Post-Coronavirus Disease 2019 Syndrome in Japan: An Observational Study Using a Medical Database.

Introduction: In Japan, the clinical information on post-COVID-19 syndrome, including nursing care requirements, is limited. The present study investigated the incidence of acute and post-COVID-19 symptoms, including nursing care requirements, when different SARS-CoV2 strains were prevalent and vaccination statuses changed to mass vaccination programs in Japan. Methods: Electronic health records of 122,045 patients diagnosed with COVID-19 between January 1, 2020, and June 30, 2022, were obtained from the Tokushukai Group Medical Database. Patient data was divided into three observation periods. Using the International Statistical Classification of Diseases and Related Health Problems 10 codes, typical symptoms of acute (within two weeks after diagnosis) and post-COVID-19 (2-12 weeks after diagnosis) were extracted. Moreover, the nursing care requirements of patients who visited the hospital before and after the COVID-19 diagnosis were examined. Results: Original and alpha strains were prevalent in Period 1, wherein most of the population was unvaccinated. The delta strain was prevalent in Period 2, wherein approximately 70% of the population was vaccinated. The omicron strain was prevalent in Period 3, wherein approximately 70% of the population completed the two vaccination doses. Headache, malaise/fatigue, depression, and disuse syndrome were detected in acute and post-COVID-19. The incidence of depression and disuse syndrome in post-COVID-19 increased with age, with the highest incidence in the 60-85-year group. Moreover, increased high-level nursing care requirements were observed after COVID-19 in the 60-85-year-age group. Conclusions: A lower incidence of acute and post-COVID-19 symptoms in Japan is linked to increased population vaccination coverage. However, differences in viral strains may be involved. Moreover, a reduction in long-term quality of life exists in older adult patients after COVID-19. These data provide fundamental information for preventing and treating post-COVID-19 syndrome in Japan.

Identification of Galectin-7 as a crucial metastatic enhancer of squamous cell carcinoma associated with immunosuppression.

Metastasis predicts poor prognosis in cancer patients. It has been recognized that specific tumor microenvironment defines cancer cell metastasis, whereas the underlying mechanisms remain elusive. Here we show that Galectin-7 is a crucial mediator of metastasis associated with immunosuppression. In a syngeneic mouse squamous cell carcinoma (SCC) model of NR-S1M cells, we isolated metastasized NR-S1M cells from lymph nodes in tumor-bearing mice and established metastatic NR-S1M cells in in vitro culture. RNA-seq analysis revealed that interferon gene signature was markedly downregulated in metastatic NR-S1M cells compared with parental cells, and in vivo NR-S1M tumors heterogeneously developed focal immunosuppressive areas featured by deficiency of anti-tumor immune cells. Spatial transcriptome analysis (Visium) for the NR-S1M tumors revealed that various pro-metastatic genes were significantly upregulated in immunosuppressive areas when compared to immunocompetent areas. Notably, Galectin-7 was identified as a novel metastasis-driving factor. Galectin-7 expression was induced during tumorigenesis particularly in the microenvironment of immunosuppression, and extracellularly released at later stage of tumor progression. Deletion of Galectin-7 in NR-S1M cells significantly suppressed lymph node and lung metastasis without affecting primary tumor growth. Therefore, Galectin-7 is a crucial mediator of tumor metastasis of SCC, which is educated in the immune-suppressed tumor areas, and may be a potential target of cancer immunotherapy.

Impact of Na Concentration on the Phase Transition Behavior and H- Conductivities in the Ba-Li-Na-H-O Oxyhydride System.

K2 NiF4 -type Ba-Li oxyhydride (BLHO) transitions to a so-called hydride superionic conductor, exhibiting a high and essentially temperature-independent hydride ion (H- ) conductivity over 0.01 S cm-1 through the disordering of H- vacancies above 300 °C. In this study, a Ba-Li-Na-H-O oxyhydride system synthesized in which lithium is partially substituted with sodium in BLHO and investigated the effects of Na content on the phase transition behavior and the conductivity. Structural refinements and differential scanning calorimetry experiments confirmed a lowering trend in the phase transition temperatures and decreasing enthalpy changes for the transition with increasing Na content. Substitution of not <40% of Li with Na lowered the degree of ordered vacancies at the H- sites at room temperature and improved conductivities by more than two orders of magnitude in the low-temperature region (T < 300 °C) before the phase transition. These findings clearly show that introducing Na into the lattice effectively stabilizes the high-conductive phase of BLHO.

