Effects of the matrix-bounded nanovesicles of high-hydrostatic pressure decellularized tissues on neural regeneration.

Decellularized tissues have been used as implantable materials for tissue regeneration because of their high biofunctionality. We have reported that high hydrostatic pressured (HHP) decellularized tissue developed in our laboratory exhibits good in vivo performance, but the details of the mechanism are still not known. Based on previous reports of bioactive factors called matrix bound nanovesicles (MBVs) within decellularized tissues, this study aims to investigate whether MBVs are also present in decellularized tissues prepared by HHP decellularization, which is different from the previously reported methods. In this study, we tried to extract bioactive factors from HHP decellularized brain and placenta, and evaluated their effects on nerves in vitro and in vivo, where its effects have been previously reported. The results confirmed that those factors can be extracted even if the decellularization method and tissue of origin differ, and that they have effects on a series of processes toward nerve regeneration, such as neurite outgrowth and nerve fiber repair.

In this study, we evaluated the neuroregenerative effects of matrix-bounded nanovesicles extracted from decellularized tissue using a high hydrostatic pressure method. The results indicate that bioactive factors, including matrix-bounded nanovesicles, can be extracted regardless of the decellularization method and tissue origin.

Cell Adhesion Behaviors on Spider Silk Fibers, Films, and Nanofibers.

Silk-based materials have garnered attention for use as medical supplies due to their mechanical toughness and low cytotoxicity. Silkworm silk has been applied as surgical sutures for decades. In contrast, the utilization of spider silk is limited mainly because of its scarcity. Although the biomimicry of spider silk has been developed using recombinant protein expression systems with the use of genetic engineering, the product often results in lower molecular weight and a lack of the N- or C-terminal regions. The incomplete sequence of the spider silk-like protein prevents the objective evaluation of the native spider silk as a medical application and retards the development of spider silk-inspired materials. Here, we reeled the native spider silk directly from live spiders and investigated the cell adhesion behavior based on three kinds of surface topography of spider silk-based substrates, namely, fibers, films, and non-woven fabrics. The cell adhesion behavior was largely influenced by the surface micro/nanostructure rather than the wettability of the surface. This study will contribute to promote the utilization of spider silk in the medical field as a candidate for promising bio-based fibers in the context of sustainable development goals.

Extraction and Biological Evaluation of Matrix-Bound Nanovesicles (MBVs) from High-Hydrostatic Pressure-Decellularized Tissues.

Decellularized tissues are widely used as promising materials in tissue engineering and regenerative medicine. Research on the microstructure and components of the extracellular matrix (ECM) was conducted to improve the current understanding of decellularized tissue functionality. The presence of matrix-bound nanovesicles (MBVs) embedded within the ECM was recently reported. Results of a previous experimental investigation revealed that decellularized tissues prepared using high hydrostatic pressure (HHP) exhibited good in vivo performance. In the current study, according to the hypothesis that MBVs are one of the functional components in HHP-decellularized tissue, we investigated the extraction of MBVs and the associated effects on vascular endothelial cells. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100-300 nm) and membranous particles containing small RNA were detected in MBVs derived from HHP-decellularized small intestinal submucosa (SIS), urinary bladder matrix (UBM), and liver. To evaluate the effect on the growth of vascular endothelial cells, which are important in the tissue regeneration process, isolated SIS-derived MBVs were exposed to vascular endothelial cells to induce cell proliferation. These results indicate that MBVs can be extracted from HHP-decellularized tissues and may play a significant role in tissue remodeling.

Electrocardiographic features of arrhythmogenic right ventricular cardiomyopathy in school-aged children.

It can be difficult to distinguish children with early-stage arrhythmogenic right ventricular cardiomyopathy (ARVC) from those with benign premature ventricular contraction (PVC). We retrospectively evaluated six school-aged children with ARVC and compared with those of 20 with benign PVC. The median age at initial presentation was 11.4 and 10.2 years in ARVC and benign PVC, respectively. None of the ARVC patients fulfilled the diagnostic criteria of ARVC at initial presentation. At ARVC diagnosis, the treadmill exercise test and Holter monitoring showed provoked PVC during exercise and pleomorphic PVC in all ARVC cases, respectively. During the observation period, terminal activation duration (TAD) was prolonged in all ARVC patients. In addition, ΔTAD (5.5 [3-10] ms) were significantly longer than those with benign PVC (p < 0.001). A new notched S-wave in V1 appeared in four (67%) ARVC patients, who had myocardial abnormalities in the right ventricle, and in zero benign PVC. Our electrocardiographic findings, such as provoked PVC during exercise, pleomorphic PVC, prolonged TAD, and a new notched S-wave in V1 could contribute to the early detection of ARVC in school-aged children.

