Prevalence and Risk Factors of Silent Cerebral Microbleeds in Patients with Coronary Artery Disease.

OBJECTIVES: Cerebral microbleeds (CMBs), which can be detected by gradient-echo T2*-weighted magnetic resonance imaging (MRI), represent small chronic brain hemorrhages caused by structural abnormalities in cerebral small vessels. CMBs are known to be a potential predictor of future stroke, and are associated with age, various cardiovascular risk factors, cognitive impairment, and the use of antithrombotic drugs. Patients with coronary artery disease (CAD) are at potentially high risk of CMBs due to the presence of coexistent conditions. However, little is known about CMBs in patients with CAD. We aimed to identify the factors associated with the presence of CMBs among patients with CAD. METHODS: We evaluated 356 consecutive patients [mean age, 72 ± 10 years; men = 276 (78%)] with angiographically proven CAD who underwent T2*-weighted brain MRI. The brain MRI was assessed by researchers blinded to the patients' clinical details. RESULTS: CMBs were found in 128 (36%) patients. Among 356 patients, 119 (33%) had previously undergone percutaneous coronary intervention (PCI), and 26 (7%) coronary artery bypass grafting (CABG). There was no significant relationship between CMBs and sex, hypertension, dyslipidemia, diabetes mellitus, anticoagulation therapy, antiplatelet therapy, or prior PCI. CMBs were significantly associated with advanced age, previous CABG, eGFR, non-HDL cholesterol, carotid artery disease, long-term antiplatelet therapy, and long-term dual antiplatelet therapy (DAPT) using univariate logistic regression analysis. The multivariate logistic regression analysis showed that long-term antiplatelet therapy (odds ratio, 1.73; 95% CI, 1.06 - 2.84; P = 0.03) or long-term DAPT (odds ratio, 2.92; 95% CI, 1.39 - 6.17; P = 0.004) was significantly associated with CMBs after adjustment for confounding variables. CONCLUSIONS: CMBs were frequently observed in patients with CAD and were significantly associated with long-term antiplatelet therapy, especially long-term DAPT.

Heterozygous disruption of ALAS1 in mice causes an accelerated age-dependent reduction in free heme, but not total heme, in skeletal muscle and liver.

5-Aminolevulinic acid (ALA) is the rate-limiting intermediate in heme biosynthesis in vertebrate species; a reaction catalyzed by the mitochondrial ALA synthase 1 (ALAS1) enzyme. Previously we reported that knockdown of the ubiquitously expressed ALAS1 gene in mice disrupts normal glucose metabolism, attenuates mitochondrial function and results in a prediabetic like phenotype when animals pass 20-weeks of age (Saitoh et al., 2018). Contrary to our expectations, the cytosolic and mitochondrial heme content of ALAS1 heterozygous (A1+/-) mice were similar to WT animals. Therefore, we speculated that regulatory "free heme" may be reduced in an age dependent manner in A1+/- mice, but not total heme. Here, we examine free and total heme from the skeletal muscle and liver of WT and A1+/- mice using a modified acetone extraction method and examine the effects of aging on free heme by comparing the amounts at 8-12 weeks and 30-36 weeks of age, in addition to the mRNA abundance of ALAS1. We found an age-dependent reduction in free heme in the skeletal muscle and liver of A1+/- mice, while WT mice showed only a slight decrease in the liver. Total heme levels showed no significant difference between young and aged WT and A1+/- mice. ALAS1 mRNA levels showed an age-dependent reduction similar to that of free heme levels, indicating that ALAS1 mRNA expression levels are a major determinant for free heme levels. The free heme pools in skeletal muscle tissue were almost 2-fold larger than that of liver tissue, suggesting that the heme pool varies across different tissue types. The expression of heme oxygenase 1 (HO-1) mRNA, which is expressed proportionally to the amount of free heme, were similar to those of free heme levels. Taken together, this study demonstrates that the free heme pool differs across tissues, and that an age-dependent reduction in free heme levels is accelerated in mice heterozygous for ALAS1, which could account for the prediabetic phenotype and mitochondrial abnormality observed in these animals.