ACE2-like enzyme B38-CAP suppresses abdominal sepsis and severe acute lung injury.

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.

Intensive single-cell analysis reveals immune-cell diversity among healthy individuals.

Immune responses are different between individuals and personal health histories and unique environmental conditions should collectively determine the present state of immune cells. However, the molecular systems underlying such heterogeneity remain elusive. Here, we conducted a systematic time-lapse single-cell analysis, using 171 single-cell libraries and 30 mass cytometry datasets intensively for seven healthy individuals. We found substantial diversity in immune-cell profiles between different individuals. These patterns showed daily fluctuations even within the same individual. Similar diversities were also observed for the T-cell and B-cell receptor repertoires. Detailed immune-cell profiles at healthy statuses should give essential background information to understand their immune responses, when the individual is exposed to various environmental conditions. To demonstrate this idea, we conducted the similar analysis for the same individuals on the vaccination of influenza and SARS-CoV-2. In fact, we detected distinct responses to vaccines between individuals, although key responses are common. Single-cell immune-cell profile data should make fundamental data resource to understand variable immune responses, which are unique to each individual.

[Role of ACE2 in COVID-19].

In the renin-angiotensin system (RAS), angiotensin II (AngII) converted by angiotensin converting enzyme (ACE) exerts a strong physiological activity via the AT1 receptor (AT1R). Thus, the ACE-AngII-AT1R axis positively regulates RAS. On the other hand, angiotensin converting enzyme 2 (ACE2) is known to negatively regulate RAS by degrading AngII into angiotensin 1-7 (Ang1-7). In the acute respiratory distress syndrome (ARDS), which is characterized by pulmonary hyperinflammation, the AngII-AT1R axis acts to exacerbate ARDS and the ACE2-AT2R axis acts protectively. More recently, ACE2 has been shown to be a receptor for SARS-CoV, the causative virus of severe acute respiratory syndrome (SARS), and SARS-CoV2, the causative virus of the 2019 coronavirus infection (COVID-19). Therefore, inhibition of the binding between ACE2 and virus spike protein is a drug discovery target for antiviral drugs against SARS-CoV and SARS-CoV2. In addition, when SARS and COVID-19 become severe, ARDS with cytokine storm is occured. We reported that soluble ACE2 protein and microbial-derived ACE2 like enzyme suppress pulmonary hyperinflammation due to SARS and COVID-19, respectively. In addition, it has been reported that the ACE2-soluble protein has an effect of suppressing the establishment of infection by inhibiting the binding between SARS-CoV2 and the cell membrane surface ACE2. Here, we describe the role of ACE2 in the pathophysiology of SARS/COVID-19 from the perspectives of inhibiting the progression to ARDS by suppressing pulmonary inflammation and suppressing the replication of the virus by inhibiting the binding of ACE2 to the spike protein.

Hydride-ion-conducting K2NiF4-type Ba-Li oxyhydride solid electrolyte.

Hydrogen transport in solids, applied in electrochemical devices such as fuel cells and electrolysis cells, is key to sustainable energy societies. Although using proton (H+) conductors is an attractive choice, practical conductivity at intermediate temperatures (200-400 °C), which would be ideal for most energy and chemical conversion applications, remains a challenge. Alternatively, hydride ions (H-), that is, monovalent anions with high polarizability, can be considered a promising charge carrier that facilitates fast ionic conduction in solids. Here, we report a K2NiF4-type Ba-Li oxyhydride with an appreciable amount of hydrogen vacancies that presents long-range order at room temperature. Increasing the temperature results in the disappearance of the vacancy ordering, triggering a high and essentially temperature-independent H- conductivity of more than 0.01 S cm-1 above 315 °C. Such a remarkable H- conducting nature at intermediate temperatures is anticipated to be important for energy and chemical conversion devices.

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.

Drug Repurposing Prediction and Validation From Clinical Big Data for the Effective Treatment of Interstitial Lung Disease.

Interstitial lung diseases (ILDs) are a group of respiratory disorders characterized by chronic inflammation and fibrosis of the pulmonary interstitial tissues. Although the etiology of ILD remains unclear, some drug treatments are among the primary causes of ILD. In the present study, we analyzed the FDA Adverse Event Reporting System and JMDC Inc. insurance claims to identify a coexisting drug that reduced the incidence of ILD associated with the use of an anti-arrhythmic agent, amiodarone, and found that the thrombin inhibitor dabigatran prevented the amiodarone-induced ILD in both clinical datasets. In an experimental validation of the hypothesis, long-term oral treatment of mice with amiodarone caused a gradual decrease in body weight caused by respiratory insufficiency. In the lungs of amiodarone-treated mice, infiltration of macrophages was observed in parallel with a delayed upregulation of the platelet-derived growth factor receptor α gene. In contrast, co-treatment with dabigatran significantly attenuated these amiodarone-induced changes indicative of ILD. These results suggest that dabigatran is effective in preventing drug-induced ILD. This combinatorial approach of drug repurposing based on clinical big data will pave the way for finding a new treatment with high clinical predictability and a well-defined molecular mechanism.