Acellular matrix derived from rat liver improves the functionality of rat pancreatic islets before or after vitrification.

Cryopreservation of pancreatic islets can overcome the severe shortage of islet donors in clinical islet transplantation, but the impaired quality of post-warm islets need improvement. This present study was conducted to investigate whether the pre- or post-treatment of rat islets with liver decellularized matrix (LDM) for vitrification can improve the viability (FDA/PI double staining) and the functionality (glucose-stimulated insulin secretion [GSIS] assay). Rat LDM was prepared by high-hydrostatic pressure, lyophilization, and re-suspension in saline. Co-culturing of isolated islets with 0 (control), 30, 60, or 90 µg/ml LDM for 24 h resulted in the comparable viability among the 4 groups (98.7-99.6%) and the higher insulin secretion potential in 30 and 60 µg/ml LDM treatment groups than the control group (stimulation index [SI]: 12.1 and 12.7, respectively, vs. 6.5 in the control group, P < 0.05). When the islets co-cultured with 60 µg/ml LDM were vitrified-warmed on a nylon mesh cryodevice, the viability and the GSIS of the post-warm islets were not improved. Post-treatment of vitrified-warmed islets with 60 µg/ml LDM during the recovery culture for 12 h resulted in the comparable clearance of degenerating cell debris from the post-warm islets, while their insulin secretion potential was improved (SI: 5.0 vs. 3.5 in the control group, P < 0.05). These findings indicate that the components in LDM can enhance the insulin secretion potential of rat islets suffering damage by enzymatic stress during the islet isolation process or by cryoinjuries during the vitrification-warming process.

Abnormal glucose metabolism in patients with Fontan circulation: Unique characteristics and associations with Fontan pathophysiology.

BACKGROUND: Fontan patients exhibit a high prevalence of abnormal glucose metabolism (AGM). We aimed to characterize AGM and clarify its association with Fontan pathophysiology. METHODS: We prospectively evaluated AGM with plasma glucose dynamics [mg/dL; fasting glucose (FPG), and maximum glucose increase (PG-spike)] during oral glucose tolerance test and hemoglobin A1c (HbA1c) in 276 consecutive Fontan patients (aged 19 ±â€¯7 years). Of these, 176 patients had serial AGM assessments with a mean interval of 6.5 years. RESULTS: Initial analysis revealed a high prevalence of impaired glucose tolerance (38.4%) and diabetes mellitus (DM) (4.7%), and positive family history, high HbA1c, and high central venous pressure independently predicted presence of DM. HbA1c was independently determined by hypersplenism and presence of DM (P < .05). Serial assessments revealed an increased PG-spike and a decreased HbA1c (P < .001 for both). Prevalence of DM increased (6.3% to 10.3%), and positive family history, high liver enzymes, and AGM predicted new onset of DM (P < .05 for all). Twenty-one patients died during 7.1-year follow-up. FPG (P < .01) and PG-spike (P < .05) independently predicted all-cause mortality. Particularly, patients with FPG ≤ 74 and/or PG-spike ≥85 had a mortality rate 8.7 times higher than those without (P = .0129). CONCLUSIONS: AGM progressed even in young adult Fontan patients, and HbA1c showed limited predictive value for progression. Oral glucose tolerance test plays important roles in uncovering unique Fontan AGM as well as predicting all-cause mortality.

Evaluating the response to cardiac resynchronization therapy performed with a new ventricular morphology-based strategy for congenital heart disease.