The clinical characteristics and outcomes of heart failure patient with chronic obstructive pulmonary disease from the Japanese community-based registry.

Both heart failure (HF) and chronic obstructive pulmonary disease (COPD) are common diseases, but few studies have assessed the relationship between COPD and outcomes in patients with acute HF, especially in relation to age or ejection fraction (EF). The Kitakawachi Clinical Background and Outcome of Heart Failure Registry was a prospective, multicenter, community-based cohort and enrolled a total of 1,102 patients with acute HF between 2015 and 2017 in this study. The primary endpoint was defined as a composite endpoint that included all-cause mortality and hospitalization for HF. We stratified patients into two groups: those aged ≥ 80 years (elderly) and < 80 years (nonelderly). HF with preserved EF (HFpEF) was defined as EF ≥ 50%, whereas HF with reduced ejection fraction (HFrEF) was defined as EF < 50%. A total of 159 patients (14.4%) with COPD and 943 patients (83.6%) without COPD were included. COPD was found to be independently associated with a higher risk of the composite endpoint (adjusted hazard ratio: 1.42, 95% confidence interval: 1.14-1.77; p = 0.003). During a subgroup analysis, COPD was exposed as an independent risk factor of the composite endpoint in nonelderly patients; however, there was not such a finding observed among elderly patients. Separately, there was a significant association with COPD and the composite endpoint in patients with HFpEF. COPD showed a significantly higher risk of the composite endpoint after discharge in acute HF. However, this heightened risk was observable only in the subgroup of nonelderly patients and those of HFpEF.

Crosstalk between Metabolic Disorders and Immune Cells.

Metabolic syndrome results from multiple risk factors that arise from insulin resistance induced by abnormal fat deposition. Chronic inflammation owing to obesity primarily results from the recruitment of pro-inflammatory M1 macrophages into the adipose tissue stroma, as the adipocytes within become hypertrophied. During obesity-induced inflammation in adipose tissue, pro-inflammatory cytokines are produced by macrophages and recruit further pro-inflammatory immune cells into the adipose tissue to boost the immune response. Here, we provide an overview of the biology of macrophages in adipose tissue and the relationship between other immune cells, such as CD4+ T cells, natural killer cells, and innate lymphoid cells, and obesity and type 2 diabetes. Finally, we discuss the link between the human pathology and immune response and metabolism and further highlight potential therapeutic targets for the treatment of metabolic disorders.

Clinical Characteristics and Outcomes of Heart Failure Patients With Long-Term Care Insurance　- Insights From the Kitakawachi Clinical Background and Outcome of Heart Failure Registry.

BACKGROUND: In Japan, the long-term care insurance (LTCI) system has an important role in helping elderly people, but there have been no clinical studies that have examined the relationship between the LTCI and prognosis for patients with acute heart failure (HF).Methods and Results:This registry was a prospective multicenter cohort, 1,253 patients were enrolled and 965 patients with acute HF aged ≥65 years were comprised the study group. The composite endpoint included all-cause death and hospitalization for HF after discharge. We divided the patients into 4 groups: (i) patients without LTCI, (ii) patients requiring support level 1 or 2, (iii) patients with care level 1 or 2, and (iv) patients with care levels 3-5. The Kaplan-Meier analysis identified a lower rate of the composite endpoint in group (i) than in the other groups. After adjusting for potentially confounding effects using a Cox proportional regression model, the hazard ratio (HR) of the composite endpoint increased significantly in groups (iii) and (iv) (adjusted HR, 1.62; 95% confidence interval [CI], 1.22-1.98 and adjusted HR, 1.62; 95% CI, 1.23-2.14, respectively) when compared with group (i). However, there was no significant difference between groups (i) and (ii). CONCLUSIONS: The level of LTCI was associated with a higher risk of the composite endpoint after discharge in acute HF patients.

Living Alone and Gender Differences in Rehospitalization for Heart Failure After Discharge Among Acute Heart Failure Patients.