Dynamics of the host chromatin three-dimensional response to influenza virus infection.

The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates the pathogenesis of immune and infectious diseases. Influenza virus is a single-stranded RNA virus, and transcription and replication of the virus genome occur in the nucleus. Since viral infection is generally associated with virus-driven hijack of the host cellular machineries, influenza virus may utilize and/or affect the nuclear system. In this review article, we focus on recent studies showing that the three-dimensional structure of chromatin changes with influenza virus infection, which affects the pathology of infection. Also, we discuss studies showing the roles of epigenetics in influenza virus infection. Understanding how this affects immune responses may lead to novel strategies to combat immune and infectious diseases.

Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation.

The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates disease pathogenesis. Here, we show that upon influenza virus infection, the H4K20me3 methyltransferase Suv4-20h2 binds the viral protein NP, which results in the inactivation of Suv4-20h2 and the dissociation of cohesin from Suv4-20h2. Inactivation of Suv4-20h2 by viral infection or genetic deletion allows the formation of an active chromatin loop at the HoxC8-HoxC6 loci coincident with cohesin loading. HoxC8 and HoxC6 proteins in turn enhance viral replication by inhibiting the Wnt-ß-catenin mediated interferon response. Importantly, loss of Suv4-20h2 augments the pathology of influenza infection in vivo. Thus, Suv4-20h2 acts as a safeguard against influenza virus infection by suppressing cohesin-mediated loop formation.

GLP-1 Receptor Signaling Differentially Modifies the Outcomes of Sterile vs Viral Pulmonary Inflammation in Male Mice.

A number of disease states, including type 2 diabetes (T2D), are associated with an increased risk of pulmonary infection. Glucagon-like peptide-1 (GLP-1) receptor agonists are used to treat T2D and exert anti-inflammatory actions through a single, well-defined GLP-1 receptor (GLP-1R). Although highly expressed in the lung, little is known about the role of the GLP-1R in the context of pulmonary inflammation. Here we examined the consequences of gain or loss of GLP-1R activity in infectious and noninfectious lung inflammation. We studied wild-type mice treated with a GLP-1R agonist, and Glp1r-/- mice, in the setting of bleomycin-induced noninfectious lung injury and influenza virus infection. Loss of the GLP-1R attenuated the severity of bleomycin-induced lung injury, whereas activation of GLP-1R signaling increased pulmonary inflammation via the sympathetic nervous system. In contrast, GLP-1R agonism reduced the pathogen load in mice with experimental influenza virus infection in association with increased expression of intracellular interferon-inducible GTPases. Notably, the GLP-1 receptor agonist liraglutide improved the survival rate after influenza virus infection. Our results reveal context-dependent roles for the GLP-1 system in the response to lung injury. Notably, the therapeutic response of GLP-1R agonism in the setting of experimental influenza virus infection may have relevance for ongoing studies of GLP-1R agonism in people with T2D susceptible to viral lung injury.

Eosinophils promote corneal wound healing via the 12/15-lipoxygenase pathway.

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.

m6 A demethylase ALKBH5 promotes proliferation of esophageal squamous cell carcinoma associated with poor prognosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal types of malignant tumors worldwide. Epitranscriptome, such as N6 -methyladenosine (m6 A) of mRNA, is an abundant post-transcriptional mRNA modification and has been recently implicated to play roles in several cancers, whereas the significance of m6 A modifications is virtually unknown in ESCC. Analysis of tissue microarray of the tumors in 177 ESCC patients showed that higher expression of m6 A demethylase ALKBH5 correlated with poor prognosis and that ALKBH5 was an independent prognostic factor of the survival of patients. There was no correlation between the other demethylase FTO and prognosis. siRNA knockdown of ALKBH5 but not FTO significantly suppressed proliferation and migration of human ESCC cells. ALKBH5 knockdown delayed progression of cell cycle and accumulated the cells to G0/G1 phase. Mechanistically, expression of CDKN1A (p21) was significantly up-regulated in ALKBH5-depleted cells, and m6 A modification and stability of CDKN1A mRNA were increased by ALKBH5 knockdown. Furthermore, depletion of ALKBH5 substantially suppressed tumor growth of ESCC cells subcutaneously transplanted in BALB/c nude mice. Collectively, we identify ALKBH5 as the first m6 A demethylase that accelerates cell cycle progression and promotes cell proliferation of ESCC cells, which is associated with poor prognosis of ESCC patients.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

ZBP1 governs the inflammasome-independent IL-1α and neutrophil inflammation that play a dual role in anti-influenza virus immunity.

