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.

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.

Diagnostic ability of Japanese version of high bleeding risk criteria for ischemic outcomes in patients with acute myocardial infarction.

The Japanese version of high bleeding risk (J-HBR) criteria was domestically proposed to identify patients at HBR after percutaneous coronary intervention (PCI). The applicability of J-HBR on bleeding events has been validated, while whether J-HBR predicts ischemic events is uncertain. This bi-center registry included 904 patients with acute myocardial infarction (MI) undergoing primary PCI. Patients were stratified by the J-HBR major (1 point) and minor (0.5 point) criteria. Patients with J-HBR ≥ 1 point were diagnosed as having HBR. The primary endpoint was major adverse cardiovascular events (MACE), a composite of cardiovascular death, recurrent MI, and ischemic stroke, after discharge. Of the 904 patients, 451 (49.9%) had the J-HBR. The primary endpoint more frequently occurred in patients with J-HBR than in those without (10.9% vs. 4.9%, p < 0.001) during the median follow-up period of 522 days. Probability of MACE was progressively increased with the increase in the number of J-HBR major and minor criteria, in which severe anemia, severe chronic kidney disease, prior heart failure, peripheral artery disease, and prior ischemic stroke were identified as significant factors associated with MACE. In patients with acute MI undergoing PCI, the J-HBR criteria were predictive for ischemic outcomes after discharge, suggesting that the J-HBR criteria may be useful to identify patients at high bleeding and ischemic risks.

Impact of myocardial bridge on non-culprit vessel lumen changes in patients with acute coronary syndrome.

This study aims to clarify the impact of myocardial bridge (MB) on the presence and progression of atherosclerosis in left descending coronary artery (LAD) in patients with acute coronary syndrome (ACS). Ninety-eight patients who underwent percutaneous coronary intervention with the diagnosis of ACS and follow-up coronary angiography but had no significant stenosis in the LAD were included. MB was defined based on coronary angiography. Quantitative coronary angiography was performed to determine the segments where MB was present and proximal to the MB (proximal segment) in patients with MB. In patients without MB, a corresponding region was quantitatively analyzed. The primary endpoint was changes in minimum lumen diameter (MLD) and percentage of diameter stenosis (%DS) in the proximal segment from baseline to follow-up angiography, namely ΔMLD and Δ%DS. MB was identified in 29 (29.6%) patients. Patients with MB had larger MLD and smaller %DS in the proximal segment than their counterpart. During the mean follow-up period of 12.9 ± 5.7 months, MLD and %DS in the proximal segment did not change significantly from baseline to follow-up in patients with and without MB. No significant between-group differences were observed in ΔMLD and Δ%DS. Baseline MLD was identified as the only factor associated with ΔMLD in the proximal segment. ACS patients who had MB but no significant stenosis in the LAD had larger MLD and smaller %DS at the segment proximal to MB compared to those without. In this selected population, serial lumen changes assessed by ΔMLD were not associated with the presence of MB in the LAD.

von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation.

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

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.

Impact of PARIS and CREDO-Kyoto Thrombotic and Bleeding Risk Scores on Clinical Outcomes in Patients With Acute Myocardial Infarction.

BACKGROUND: The PARIS and CREDO-Kyoto risk scores were developed to identify patients at risks of thrombotic and bleeding events individually after percutaneous coronary intervention (PCI). However, these scores have not been well validated in different cohorts.Methods and Results:This 2-center registry enrolled 905 patients with acute myocardial infarction (MI) undergoing primary PCI. Patients were divided into 3 groups according to the PARIS and CREDO-Kyoto thrombotic and bleeding risk scores. The study endpoints included ischemic (cardiovascular death, recurrent MI, and ischemic stroke) and major bleeding events. Of 905 patients, 230 (25%) and 219 (24%) had high thrombotic and bleeding risks, respectively, with the PARIS scores, compared with 78 (9%) and 50 (6%) patients, respectively, with the CREDO-Kyoto scores. According to the 2 scores, >50% of patients with high bleeding risk had concomitant high thrombotic risk. During the mean follow-up period of 714 days, 163 (18.0%) and 95 (10.5%) patients experienced ischemic and bleeding events, respectively. Both PARIS and CREDO-Kyoto scores were significantly associated with ischemic and bleeding events after primary PCI. For ischemic events, the CREDO-Kyoto rather than PARIS thrombotic risk score had better diagnostic ability. CONCLUSIONS: In the present Japanese cohort of acute MI patients undergoing contemporary primary PCI, the PARIS and CREDO-Kyoto thrombotic and bleeding risk scores were discriminative for predicting ischemic and bleeding events.