Home treatment for heart failure (HF) is one of the most important problems in patients after discharge as a secondary preventive measure for rehospitalization for HF. However, there are no detailed studies on gender differences in sociopsychological factors such as living alone for HF rehospitalization among patients with acute HF (AHF).This prospective multicenter cohort study enrolled patients with AHF between April 2015 and August 2017. Patients of each gender with first AHF were divided into those living alone and those not living alone. The primary endpoint was defined as rehospitalization for HF after discharge. Cox proportional hazard analysis was performed to determine the association between living alone and the endpoint.Overall, 581 patients were included in this study during the 3-year follow-up. The proportion of rehospitalization for HF was significantly higher in patients living alone than in those not living alone among male patients. However, female patients showed no difference in endpoints between the two groups. The difference was independently maintained even after adjusting for differences in social backgrounds in male patients (adjusted hazard ratio (HR) 2.02; 95% confidence interval (CI), 1.07-3.70). In female patients, the HR for rehospitalization for HF showed no difference between the two groups (adjusted HR, 0.99; 95% CI, 0.56-1.69).In this study population, male patients living alone after first AHF discharge had a higher risk of rehospitalization for HF than those not living alone, but these differences were not observed in female patients.

Interleukin-12p40 variant form reduces Interleukin-12p80 secretion.

IL-12 is a key cytokine for the promotion of CD4+ T cells differentiation to type 1 helper T cells. IL-12 is a heterodimer (IL-12p70) consisting of p40 and p35 subunits, and is mainly secreted from activated antigen-presenting cells, such as macrophages and dendritic cells (DCs). In this study, we found that activated mouse bone marrow-derived DCs (BMDCs) produced a p40 splice variant form mRNA in addition to the conventional p40 mRNA. This p40 variant mRNA was produced by alternative splicing in exon 5, and possessed a premature stop codon. As a result, the p40 variant protein contained 157 amino acids of the N-terminal part of p40 and an additional 10 novel amino acids. When the p40 variant was expressed in HEK-293T cells, it was not secreted from the cells. To investigate the function of the p40 variant, it was co-expressed with p40 and/or p35. The p40 variant did not affect the secretion of IL-12p40 or IL-12p70, or the function of the secreted p70. In contrast, the secretion of IL-12p80, a homodimeric IL-12 with two p40 subunits, was significantly decreased when the p40 variant was expressed. This new splicing variant p40 may act to fine-tune the function of IL-12p80.

Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.

Poor embryo development in post-ovulatory in vivo-aged mouse oocytes is associated with mitochondrial dysfunction, but mitochondrial transfer from somatic cells is not sufficient for rejuvenation.

STUDY QUESTION: Does in vivo aging of mouse oocytes affect mitochondrial function? SUMMARY ANSWER: Mitochondrial function was impaired in post-ovulatory in vivo-aged mouse oocytes and microinjection of somatic cell mitochondria did not rescue poor fertilization and embryonic development rates. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in oocyte quality associated with oocyte aging remain unknown, although studies have suggested that the decline is regulated by mitochondrial dysfunction. However, only a limited number of studies have provided direct evidence implicating mitochondrial dysfunction in oocyte quality during the aging of oocytes. STUDY DESIGN, SIZE, DURATION: We used post-ovulatory, in vivo-aged mouse oocytes as a model for studying low-quality oocytes in oocyte aging. PARTICIPANTS/MATERIALS, SETTING, METHOD: Superovulated oocytes released from the oviduct at 14 h and 20-24 h post-hCG injection were designated as 'fresh' and 'aged' oocytes, respectively. Membrane potentials and oxygen consumption in single oocytes were evaluated as measures of mitochondrial function in fresh and aged oocytes. Mitochondrial transcriptional factor A (TFAM) expression levels were examined by western blotting, and colocalization of mitochondria and TFAM was analyzed by measuring immunofluorescence in fresh and aged oocytes. IVF and blastocyst formation rates were calculated after oocyte microinjection with mitochondria derived from liver cells. MAIN RESULTS AND THE ROLE OF CHANCE: The average mitochondrial membrane potential in fresh oocytes was significantly higher than that in aged oocytes (P < 0.05). The average oxygen consumption rate in aged oocytes was significantly lower than that in fresh oocytes (P < 0.05). Although total TFAM expression was unchanged, its colocalization with mitochondria decreased in aged oocytes. IVF and blastocyst formation rates for mitochondrion-injected aged oocytes were not significantly different from those for buffer-injected aged oocytes. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: A limitation of this study is that we did not examine the effects of microinjecting mitochondria from other somatic cell types into aged oocytes on their fertilization and embryonic development rates. WIDER IMPLICATIONS OF THE FINDINGS: The results from the present study showed that poor embryonic development was associated with impairment of mitochondrial functions in in vivo-aged oocytes. However, the microinjection of mitochondria from liver cells did not improve the low fertilization and embryonic development rates of aged oocytes. It remains to be demonstrated whether oocyte quality can be rescued by the transfer of cytosolic factors or cellular organelles, such as the endoplasmic reticulum or mitochondria, from specific cell types. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Grants-in-Aid for General Science Research to Toshifumi Takahashi (No. 25462550) and Hideki Igarashi (No. 26462474). The funding source played no role in study design in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The authors have no conflict of interest to disclose.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