BACKGROUND: In cardiac resynchronization therapy (CRT) for patients with congenital heart disease (CHD) and a ventricular morphology other than a systemic left ventricle (LV), we previously proposed pacing sites that are different from those used for a systemic LV. The leads should be placed laterally on opposite sides of both ventricles in patients with short-axis dyssynchrony and a single ventricular physiology with two ventricles, whereas they should be placed at the farthest sites along the longitudinal direction in the right ventricle (RV) in patients with long-axis dyssynchrony of the RV. Moreover, in patients with interventricular dyssynchrony and a biventricular physiology with a systemic RV, they should be placed at sites that both ventricles can contract simultaneously. We retrospectively investigated 27 consecutive procedures in 24 patients with CHD who underwent CRT to evaluate the effectiveness of a new ventricular morphology-based CRT strategy. The responder rate was 63% (17/27). The reasons for a non-response to CRT in 10 cases were as follows: non-optimal lead positions during CRT, 4; no systemic ventricular conduction delay or heart failure symptoms before the CRT, 5; short follow-up periods after the CRT, 2; and an extremely dilated systemic RV, 1. The responder rate became 88% (14/16), after excluding the procedures without a ventricular conduction delay or heart failure symptoms and those with non-optimal lead positions. This new strategy for CRT can provide favorable results for CHD patients with a systemic ventricular conduction delay and heart failure.

Ruptured mitral valves chordae tendineae around a convalescent infant with acute Kawasaki disease.

Severe valvulitis owing to acute Kawasaki disease leading to severe mitral regurgitation is a rare event in infants. Further, there is less information about underlying ruptured mitral chordae tendineae causing severe mitral regurgitation. We encountered ruptured mitral chordae tendineae in three female patients after Kawasaki disease. The age at the onset of Kawasaki disease ranged from 3 to 8 months, and detection of ruptured mitral chordae tendineae was from 24 to 90 days. Two patients had acute heart failure, and one was asymptomatic. One patient underwent mitral annuloplasty, and the others responded to medication. These ruptured mitral chordae tendineae occurred after the remission of the initial acute Kawasaki disease, in the early course and the convalescent of acute Kawasaki disease. Further, the recurrent fever was also detected in them. The ruptured mitral chordae tendineae in an infant within 6 months can be detected by systolic heart murmur around the convalescent stage of acute Kawasaki disease, although the prevalence is very low.

Extremely short setting of optimal sensed atrioventricular interval in patients after Fontan procedure with implanted dual-chamber pacemaker.

BACKGROUND: Atrioventricular interval optimisation is important in patients with dual-chamber pacing, especially with heart failure. In patients with CHD, especially in those with Fontan circulation, the systemic atrial contraction is supposed to be more important than in patients without structural heart disease. METHODS: We retrospectively evaluated two patients after Fontan procedure with dual-chamber pacemaker with a unique setting of optimal sensed atrioventricular interval. RESULTS: The optimal sensed atrioventricular interval determined by echocardiogram was extremely short sensed atrioventricular interval at 25 and 30 ms in both cases; however, the actual P wave and ventricular pacing interval showed 180 and 140 ms, respectively. In both cases, the atrial epicardial leads were implanted on the opposite site of the origin of their own atrial rhythm. The time differences between sensed atrioventricular interval and actual P wave and ventricular pacing interval occurred because of the site of the epicardial atrial pacing leads and the intra-atrial conduction delay. CONCLUSION: We need to consider the origin of the atrial rhythm, the site of the epicardial atrial lead, and the atrial conduction delay by using electrocardiogram and X-ray when we set the optimal sensed atrioventricular interval in complicated CHD.

Cellular fraction analysis of pericardial effusion helps the diagnosis of eosinophilic myocarditis.

Eosinophilic myocarditis is rare in children, and consequently, it is difficult to diagnose eosinophilic myocarditis rapidly. We report the clinical course of acute eosinophilic myocarditis with pericarditis in two adolescent boys and their associated electrocardiograms. The two patients, 13- and 14-year-old boys, developed cardiomegaly and chest pain with vomiting. On examination by two-dimensional echocardiography, thickening of the ventricular septum and a pericardial effusion were detected. The eosinophil count had increased by the pericardial effusion. Acute eosinophilic myocarditis often complicates a moderate to severe pericardial effusion owing to acute pericarditis. A cellular fraction analysis of the pericardial effusion is easy and useful for the diagnosis of eosinophilic myocarditis. Some serial changes in the electrocardiogram occur during each stage of acute eosinophilic myocarditis. They are induced by eosinophilic granules, which are capable of inducing tissue damage and dysfunction, and those changes in the electrocardiogram resemble the changes after an acute myocardial infarction. It is important to know the characteristics of eosinophilic myocarditis in order to prevent lethal complications.

Clinical Characteristics of Adult Patients With Congenital Heart Disease Hospitalized for Acute Heart Failure.