Influence of myocardial bridge on atherosclerotic plaque distribution and characteristics evaluated by near-infrared spectroscopy intravascular ultrasound.

BACKGROUND: This study aims to clarify whether myocardial bridge (MB) could influence atherosclerotic plaque characteristics assessed using near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS) imaging. METHODS: One hundred and sixteen patients who underwent percutaneous coronary intervention (PCI) using NIRS-IVUS imaging were included. MB was defined as an echo-lucent band surrounding left anterior descending artery (LAD). In MB patients, LAD was divided into three segments: proximal, MB, and distal segments. In non-MB patients, corresponding three segments were defined based on the average length of the above segments. Segmental maximum plaque burden and lipid content derived from NIRS-IVUS imaging in the section of maximum plaque burden were evaluated in each segment. Lipid content of atherosclerotic plaque was evaluated as lipid core burden index (LCBI) and maxLCBI4mm. LCBI is the fraction of pixels indicating lipid within a region multiplied by 1000, and the maximum LCBI in any 4-mm region was defined as maxLCBI4mm. RESULTS: MB was identified in 42 patients. MB was not associated with maximum plaque burden in proximal segment. LCBI and maxLCBI4mm were significantly lower in patients with MB than those without in proximal segment. Multivariable analysis demonstrated both MB and maximum plaque burden in proximal segment to be independent predictors of LCBI in proximal segment. CONCLUSIONS: Lipid content of atherosclerotic plaque assessed by NIRS-IVUS imaging was significantly smaller in patients with MB than those without. MB could be considered as a predictor of lipid content of atherosclerotic plaque when assessed by NIRS-IVUS imaging.

Impact of perioperative antithrombotic strategies on clinical events in non-cardiac surgery.

Antithrombotic therapy including antiplatelet agents and anticoagulants are prescribed for secondary prevention in patients with established cardiovascular disease. Although antithrombotic therapy is often interrupted before non-cardiac surgery with or without perioperative bridging anticoagulation, the impact on thrombotic and bleeding events remains uncertain. A total of 360 patients chronically treated with antithrombotic therapy for secondary prevention underwent elective non-cardiac surgery under general anesthesia, with the complete interruption of antithrombotic agents. The study endpoints included all-cause death, thrombotic events, and major bleeding complications after surgical procedures. Of 360 patients, 190 (52.8%) and 200 (55.6%) received antiplatelet and anticoagulation perioperatively. Atrial fibrillation (32.8%) and coronary artery disease (22.5%) were the major indications for antithrombotic regimens. Antithrombotic therapy was interrupted from 5 [2, 7] days before the surgery to 4 [2, 7] days postoperatively. Perioperative bridging therapy with unfractionated heparin was employed in 113 (31.4%) patients. During the hospitalization, one (0.3%) patient died due to non-cardiovascular causes. Thrombotic events and major bleeding occurred in two (0.6%) and eight (2.2%) patients. Bridging therapy with heparin was significantly associated with an increased risk of bleeding events (5.3% vs. 0.8%, p = 0.02). Pre-operative bridging therapy with heparin and operative duration were significantly associated with bleeding complications. In the present study, complete interruption of antithrombotic therapy resulted in a few thrombotic events in patients undergoing elective non-cardiac surgery. Bridging therapy with heparin and longer operative duration were significantly associated with post-operative bleeding complications.