SOD1 deficiency induces the systemic hyperoxidation of peroxiredoxin in the mouse.

A deficiency of superoxide dismutase 1 (SOD1) or peroxiredoxin (Prx) 2 causes anemia in mice due to elevated oxidative stress. In the current study, we investigated whether intrinsic oxidative stress caused by a SOD1 deficiency affected the redox status of Prx2 and other isoforms in red blood cells (RBCs) and several organs of mice. We observed a marked elevation in hyperoxidized Prx2 levels in RBCs from SOD1-deficient mice. Hyperoxidized Prx2 reportedly undergoes a rhythmic change in isolated RBCs under culture conditions. We confirmed such changes in RBCs from wild-type mice but observed no evident changes in SOD1-deficient RBCs. In addition, an elevation in hyperoxidized Prxs, notably Prx2 and Prx3, was observed in several organs from SOD1-deficient mice. However, a SOD1 deficiency had no impact on the wheel-running activity of the mice. Thus, although the redox status of some Prxs is systemically shifted to a more oxidized state as the result of a SOD1 deficiency, which is associated with anemia and some diseases, a redox imbalance appears to have no detectable effect on the circadian activity of mice.

Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.

Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.

Heme regulates B-cell differentiation, antibody class switch, and heme oxygenase-1 expression in B cells as a ligand of Bach2.

Heme binds to proteins to modulate their function, thereby functioning as a signaling molecule in a variety of biologic events. We found that heme bound to Bach2, a transcription factor essential for humoral immunity, including antibody class switch. Heme inhibited the DNA binding activity of Bach2 in vitro and reduced its half-life in B cells. When added to B-cell primary cultures, heme enhanced the transcription of Blimp-1, the master regulator of plasma cells, and skewed plasma cell differentiation toward the IgM isotype, decreasing the IgG levels in vitro. Intraperitoneal injection of heme in mice inhibited the production of antigen-specific IgM when heme was administered simultaneously with the antigen but not when it was administered after antigen exposure, suggesting that heme also modulates the early phase of B-cell responses to antigen. Heme oxygenase-1, which is known to be regulated by heme, was repressed by both Bach2 and Bach1 in B cells. Furthermore, the expression of genes for heme uptake changed in response to B-cell activation and heme administration. Our results reveal a new function for heme as a ligand of Bach2 and as a modulatory signal involved in plasma cell differentiation.

Method of detecting genetically modified chicken containing human erythropoietin gene.