BACKGROUND: Heart failure (HF) is an important complication in adults with congenital heart disease (CHD), but because only a few studies have focused on acute HF hospitalization in adults with CHD, we study aimed to define the clinical characteristics of such patients and examine the differences in acute HF between adults with CHD and acquired heart disease.Methods and Results:We retrospectively evaluated 50 adults with CHD admitted for treatment of acute HF and compared their data with those from Japanese HF registries. Patient mean age was 37±15 years and 58% were male. In total, 86% of the patients had complex forms of CHD and 62% had undergone corrective surgery, including the Fontan procedure; 66% of patients showed right heart hemodynamic abnormality. In-hospital mortality was 4%, which was comparable to the Japanese HF registries. Survival rate was 93% at 1 year and 75% at 3 years, which was similarly poor to the rates of HF secondary to acquired heart disease. CONCLUSIONS: We clarified the clinical characteristics of adults with CHD requiring HF hospitalization. Young adults with complex CHD were hospitalized for management of acute right HF. Short-term and mid-term outcomes were similarly poor compared with acute HF secondary to acquired heart disease.

Metals Differentially Activate Ovarian Cancer G Protein-Coupled Receptor 1 in Various Species.

Human, mouse, and zebrafish ovarian cancer G protein-coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.

Hemodynamic determinants of mortality after Fontan operation.

BACKGROUND: Elevated central venous pressure (CVP), low cardiac output, and mild hypoxia are common early and late after Fontan operations. However, the association of these characteristics with late mortality is unclear. We aimed to elucidate the hemodynamic determinants of mortality after Fontan operation. METHOD: We evaluated early (group early; 0.5-5years postoperatively, n=387) and late (group late; ≥15years postoperatively, n=161) Fontan hemodynamics that included CVP (mm Hg), cardiac index (CI; L/min per m2), systemic ventricular end-diastolic volume index (mL/m2), ejection fraction (EF; %), and arterial blood oxygen saturation (%). We examined the effect of these variables on 5-year all-cause mortality. RESULTS: Mortality was higher in group late than in group early (17 vs 11, P<.0001). In both groups, higher CVP (hazard ratio [HR]1.46 and 1.38, respectively; P<.001-.0001) and lower arterial blood oxygen saturation (HR 1.12, P<.001 for both) were associated with increased mortality. Greater end-diastolic volume index (HR per 20: 1.73) and lower EF (HR per 10%: 3.38) were associated with increased mortality only in group early (P<.0001 for both). In contrast, only in group late was higher CI associated with increased mortality (HR 2.50, 95% CI 1.30-4.55, P<.01). Seven patients in group late with both high CVP (≥14) and CI (≥3.0) had the highest mortality (HR 18.1, 5.55-52.4, P<.0001). CONCLUSIONS: Elevated CVP and low arterial blood oxygen saturation correlate with mortality in both early and late Fontan survivors. End-diastolic volume index and EF are associated with mortality only in the earlier cohort, whereas interestingly, elevated cardiac output is associated with increased mortality in the later cohort.

Manganese and cobalt activate zebrafish ovarian cancer G-protein-coupled receptor 1 but not GPR4.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.

Mid-Term Follow-up of School-Aged Children With Borderline Long QT Interval.

BACKGROUND: There are no definitive diagnostic criteria or follow-up strategies for long QT syndrome (LQTS) in children with a borderline long QT interval (b-LQT).Methods and Results:We retrospectively evaluated the clinical course, genetic testing results, corrected QT interval (QTc), and LQTS score of 59 school-aged children (5-18 years old) with a b-LQT (400≤QTc<500 ms). Syncope, but neither aborted cardiac arrest nor sudden cardiac death, occurred in 2 patients during the follow-up (6±3.4 years) with LQTS scores ≥4.5 points. The genetic testing results were positive in 92%, 57%, and 67% of patients with high, intermediate, and low probabilities of LQTS, respectively. The maximum and mean QTc during the follow-up significantly differed among the categories with a probability of LQTS, but not the minimum QTc. However, the QTc at rest and at the recovery point after exercise stress testing dramatically changed at the last follow-up. Consequently, the probability of LQTS changed in half of the patients. CONCLUSIONS: The LQTS score is a reasonable indicator for evaluating school-aged children with a b-LQT, and patients with a low LQTS score appear to be at low risk for cardiac events. However, the LQTS score can change during follow-up. Therefore, when there is doubt or concern for patients with a b-LQT, it is preferable to continue following them. Guidelines on follow-up strategies are desired for b-LQT.

