in 2000 revealed its counterbalancing effects on ACE, producing Ang (1–7). A decade later, the efficacy of rhACE2 in an animal model of diabetic nephropathy was confirmed,⁴⁵ leading to an increasing volume of research employing rhACE2 in various disease models.^163,164 This momentum has translated into several clinical studies.^165,166 In essence, we find ourselves still in the early stages of ACE2 medication development, grappling with certain unsolved intricacies. One such challenge is the need for a more comprehensive understanding of ACE2 regulation, a hurdle that could potentially be overcome through the application of multiomics or advanced AI techniques.¹⁶⁷

Author Contributions

Z.W., H.F., and J.W., conceptualization; Z.W., writing-original draft; J.W., supervision; Z.W., H.F., and J.W., writing-reviewing and editing.

This work was supported by Grants from Natural Sciences and Engineering Research Council (NSERC) of Canada.

The authors declare no competing financial interest.