Genetically modified (GM) chickens carrying the human erythropoietin (hEpo) gene have been developed to produce recombinant hEpo protein in eggs. However, such animals have not been approved as food sources in Japan. We developed a method for detecting the hEpo gene in chicken meat using a real-time polymerase chain reaction (real-time PCR). The hEpo gene was clearly detected in genomic DNA extracted from magnum and heart of a chimeric chicken containing the hEpo gene. A plasmid containing the hEpo gene was used as a standard reference molecule as well. The results clearly showed that our method was capable of detecting the hEpo gene contained in the plasmid in the presence of genomic DNA extracted from a raw chicken meat sample. We successfully used this method to test six samples of raw chicken meat and six samples of chicken meat in processed foods. This method will be useful for monitoring chicken meat that might have originated from GM chickens carrying the hEpo gene to assure food safety.

[Study on recent status of development of genetically modified animals developed not for food purposes].

Genetically modified (GM) animals can be classified into two groups, those developed for food purposes and those developed not for food purposes. We investigated the recent status of development of GM animals developed not for food purposes. Among the GM animals developed not for food purposes, GM fish, chickens, and pigs were selected because many articles have been published on these organisms. Relevant articles published between 2008 and 2011 were surveyed using PubMed and transgenic fish, chicken, or pig as keywords. Then, studies on organisms that could potentially contaminate the food chain with products from these GM animals were selected and analyzed. Fifteen articles on GM fish were found. These articles were classified into four categories: bioreactor (n = 4), resistance to microorganisms (n = 6), resistance to environmental stresses (n = 1), and detection of chemicals (n = 4). Zebrafish were used in 8 of the articles. Six, three, and three articles were reported from Taiwan, Canada and China. Seven articles on GM chickens were found. These articles were classified into two categories: bioreactor (n = 5), and resistance to pathogens (n = 2). Two articles were reported from Japan and Korea, each. As for GM pigs, 43 articles were found. These articles were classified into three categories: xenotransplantation (n = 36), bioreactor (n = 6), and environmental cleanup (n = 1). Nineteen, seven, six, and five articles were reported from USA, Germany, Korea and Taiwan, respectively. Understanding the recent development of GM animals produced not for food purpose is important for assuring the safety of food.

[General anesthesia in a orthopedic case with the Kommerell's diverticulum of right-sided aortic arch diagnosed on preoperative examination].

Kommerell's diverticulum is a rare anomaly of the aortic arch. A 59-year-old man was scheduled for open reduction and internal fixation of his right proximal tibial fracture under general anesthesia. We diagnosed right-sided aortic arch by the chest X-ray and thoracic computed tomography. His trachea and esophagus were compressed by the aortic arch. He had complained of no dyspnea or dysphagia. Respiratory difficulty might be caused by muscle relaxants, intermittent positive pressure ventilation, change of intrathoracic pressure, postural change and overloaded infusion during general anesthesia in a case of right-sided aortic arch. We performed lumbar epidural anesthesia and inserted an i-gel after general anesthesia induction preserving spontaneous respiration in preparation for controlled ventilation or tracheal intubation via an i-gel. We could accomplish the operation uneventfully and he was discharged on POD 53. A supraglottic airway such as an i-gel was a useful device in the present case of right-sided aortic arch with Kommerell's diverticulum.

Impact of a SLC24A5 variant on the retinal pigment epithelium of a Japanese patient with oculocutaneous albinism type 6.

Oculocutaneous albinism (OCA) 6 is a non-syndromic type of OCA that has distinct ocular symptoms and variable cutaneous hypopigmentation. The causative gene of OCA6 is SLC24A5, which encodes NCKX5, a K+ -dependent Na+ /Ca2+ exchanger 5. NCKX5 is involved in the maturation of melanosomes, but its function is still unclear. In this study, we characterized a Japanese patient with OCA6. Genetic analysis revealed compound heterozygous variants in SLC24A5, c.590 + 1dupG, and c.598G>A (p.G200R). To clarify the functional significance of the missense variant, we generated a knock-in (KI) mouse model carrying the mouse homolog of the G200R variant using the CRISPR/Cas9 system. Chemical analysis showed decreased amounts of eumelanin in the hair and skin of KI mice, while levels of benzothiazine units in pheomelanin were significantly increased in their hair. Retinal pigment was also decreased in KI mice. Notably, a histopathologic study revealed a significant pigment loss in the retinal pigment epithelium (RPE) but not in the choroid. Immunohistochemically, the expression of NCKX5 in the RPE was decreased but was maintained in the choroid of KI mice. These findings could explain the difference in phenotypic severity between eye symptoms and hypopigmentation in the skin/hair.

