IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis.

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.

Association between defibrillation-to-adrenaline interval and short-term outcomes in patients with out-of-hospital cardiac arrest and an initial shockable rhythm.

Aim: The optimal timing of adrenaline administration after defibrillation in patients with out-of-hospital cardiac arrest (OHCA) and an initial shockable rhythm is unknown. We investigated the association between the defibrillation-to-adrenaline interval and clinical outcomes. Methods: Between 2011 and 2020, we enrolled 1,259,960 patients with OHCA into a nationwide prospective population-based registry in Japan. After applying exclusion criteria, 20,905 patients with an initial shockable rhythm documented at emergency medical services (EMS) arrival who received adrenaline after defibrillation were eligible for this study. Multivariable logistic regression analysis was used to predict favourable short-term outcomes: prehospital return of spontaneous circulation (ROSC), 30-day survival, or a favourable neurological outcome (Cerebral Performance Category 1 or 2) at 30 days. Patients were categorised into 2-minute defibrillation-to-adrenaline intervals up to 18 min, or more than 18 min. Results: At 30 days, 1,618 patients (8%) had a favourable neurological outcome. The defibrillation-to-adrenaline interval in these patients was significantly shorter than in patients with an unfavourable neurological outcome [8 (5-12) vs 11 (7-16) minutes; P < 0.001]. The proportion of patients with prehospital ROSC, 30-day survival, or a favourable neurological outcome at 30 days decreased as the defibrillation-to-adrenaline interval increased (P < 0.001 for trend). Multivariable analysis revealed that a defibrillation-to-adrenaline interval of > 6 min was an independent predictor of worse prehospital ROSC, 30-day survival, or neurological outcome at 30 days when compared with an interval of 4-6 min. Conclusion: A longer defibrillation-to-adrenaline interval was significantly associated with worse short-term outcomes in patients with OHCA and an initial shockable rhythm.

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes.

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Diagnostic Method for COVID-19 Using Sugar Chain-Immobilized Nanoparticles and Saliva Specimens.

Identification of viruses that infects animals or plants, and determination of their quantity are essential for the diagnosis of infectious disease and for the determination of a strategy in the treatment of virus-derived diseases. However, the concentration of viruses existing in a living body (in bodily fluid), food, drinking water, river water, and so on. is not high enough to be detected using conventional diagnostic methods. For example, since the concentration of influenza virus released from an infected person is less than the detection limit of conventional simple examination kits (rapid kit) or even a PCR process at the initial stage of infection, it is difficult to detect the presence of influenza virus which will lead to influenza disease. Our technology allows for safe, efficient, and selective concentration of viruses without troublesome ultracentrifugation, using sugar chain-immobilized metal nanoparticles based on the binding interaction between viruses and sugar chains. For COVID-19, we have developed and commercialized two molecular diagnosis kits: SUDx SARS-CoV-2 detection kit, and SGNP nCoV/Flu PCR detection kit, for the Japanese market in 2020.

Vascular endothelial growth factor receptor-1 regulates postnatal angiogenesis through inhibition of the excessive activation of Akt.

Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs "fine tuning" of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1(+/-) mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1(+/-) Akt1(+/-) mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.

Initial medical management in acute type A aortic dissection patients with a thrombosed false lumen in the ascending aorta combining intramural hematoma and retrograde dissection from the descending to the ascending aorta.

BACKGROUND: The feasibility of medical management for select patients with acute type A aortic dissection has been reported from a few institutions. In this study, we retrospectively investigated the safety and feasibility of our conservative approach for patients with type A aortic dissection in daily practice. METHODS: From January 2013 to December 2017, 131 consecutive patients were admitted to our institution for acute aortic dissection, including 58 patients of type A. Initial medical management was attempted in select patients who were clinically stable and had a thrombosed false lumen of the ascending aorta without ulcer-like projections in the ascending aorta. RESULTS: Except for nine patients contraindicated for surgery, urgent surgery was performed in 26 patients (SRG group), while 23 patients (MED group) were treated with the initial medical management. The maximum diameter of the ascending aorta was significantly larger in the SRG group than in the MED group. In the MED group, the heart rate and blood pressures were well-controlled at admission to the intensive-care unit, and the systolic blood pressure was further reduced at 24 h after. The in-hospital mortality rates of the MED and SRG groups were 0% and 15%, respectively. During the follow-up period, the survival rate was significantly higher in the MED group than in the SRG group, and the aortic event-free survival at one year was 80%. CONCLUSIONS: The initial medical management for select patients with a thrombosed false lumen in the ascending aorta was a safe and feasible strategy in real-world practice.