Systemic Endothelial Function, Plasma Xanthine Oxidoreductase Activity, and Blood Pressure Variability in Patients with Stable Coronary Artery Disease.

Background and Objectives: Although previous studies showed that an activity of xanthine oxidoreductase (XOR), a rate-limiting enzyme in purine metabolism, beyond the serum uric acid level, was associated with the development of coronary artery disease (CAD), the underlying mechanisms are unclear. Because endothelial dysfunction and a greater blood pressure (BP) variability may play a role, we investigated the relations among the endothelial function, XOR, and BP variability. Materials and Methods: This was a post-hoc study using pooled data of patients with a stable CAD from two prospective investigations, in which the systemic endothelial function was assessed with the reactive hyperemia index (RHI) and the XOR activity was measured. The BP variability was evaluated using BP measurements during the three- and four-day hospitalization. Results: A total of 106 patients with a stable CAD undergoing a percutaneous coronary intervention were included. Of the 106 patients, 46 (43.4%) had a systemic endothelial dysfunction (RHI < 1.67). The multivariable analysis identified a higher body mass index (BMI), female gender, and diabetes as factors associated with an endothelial dysfunction. A higher BMI was also related to an elevated XOR activity, in addition to current smoking. No significant correlation was observed between the RHI and XOR activity. Similarly, the in-hospital BP variability was associated with neither the endothelial function nor XOR. Conclusions: Among patients with a stable CAD, several factors were identified as being associated with a systemic endothelial dysfunction or an elevated XOR activity. However, no direct relations between the endothelial function, XOR, and BP variability were found.

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.

Phosphorylation-dependent osterix degradation negatively regulates osteoblast differentiation.

Proteasome inhibitors exert an anabolic effect on bone formation with elevated levels of osteoblast markers. These findings suggest the important role of the proteasomal degradation of osteogenic regulators, while the underlying molecular mechanisms are not fully understood. Here, we report that the proteasome inhibitors bortezomib and ixazomib markedly increased protein levels of the osteoblastic key transcription factor osterix/Sp7 (Osx). Furthermore, we revealed that Osx was targeted by p38 and Fbw7 for proteasomal degradation. Mechanistically, p38-mediated Osx phosphorylation at S73/77 facilitated Fbw7 interaction to trigger subsequent Osx ubiquitination. Consistent with these findings, p38 knockdown or pharmacological p38 inhibition resulted in Osx protein stabilization. Treatment with p38 inhibitors following osteogenic stimulation efficiently induced osteoblast differentiation through Osx stabilization. Conversely, pretreatment of p38 inhibitor followed by osteogenic challenge impaired osteoblastogenesis via suppressing Osx expression, suggesting that p38 exerts dual but opposite effects in the regulation of Osx level to fine-tune its activity during osteoblast differentiation. Furthermore, Fbw7-depleted human mesenchymal stem cells and primary mouse calvarial cells resulted in increased osteogenic capacity. Together, our findings unveil the molecular mechanisms underlying the Osx protein stability control and suggest that targeting the Osx degradation pathway could help enhance efficient osteogenesis and bone matrix regeneration.

Utility of near-infrared spectroscopy to detect the extent of lipid core plaque leading to periprocedural myocardial infarction.