Altered composition of fatty acids exacerbates hepatotumorigenesis during activation of the phosphatidylinositol 3-kinase pathway.

BACKGROUND & AIMS: Some clinical findings have suggested that systemic metabolic disorders accelerate in vivo tumor progression. Deregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is implicated in both metabolic dysfunction and carcinogenesis in humans; however, it remains unknown whether the altered metabolic status caused by abnormal activation of the pathway is linked to the protumorigenic effect. METHODS: We established hepatocyte-specific Pik3ca transgenic (Tg) mice harboring N1068fs*4 mutation. RESULTS: The Tg mice exhibited hepatic steatosis and tumor development. PPARγ-dependent lipogenesis was accelerated in the Tg liver, and the abnormal profile of accumulated fatty acid (FA) composition was observed in the tumors of Tg livers. In addition, the Akt/mTOR pathway was highly activated in the tumors, and in turn, the expression of tumor suppressor genes including Pten, Xpo4, and Dlc1 decreased. Interestingly, we found that the suppression of those genes and the enhanced in vitro colony formation were induced in the immortalized hepatocytes by the treatment with oleic acid (OA), which is one of the FAs that accumulated in tumors. CONCLUSIONS: Our data suggest that the unusual FA accumulation has a possible role in promoting in vivo hepato-tumorigenesis under constitutive activation of the PI3K pathway. The Pik3ca Tg mice might help to elucidate molecular mechanisms by which metabolic dysfunction contributes to in vivo tumor progression.

Systemic heme oxygenase-1 transgenic overexpression aggravates pressure overload-induced cardiac hypertrophy in mice.

BACKGROUND/AIMS: Heme oxygenase-1(HO-1) has been reported to protect against cardiac hypertrophy in cultured neonatal cardiomyocytes treated with HO-1 inducer, cardiac specific HO-1 transgenic mice, or animals treated with HO-1 inducer. The aim of the present study is to examine the effects of systemic HO-1 transgenic overexpression on pressure overload-induced cardiac hypertrophy in mice. METHODS: Pressure-overload cardiac hypertrophy was induced by transverse aortic constriction (TAC) in WT (wild type) and systemic HO-1 transgenic overexpression (TG) mice. RESULTS: We found that systemic HO-1 transgenic overexpression aggravated pressure overload-induced cardiac hypertrophy. Pressure-overload induced the more increases of heart weight/ body weigh index, left ventricular weight/ body weight index, ß-MHC protein expression, cardiac interstitial fibrosis in TG mice than in WT mice. Pressure-overload increased cardiac HO-1 protein expression in WT but not TG mice, but the cardiac HO-1 protein level was still higher in TAC-treated TG mice than in TAC-treated WT mice. The basal cardiac calcineurin protein level in TG mice was lower than that in WT mice. Pressure-overload increased calcineurin protein expression in both WT and TG mice; however, pressure-overload induced more calcineurin protein expression in TG mice than in WT mice. CONCLUSION: This study shows for the first time that systemic HO-1 transgenic overexpression aggravates pressure overload-induced cardiac hypertrophy.

Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5-aminolevulinate synthase gene in mouse.

The first step of heme biosynthesis in animals is catalyzed by 5-aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS-N) plays in vivo, we prepared a green fluorescent protein (GFP) knock-in mouse line in which the Alas1 gene (encoding ALAS-N) is replaced with a gfp gene. We found that mice bearing a homozygous knock-in allele (Alas1(GFP/GFP)) were lethal by embryonic day 8.5, demonstrating that ALAS-N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse (Alas1(+/GFP)) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS-N mRNA in various tissues of Alas1(+/GFP) mice suggested that the destabilization of ALAS-N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS-N expression level and that this control is essential for heme homeostasis in animals.