Cellular senescence impairs circadian expression of clock genes in vitro and in vivo.

Circadian rhythms are regulated by a set of clock genes that form transcriptional feedback loops and generate circadian oscillation with a 24-hour cycle. Aging alters a broad spectrum of physiological, endocrine, and behavioral rhythms. Although recent evidence suggests that cellular aging contributes to various age-associated diseases, its effects on the circadian rhythms have not been examined. We report here that cellular senescence impairs circadian rhythmicity both in vitro and in vivo. Circadian expression of clock genes in serum-stimulated senescent cells was significantly weaker compared with that in young cells. Introduction of telomerase completely prevented this reduction of clock gene expression associated with senescence. Stimulation by serum activated the cAMP response element-binding protein, but the activation of this signaling pathway was significantly weaker in senescent cells. Treatment with activators of this pathway effectively restored the impaired clock gene expression of senescent cells. When young cells were implanted into young mice or old mice, the implanted cells were effectively entrained by the circadian rhythm of the recipients. In contrast, the entrainment of implanted senescent cells was markedly impaired. These results suggest that senescence decreases the ability of cells to transmit circadian signals to their clocks and that regulation of clock gene expression may be a novel strategy for the treatment of age-associated impairment of circadian rhythmicity.

Critical roles of muscle-secreted angiogenic factors in therapeutic neovascularization.

The discovery of bone marrow-derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 "no-option" patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1beta (IL-1beta). We then examined an experimental model of limb ischemia using IL-1beta-deficient mice. Implantation of IL-1beta-deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1beta-deficient mononuclear cells increased expression of IL-1beta and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1beta markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Angiotensin II induces premature senescence of vascular smooth muscle cells and accelerates the development of atherosclerosis via a p21-dependent pathway.

BACKGROUND: Angiotensin II (Ang II) has been reported to contribute to the pathogenesis of various human diseases including atherosclerosis, and inhibition of Ang II activity has been shown to reduce the morbidity and mortality of cardiovascular diseases. We have previously demonstrated that vascular cell senescence contributes to the pathogenesis of atherosclerosis; however, the effects of Ang II on vascular cell senescence have not been examined. METHODS AND RESULTS: Ang II significantly induced premature senescence of human vascular smooth muscle cells (VSMCs) via the p53/p21-dependent pathway in vitro. Inhibition of this pathway effectively suppressed induction of proinflammatory cytokines and premature senescence of VSMCs by Ang II. Ang II also significantly increased the number of senescent VSMCs and induced the expression of proinflammatory molecules and of p21 in a mouse model of atherosclerosis. Loss of p21 markedly ameliorated the induction of proinflammatory molecules by Ang II, thereby preventing the development of atherosclerosis. Replacement of p21-deficient bone marrow cells with wild-type cells had little influence on the protective effect of p21 deficiency against the progression of atherogenesis induced by Ang II. CONCLUSIONS: We demonstrated that Ang II promotes vascular inflammation by inducing premature senescence of VSMCs both in vitro and in vivo. Our results suggest a critical role of p21-dependent premature senescence of VSMCs in the pathogenesis of atherosclerosis.

Highly sensitive detection of influenza virus in saliva by real-time PCR method using sugar chain-immobilized gold nanoparticles; application to clinical studies.

A highly sensitive and convenient method for detecting influenza virus was developed using modified end-point melt curve analysis of a RT-qPCR SYBR Green method and influenza virus-binding sugar chain-immobilized gold-nanoparticles (SGNP). Because SGNPs capture influenza viruses, the virus-SGNP complex was separated easily by centrifugation. Viral RNA was detected at very low concentrations, suggesting that SGNP increased sensitivity compared with standard methods. This method was applied to clinical studies. Influenza viruses were detected in saliva of patients or inpatients who had been considered influenza-free by a rapid diagnostic assay of nasal swabs. Furthermore, the method was applied to a human trial of prophylactic anti-influenza properties of yogurt containing Lactobacillus acidophilus L-92. The incidence of influenza viruses in saliva of the L-92 group was found to be significantly lower compared to the control group. Thus, this method was useful for monitoring the course of anti-influenza treatment or preventive measures against nosocomial infection.

