S100a6 knockdown promotes the differentiation of dental epithelial cells toward the epidermal lineage instead of the odontogenic lineage.

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development.

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Expression patterns of keratin family members during tooth development and the role of keratin 17 in cytodifferentiation of stratum intermedium and stellate reticulum.

Keratins are typical intermediate filament proteins of the epithelium that exhibit highly specific expression patterns related to the epithelial type and stage of cellular differentiation. They are important for cytoplasmic stability and epithelial integrity and are involved in various intracellular signaling pathways. Several keratins are associated with enamel formation. However, information on their expression patterns during tooth development remains lacking. In this study, we analyzed the spatiotemporal expression of keratin family members during tooth development using single-cell RNA-sequencing (scRNA-seq) and microarray analysis. scRNA-seq datasets from postnatal Day 1 mouse molars revealed that several keratins are highly expressed in the dental epithelium, indicating the involvement of keratin family members in cellular functions. Among various keratins, keratin 5 (Krt5), keratin 14 (Krt14), and keratin 17 (Krt17) are highly expressed in the tooth germ; KRT17 is specifically expressed in the stratum intermedium (SI) and stellate reticulum (SR). Depletion of Krt17 did not affect cell proliferation in the dental epithelial cell line SF2 but suppressed their differentiation ability. These results suggest that Krt17 is essential for SI cell differentiation. Furthermore, scRNA-seq results indicated that Krt5, Krt14, and Krt17 exhibited distinct expression patterns in ameloblast, SI, and SR cells. Our findings contribute to the elucidation of novel mechanisms underlying tooth development.

Deficiency of G protein-coupled receptor Gpr111/Adgrf2 causes enamel hypomineralization in mice by alteration of the expression of kallikrein-related peptidase 4 (Klk4) during pH cycling process.

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

Single-Crystal Structures of Benzenehexathiol and Its Disulfide Forms.

Oligothiols are useful as building blocks in the construction of disulfide-based macrocycles and polymers or as ligands for coordination polymers. Above all, benzenehexathiol (BHT) is a particularly important molecule, as it is used to construct conductive two-dimensional MOFs. Despite the desire to clarify its structure and isolate it to high purity, the chemical instability of BHT has hampered single-crystal X-ray structure analysis of intact BHT. In addition, the synthesis of discrete disulfide molecules of BHT has not been reported. Here, we succeed in obtaining the single crystals of intact BHT, which is analyzed by single crystal X-ray structure analysis. Furthermore, the structures of a group of molecules with intermolecular disulfide bonds (BHT·4im and BHT2·2TBA, im = imidazole, TBA = tetrabutylammonium cation) obtained by processing BHT in the presence of bases are determined.

Enamel defects of Axenfeld-Rieger syndrome and the role of PITX2 in its pathogenesis.

OBJECTIVES: To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld-Rieger syndrome (ARS) with a heterozygous microdeletion involving paired-like homeodomain 2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects. SUBJECTS AND METHODS: Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis (CMA) were used to screen the disease-causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro-computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/ß-catenin pathway in proliferation of ameloblast precursor cells was investigated. RESULTS: An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stifmulation partially reversed the proliferation ability after Pitx2 knockdown. CONCLUSIONS: Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/ß-catenin signaling pathway.

Japanese Laws and the Current Status of Regenerative Medicine in the Tohoku Region.

AIM: The aim of this study was to review Japanese laws regarding regenerative medicine and the current status of clinical application of regenerative medicine, to learn about the advantages and problems, and to thereby serve as a reference for measures necessary for the development of regenerative medicine. BACKGROUND: Regenerative medicine started in 1957 with the transplantation of hematopoietic stem cells, followed by the establishment of embryonic stem cells in 1981 and induced pluripotent stem cells in 2006, and continues to evolve progressively. At the same time, however, problems have emerged due to lax legal regulations, such as the use of treatments that lack scientific evidence. REVIEW RESULTS: The Japanese government enacted two laws to regulate regenerative medicine: the Law to Ensure the Safety of Regenerative Medicine and the Amend the Pharmaceutical Affairs Law in 2013. These laws were enacted with the aim of providing safe regenerative medicine promptly and smoothly and developing many regenerative medicine products. In these laws, regenerative medicine is defined as medical treatment that restores lost functions of damaged organs and tissues with the help of cellular and tissue-based products. Nowadays, there are two major methods of regenerative medicine. One representative method involves the transplantation of devices that activates self-regenerative ability by introducing living cells into patients' body. The other method is the activation and differentiation of endogenous stem cells with cell growth and differentiation factors. CONCLUSION: The current status of regenerative medicine in the Tohoku region after the enactment of these laws is described in detail. This clarified the advantages and disadvantages associated with regenerative medicine as it is currently practiced in Japan. CLINICAL SIGNIFICANCE: Development of regenerative medicine in dentistry will be advanced by learning about its clinical application in medicine.

