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.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function.

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

[Laparoscopic Pyelolithotomy in a Horseshoe Kidney : A Case Report and Review of the Literature].

A 76-year-old man was introduced to our department with a right kidney stone. On the basis of further examination, he was diagnosed with a 23 mm right kidney stone accompanied with a horseshoe kidney. Retrograde pyelography and diuretic renogram revealed a non-obstructed right ureteropelvic junction. Finally, we chose laparoscopic pyelolithotomy via peritoneal approach because the stone was large and accompanied with a horseshoe kidney. The surgery took 165 minutes and the estimated blood loss was 25 ml. There were no minor or major complications. Because horseshoe kidney has anatomical abnormalities, it seems to be necessary to consider a different treatment strategy from that of an upper urinary tract stone in a healthy kidney. We assume that laparoscopic pyelolithotomy is an effective and safe procedure for renal pelvic stones in the case of a horseshoe kidney.

ELABELA-APJ axis protects from pressure overload heart failure and angiotensin II-induced cardiac damage.

AIMS: Elabela/Toddler/Apela (ELA) has been identified as a novel endogenous peptide ligand for APJ/Apelin receptor/Aplnr. ELA plays a crucial role in early cardiac development of zebrafish as well as in maintenance of self-renewal of human embryonic stem cells. Apelin was the first identified APJ ligand, and exerts positive inotropic heart effects and regulates the renin-angiotensin system. The aim of this study was to investigate the biological effects of ELA in the cardiovascular system. METHODS AND RESULTS: Continuous infusion of ELA peptide significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and impaired contractility in mice. ELA treatment reduced mRNA expression levels of genes associated with heart failure and fibrosis. The cardioprotective effects of ELA were diminished in APJ knockout mice, indicating that APJ is the key receptor for ELA in the adult heart. Mechanistically, ELA downregulated angiotensin-converting enzyme (ACE) expression in the stressed hearts, whereas it showed little effects on angiotensin-converting enzyme 2 (ACE2) expression, which are distinct from the effects of Apelin. FoxM1 transcription factor, which induces ACE expression in the stressed hearts, was downregulated by ELA but not by Apelin. ELA antagonized angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. CONCLUSION: The ELA-APJ axis protects from pressure overload-induced heart failure possibly via suppression of ACE expression and pathogenic angiotensin II signalling. The different effects of ELA and Apelin on the expression of ACE and ACE2 implicate fine-tuned mechanisms for a ligand-induced APJ activation and downstream signalling.

Apelin is a positive regulator of ACE2 in failing hearts.

Angiotensin converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system (RAS), catalyzing the conversion of Angiotensin II to Angiotensin 1-7. Apelin is a second catalytic substrate for ACE2 and functions as an inotropic and cardioprotective peptide. While an antagonistic relationship between the RAS and apelin has been proposed, such functional interplay remains elusive. Here we found that ACE2 was downregulated in apelin-deficient mice. Pharmacological or genetic inhibition of angiotensin II type 1 receptor (AT1R) rescued the impaired contractility and hypertrophy of apelin mutant mice, which was accompanied by restored ACE2 levels. Importantly, treatment with angiotensin 1-7 rescued hypertrophy and heart dysfunctions of apelin-knockout mice. Moreover, apelin, via activation of its receptor, APJ, increased ACE2 promoter activity in vitro and upregulated ACE2 expression in failing hearts in vivo. Apelin treatment also increased cardiac contractility and ACE2 levels in AT1R-deficient mice. These data demonstrate that ACE2 couples the RAS to the apelin system, adding a conceptual framework for the apelin-ACE2-angiotensin 1-7 axis as a therapeutic target for cardiovascular diseases.

The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza.

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.