Diagnostic performance of cardiac fusion images from myocardial perfusion imaging and multislice computed tomography coronary angiography for assessment of hemodynamically significant coronary artery lesions: an observational study.

BACKGROUND: In detecting coronary artery disease (CAD), fusion images obtained by combining myocardial perfusion imaging (MPI) and computed tomography coronary angiography (CTCA) have shown a higher accuracy and clinical usefulness than these modalities used separately or a simple comparison of individual images. However, the clinical use of fusion images has been restricted by the necessity of obtaining images with an integral type device or with devices made by the same manufacturer. Thus, we evaluated the detection of hemodynamically significant CAD by fusion images created with a newly developed general-purpose application that can be used with any type of device. METHODS AND RESULTS: In 49 patients, MPI during exercise and at rest and CTCA were obtained separately and combined into fusion images using the new application. As the reference standard, a comparative interpretation of MPI and the conventional coronary arteriography (CAG) was adopted. Hemodynamically significant CAD were diagnosed when MPI showed a reversible perfusion defect in a region with greater than 50% luminal stenosis on CAG. The capability of fusion images to detect CAD was compared with that of CTCA images alone. Fusion images showed a higher ability to detect CAD (sensitivity 80%, specificity 94%, positive predictive value 77%, and negative predictive value 95%) than CTCA alone (77, 77, 46, and 93%, respectively; fusion vs. CTCA: specificity P=0.0002, positive predictive value P=0.0001). CONCLUSION: Fusion images obtained with a general-purpose application were superior to CTCA images alone for detecting hemodynamically significant CAD.

Suspected takotsubo cardiomyopathy caused by withdrawal of bupirenorphine in a child.

A 2-year-old Japanese girl had transient left ventricular apical ballooning on echocardiography and ST-segment elevation and T-wave inversion on electrocardiogram after withdrawal of bupirenorphine and midazolam. The findings improved within 2 weeks. There are many case reports of adults with takotsubo cardiomyopathy but none in children. Takotsubo cardiomyopathy is not well known by pediatric cardiologists, so pediatric cases may have been overlooked. Awareness of a phenomenon similar to takotsubo cardiomyopathy, even in young children, may be important.

Incidences of nasopharyngeal colonization of respiratory bacterial pathogens in Japanese children attending day-care centers.

BACKGROUND: In Japan, many younger children attending day-care centers tend to frequently experience acute respiratory infections and prolonged otitis media. OBJECTIVES: To evaluate the carriage rate of respiratory bacterial pathogens in children attending day-care centers in our district. METHODS: Nasopharyngeal cultures of 156 healthy children between the ages of 1 month and 5 years were conducted at two day-care centers in Japan, in April 1999. The carriage rates of four major pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus) and the antibiotic susceptibilities of the isolates were examined. RESULTS: Streptococcus pneumoniae, H. influenzae, M. catarrhalis and S. aureus were detected in 94 (60.3%), 83 (53.2%), 54 (34.6%) and 28 (17.9%) children, respectively. A total of 141 (90.4%) children carried at least one pathogen among these four pathogens and 87 (55.8%) children carried more than one pathogen. Fifty-seven of the 94 (60.6%) S. pneumoniae isolates were penicillin-intermediately or highly resistant strains of S. pneumoniae (PISP/PRSP). Beta-lactamase producing H. influenzae was not detected. Twelve of the 28 (42.9%) S. aureus isolates were methicillin-resistant. The incidence of colonization by PISP/PRSP in children younger than 3 years (43/69, 62.3%) was significantly higher than that in children aged 3-5 years (14/87, 16.1%) (P < 0.0001). CONCLUSIONS: We conclude that the rates of colonization by respiratory bacterial pathogens, especially by antibiotic-resistant strains, were high in children attending day-care centers in our district, suggesting their horizontal spread among children in day-care centers. Considering that the majority of children attending day- care centers carried one or more of the bacterial pathogens, the judicious use of antimicrobials will be required to prevent the increase of antibiotic-resistant rates among the colonizing pathogens.