Interventricular dyssynchrony in a patient with a biventricular physiology and a systemic right ventricle.

We describe of a unique pattern of cardiac dyssynchrony in a patient with a biventricular physiology and systemic right ventricle (RV): the interventricular dyssynchrony due to a contraction delay between the left ventricle and RV. In the present case, the cineangiography and intracardiac electrocardiography of the RV did not reveal intraventricular dyssynchrony of the RV, but revealed interventricular dyssynchrony. In addition, the pressure curves of the ventricles exhibited time phase differences between the two ventricles. The cardiac resynchronization therapy determining the pacing lead positions based on the idea of interventricular dyssynchrony induced reverse cardiac remodeling in this patient with systemic RV dysfunction.

Prominent QTc prolongation in a patient with a rare variant in the cardiac ryanodine receptor gene.

We report the case of a 12-year-old female patient with a history of four syncopal episodes related to exercise over 2 years and who showed prominent QTc prolongation on electrocardiogram; therefore, she was clinically diagnosed with long QT syndrome type-1. However, genetic analysis did not identify any LQT-related genes but showed a rare missense variant in the cardiac ryanodine receptor gene. From the results of drug-loading tests, administration of oral propranolol was initiated; thereafter, she experienced no syncopal episodes. This is a case report demonstrating the "overlapping clinical features" of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia.

Easy detection of hormone secretion from LßT2 cells by using Gaussia luciferase.

Reproduction is regulated by gonadotropins secreted from gonadotrophs. The production and secretion of gonadotropins are mainly regulated by gonadotropin-releasing hormone (GnRH). Agonists or antagonists that influence GnRH action on gonadotrophs are important to regulate reproduction; however, these factors have not been fully characterized due to the lack of simple and easy-to-use techniques to detect gonadotropin secretion from gonadotropin-producing cells. In the present study, we found that Gaussia luciferase (Gluc), which was expressed in LßT2 cells, can be secreted like a luteinizing-hormone (LH) upon stimulation with GnRH. The Gluc secreted into the medium was easily monitored as luminescence signals. The detection range of the GnRH-induced Gluc activity was comparable to that of the enzyme-linked immunosorbent assay for LH. In addition, when the Gluc was expressed in AtT20 cells, which produce adrenocorticotropic hormone (ACTH), the Gluc activity in the medium increased in parallel with the ACTH secretion upon stimulation with corticotropin-releasing hormone. Thus, the Gluc assay in the present study can be easily used for high-throughput screening of factors that influence LH or ACTH secretion from LßT2 or AtT20 cells, respectively.

Determinants of Aortic Size and Stiffness and the Impact on Exercise Physiology in Patients After the Fontan Operation.

The pathophysiology of congenital heart disease includes aortic dilation and increased stiffness. However, the clinical determinants and significance remain unclear in patients after the Fontan operation.Size and stiffness index (SI) of the ascending and descending aorta (aAO and dAO, respectively) were assessed using angiography in 130 consecutive Fontan patients and 30 age-matched controls. Compared with controls, Fontan patients showed a dilated aAO and smaller dAO (P < 0.0001) with greater SI (3.2 ± 0.7 versus 2.2 ± 0.3 for aAO and 2.7 ± 0.6 versus 2.2 ± 0.3 for dAO, P < 0.0001 for both). aAO was stiffer than dAO (P < 0.0001) and the greater aAO size was independently determined by the presence of pulmonary atresia, older age at Fontan operation, and low arterial oxygen saturation (P < 0.05-0.01). High plasma levels of brain natriuretic peptide (BNP) and glucose were independently associated with aAO SI (P < 0.05-0.01) and the SI ratio of aAO to dAO SI, whereas body mass index, plasma levels of highsensitivity C-reactive protein, and dAO size were independently associated with dAO SI (P < 0.05-0.01). A greater aAO and aAO/dAO ratio predicted an impaired exercise blood pressure response (P < 0.0001). Furthermore, in addition to age at Fontan operation and BNP level, the aAO SI independently predicted a lower peak oxygen uptake (P < 0.05).Fontan patients have a stiffer dilated aAO with rapidly tapering smaller dAO that predicts exercise pathophysiology. In addition to intrinsic aortic structural abnormalities, heart failure severities as well as traditional cardiovascular risk factors are also involved in the aortic structural and functional abnormalities.

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.