The tooth-specific basic helix-loop-helix factor AmeloD promotes differentiation of ameloblasts.

Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis.

Esophageal thermal lesions in radiofrequency ablation for atrial fibrillation: A prospective comparative study of thermal sensors.

BACKGROUND: Esophageal thermal lesion (ETL) is a complication of radiofrequency ablation for atrial fibrillation (RFAF). To prospectively compare the incidence of ETL, we used two linear, five- and three-sensor esophageal thermal monitoring catheters (ETMC5 and ETMC3). We also evaluated the predictors of ETL. METHODS: Patients receiving their first RFAF (n = 106) were randomized into two groups, ETMC5 (n = 52) and ETMC3 (n = 54). Ablation was followed by esophagogastroduodenoscopy within 3 days. RESULTS: Esophageal thermal lesion was detected in 7/106 (6.6%) patients (ETMC5: 3/52 [5.8%] vs. ETMC3: 4/54 [7.4%]; p = 1.0). The maximum temperature and number of measurements > 39.0°C did not differ between the groups (ETMC5: 40.5°C and 5.4 vs. ETMC3: 40.6°C and 4.9; p = .83 and p = .58, respectively). In ETMC5 group, the catheter had to be moved significantly less often (0.12 vs. 0.42; p = .0014) and fluoroscopy time was significantly shorter (79.2 min vs. 101.7 min; p = .0038) compared with ECMC3 group. The total number of ablations in ETMC5 group was significantly greater (50.2 vs. 37.7; p = .030) and ablation time was significantly longer (52.1 min vs. 40.1 min; p = .0039). Only body mass index (BMI) was significantly different between patients with and without ETL (21.4 ± 2.5 vs. 24.3 ± 3.4; p = .022). CONCLUSIONS: The incidence of ETL was comparable between ETMC5 and ETMC3 groups; however, fluoroscopy time, total ablation time, and total number of ablations differed significantly. Lower BMI may increase the risk of developing ETL.

G protein-coupled receptor Gpr115 (Adgrf4) is required for enamel mineralization mediated by ameloblasts.

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

Transcriptional regulation of the basic helix-loop-helix factor AmeloD during tooth development.

The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.

Sp6/Epiprofin is a master regulator in the developing tooth.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

The transcription factor AmeloD stimulates epithelial cell motility essential for tooth morphology.

The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic helix-loop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.

The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development.

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in Nkx2-3, encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that Nkx2-3 is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of Bmp2 and Bmpr2 in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

Idiopathic basal crux ventricular arrhythmias with left bundle branch block and superior axis: A comparison with inferior-septal valvular arrhythmias.

INTRODUCTION: Left bundle branch block (LBBB) with superior axis is common in patients with idiopathic-ventricular arrhythmia (VA) originating from the tricuspid annulus (TA) and rarely from the cardiac basal crux and mitral annulus (MA). We described the electrocardiography and electrophysiological findings of idiopathic-VA presenting with LBBB and superior axis. METHODS AND RESULTS: We described 42 idiopathic-VA patients who had an LBBB and superior axis; 15 basal crux-VA, 17 TA-VA, and 10 MA-VA. No patient had a structural heart disease. Among patients with idiopathic-VA referred for ablation, we investigated the electrocardiogram and clinical characteristics of basal crux-VA as compared with other LBBB and superior axis-VA. The left ventricular ejection fraction with MA-VA was significantly lower in comparison with basal crux-VA (P = .01). All patients had a positive R wave in lead I and aVL. The maximum deflection index with basal crux-VA was significantly higher in comparison with TA-VA or MA-VA (P = .01). Patients with basal crux-VA presented with QS wave in lead II more frequently as compared with TA-VA or MA-VA (P = .001). All MA-VA patients had Rs wave in V6, and basal crux-VA, and TA-VA patients had a monophasic R wave or Rs wave in V6. Basal crux-VA patients underwent ablation in the middle cardiac vein (MCV) or coronary sinus (success rate: 94%, recurrence rate: 6%). CONCLUSIONS: We could distinguish basal crux-VA, TA-VA, and MA-VA, using a combination of clinical and electrocardiographic findings. These findings might be useful for counseling patients about an ablation strategy. Ablation via the MCV is effective for eliminating basal crux-VA.

Effects of Aging on the Coagulation Fibrinolytic System in Outpatients of the Cardiovascular Department.