Influenza A virus (IAV) triggers the infected lung to produce IL-1 and recruit neutrophils. Unlike IL-1ß, however, little is known about IL-1α in terms of its mechanism of induction, action and physiological relevance to the host immunity against IAV infection. In particular, whether Z-DNA-binding protein 1 (ZBP1), a key molecule for IAV-induced cell death, is involved in the IL-1α induction, neutrophil infiltration and the physiological outcome has not been elucidated. Here, we show in a murine model that the IAV-induced IL-1α is mediated solely by ZBP1, in an NLRP3-inflammasome-independent manner, and is required for the optimal IL-1ß production followed by the formation of neutrophil extracellular traps (NETs). During IAV infection, ZBP1 displays a dual role in anti-IAV immune responses mediated by neutrophils, resulting in either protective or pathological outcomes in vivo. Thus, ZBP1-mediated IL-1α production is the key initial step of IAV-infected NETs, regulating the duality of the consequent lung inflammation.

Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin†A1.

Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.

Loss of Apelin Augments Angiotensin II-Induced Cardiac Dysfunction and Pathological Remodeling.

Apelin is an inotropic and cardioprotective peptide that exhibits beneficial effects through activation of the APJ receptor in the pathology of cardiovascular diseases. Apelin induces the expression of angiotensin-converting enzyme 2 (ACE2) in failing hearts, thereby improving heart function in an angiotensin 1⁻7-dependent manner. Whether apelin antagonizes the over-activation of the renin⁻angiotensin system in the heart remains elusive. In this study we show that the detrimental effects of angiotensin II (Ang II) were exacerbated in the hearts of aged apelin-gene-deficient mice. Ang II-mediated cardiac dysfunction and hypertrophy were augmented in apelin knockout mice. The loss of apelin increased the ratio of angiotensin-converting enzyme (ACE) to ACE2 expression in the Ang II-stressed hearts, and Ang II-induced cardiac fibrosis was markedly enhanced in apelin knockout mice. mRNA expression of pro-fibrotic genes, such as transforming growth-factor beta (TGF-ß) signaling, were significantly upregulated in apelin knockout hearts. Consistently, treatment with the ACE-inhibitor Captopril decreased cardiac contractility in apelin knockout mice. In vitro, apelin ameliorated Ang II-induced TGF-ß expression in primary cardiomyocytes, accompanied with reduced hypertrophy. These results provide direct evidence that endogenous apelin plays a crucial role in suppressing Ang II-induced cardiac dysfunction and pathological remodeling.

Apelin and Elabela/Toddler; double ligands for APJ/Apelin receptor in heart development, physiology, and pathology.

Apelin is an endogenous peptide ligand for the G protein-coupled receptor APJ/AGTRL1/APLNR and is widely expressed throughout human body. In adult hearts Apelin-APJ/Apelin receptor axis is potently inotropic, vasodilatory, and pro-angiogenic and thereby contributes to maintaining homeostasis in normal and pathological hearts. Apelin-APJ/Apelin receptor is also involved in heart development including endoderm differentiation, heart morphogenesis, and coronary vascular formation. APJ/Apelin receptor had been originally identified as an orphan receptor for its sequence similarity to Angiotensin II type 1 receptor, and it was later deorphanized by identification of Apelin in 1998. Both Apelin and Angiotensin II are substrates for Angiotensin converting enzyme 2 (ACE2), which degrades the peptides and thus negatively regulates their agonistic activities. Elabela/Toddler, which shares little sequence homology with Apelin, has been recently identified as a second endogenous APJ ligand. Elabela plays crucial roles in heart development and disease conditions presumably at time points or at areas of the heart different from Apelin. Apelin and Elabela seem to constitute a spatiotemporal double ligand system to control APJ/Apelin receptor signaling in the heart. These expanding knowledges of Apelin systems would further encourage therapeutic applications of Apelin, Elabela, or their synthetic derivatives for cardiovascular diseases.