BACKGROUND: Although clinical trials demonstrate that the elderly with atrial fibrillation have risks of thrombosis and bleeding, the relationship between aging and coagulation fibrinolytic system in "real-world" cardiology outpatients is uncertain. METHODS AND RESULTS: We retrospectively evaluated 773 patients (mean age: 58 years; 52% men; Asian ethnicity). To thoroughly investigate markers of coagulation and fibrinolysis, we simultaneously measured levels of D-dimer, prothrombin-fragment1+2 (F1+2), plasmin-α2 plasmin inhibitor complex (PIC), and thrombomodulin (TM). There were correlations between aging and levels of F1+2, D-dimer, PIC, and TM (R=0.61, 0.57, 0.49, and 0.30, respectively). We compared 3 age groups, which were defined as the Y group (<64 years), M group (65-74 years), and the O group (>75 years). Levels of markers were higher in older individuals (D-dimer: 1.0±0.8 vs. 0.8±0.8 vs. 0.6±0.4 µg/ml, F1+2: 281.8±151.3 vs. 224.6±107.1 vs. 155.5±90.0 pmol/L, PIC: 0.9±0.3 vs. 0.8±0.3 vs. 0.6±0.5 µg/ml, and TM: 2.9±0.8 vs. 2.7±0.7 vs. 2.5±0.7FU/ml). We performed logistic regression analysis to determine F1+2 and PIC levels. Multivariate analysis revealed that aging was the most important determinant of high F1+2 and PIC levels. CONCLUSIONS: Hypercoagulable states develop with advancing age in "real-world" cardiology outpatients. (Circ J 2016; 80: 2133-2140).

Novel hedgehog agonists promote osteoblast differentiation in mesenchymal stem cells.

Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh-Ag 1.3 and 1.7 were identified in a high-throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh-Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose-dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt-related transcription factor (Runx)2, was induced by Hh-Ag 1.7, which also rescued the osteoblast differentiation defect of RD-127, a mesenchymal cell line from Runx2-deficient mice. Hh-Ags also activated canonical Wnt signaling and synergized with low doses of BMP-2 to enhance osteoblastic potential. Thus, Hh-Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects.

J-Wave in Patients With Syncope.

BACKGROUND: Syncope is a common occurrence. The presence of J-wave, also known as early repolarization, on electrocardiogram is often seen in the general population, but the relationship between syncope and J-wave is unclear. METHODS AND RESULTS: After excluding 67 patients with structural heart disease from 326 with syncope, we classified 259 patients according to the presence or absence of J-wave (≥1 mm) in at least 2 inferior or lateral leads. Head-up tilt test (HUT) was performed for 30 min. If no syncope or presyncope occurred, HUT was repeated after drug loading. Before tilt, 97/259 (37%) had J-wave (57 male, 47.6±22.5 years) and 162 patients had no remarkable change (89 male, 51.1±21.2 years). HUT-positive rate was higher in patients with J-wave, compared with patients without (P<0.0001). The combination of J-wave and descending/horizontal ST segment in the inferior leads was more strongly associated with positive HUT than J-wave with ascending ST segment (odds ratio, 3.23). CONCLUSIONS: Prevalence of J-wave in the inferior or lateral leads was high in patients with syncope and was associated with HUT-induced neurally mediated reflex syncope (NMRS). Furthermore, the combination of J-wave and descending/horizontal ST segment in the inferior leads could be associated with a much higher risk of NMRS.

Daily Dysfunction of Autonomic Regulation Based on Ambulatory Blood Pressure Monitoring in Patients with Neurally Mediated Reflex Syncope.

BACKGROUND: The onset of neurally mediated reflex syncope (NMRS) is associated with dysfunction of the autonomic regulatory system. Yet relatively little is known about the daily conditions of the autonomic regulation system in patients with NMRS. This study elucidated characteristics of daily autonomic function using ambulatory blood pressure monitoring (ABPM) and evaluated the utility of ABPM for NMRS diagnosis. METHODS: Patients with syncope underwent the head-up tilt test (HUT) (80°, 30 minutes). If no syncope occurred, the HUT was repeated with drug loading. ABPM was performed on a different day. RESULTS: The enrolled subjects were 152 consecutive patients with syncope and 12 controls. Sixty-four patients with other diseases related to autonomic dysfunction were excluded. HUT with/without drug loading was positive in 40 patients (Group P) and negative in 48 patients (Group N). The average systolic blood pressure (SBP) in daytime was lower in Groups P and N than in the control group (Group C) (P < 0.05). The average diastolic blood pressure in daytime was also lower in Group P than in Group C (P < 0.05). The average standard deviation-SBP at nighttime was higher in Groups P and N than in Group C (P < 0.05). In heart rate variability analysis, Group P had higher high frequency normalized unit in daytime than Groups C and N (P < 0.05, P < 0.1). Low frequency/high frequency was lower in Group P than in Group N in both daytime and nighttime (P < 0.1, P < 0.05). CONCLUSION: This study suggests that patients with NMRS present with daily vagal hyperactivity and sympathetic dysfunction. ABPM may support the diagnosis of NMRS.