OBJECTIVES: The aim of this study was to investigate whether lipid core plaque (LCP) in the entire stented segment detected by near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS) could predict procedural myocardial infarction (PMI) in patients undergoing percutaneous coronary artery intervention (PCI). BACKGROUND: NIRS-IVUS can identify LCP, described as high lipid core burden index (LCBI). Previously, the highest LCBI contained only in the 4-mm segment (maxLCBI4mm ) was reported to predict PMI. METHODS: Patients who underwent NIRS-IVUS examination during PCI for coronary artery disease at Chiba University Hospital were included. The extent of LCP in the stented segment derived from NIRS-IVUS analysis was presented as LCBI, maxLCBI4mm , and LCP area index (LAI), reflecting the total amount of LCP in the entire stented segment calculated as LCBI×lesion length. PMI was defined as an elevation of creatine kinase MB > 3 times upper reference level (URL), and periprocedural myocardial injury (PMInj) was defined as an elevation of troponin I>5 times URL within 12 to 24 h after PCI. RESULTS: Out of 141 enrolled patients, PMI occurred in 20 (14.2%) and PMInj occurred in 62 (44.0%) patients. Receiver-operating characteristic curve analysis revealed LAI was the strongest predictor for both PMI and PMInj (area under curve 0.771, p < 0.001, and 0.717, p < 0.001, respectively). Multiple logistic regression analysis determined high LAI value as the independent predictor of both PMI and PMInj. CONCLUSIONS: Greater extent of LCP in the entire stented segment detected by NIRS-IVUS was significantly associated with PMI as well as PMInj in patients undergoing PCI.

Greater coronary lipid core plaque assessed by near-infrared spectroscopy intravascular ultrasound in patients with elevated xanthine oxidoreductase: a mechanistic insight.

Elevated serum uric acid level was reportedly associated with greater coronary lipid plaque. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme in purine metabolism and believed to play an important role in coronary atherosclerosis. However, the relation of XOR to coronary lipid plaque and its mechanism are unclear. Patients with stable coronary artery disease undergoing elective percutaneous coronary intervention under near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) guidance were prospectively enrolled. They were divided into three groups according to serum XOR activities: low, normal, and high. Coronary lipid core plaques in non-target vessels were evaluated by NIRS-IVUS with lipid core burden index (LCBI) and a maximum LCBI in 4 mm (maxLCBI4mm). Systemic endothelial function and inflammation were assessed with reactive hyperemia index (RHI) and high-sensitivity C-reactive protein, neutrophil-to-lymphocyte ratio, and platelet-to-lymphocyte ratio. Of 68 patients, 26, 31, and 11 were classified as low, normal, and high XOR activity groups. LCBI (474.4 ± 171.6 vs. 347.4 ± 181.6 vs. 294.0 ± 155.9, p = 0.04) and maxLCBI4mm (102.1 ± 56.5 vs. 65.6 ± 48.5 vs. 55.6 ± 37.8, p = 0.04) were significantly higher in high XOR group than in normal and low XOR groups. Although RHI was significantly correlated with body mass index, diabetes, current smoking, and high-density lipoprotein cholesterol, no relation was found between XOR activity and RHI. There were also no relations between XOR activity and C-reactive protein, neutrophil-to-lymphocyte ratio, or platelet-to-lymphocyte ratio. In conclusion, elevated XOR activity was associated with greater coronary lipid core plaque in patients with stable coronary artery disease, without significant relations to systemic endothelial function and inflammation.

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.

In-Hospital Blood Pressure Variability: A Novel Prognostic Marker of Renal Function Decline and Cardiovascular Events in Patients with Coronary Artery Disease.

INTRODUCTION: Several measures of blood pressure (BP) variability have been associated with kidney disease and cardiovascular events. Although BP is routinely measured during hospitalization in daily practice, the prognostic impact of in-hospital BP and its variability are uncertain. METHODS: A total of 226 participants who underwent elective percutaneous coronary intervention (PCI) for stable coronary artery disease (CAD) were included. BP was measured by trained nurses during the 4-day hospitalization for PCI. BP variability was assessed by standard deviation (SD) and coefficient variation of systolic BP. Estimated glomerular filtration rate (eGFR) was calculated at baseline and follow-up (≥6 months). The cardiovascular end point was defined as a composite of cardiovascular death, acute coronary syndrome, stroke, heart failure hospitalization, and any coronary revascularization. RESULTS: In-hospital BP was measured 9.5 ± 0.8 times. During a median follow-up period of 1.7 years, mean eGFR change was -1.7 mL/min/1.73 m2 per year, and 35 (15.5%) participants met the cardiovascular end point. Mean systolic BP and SD were negatively correlated with eGFR change. In the receiver operating characteristic curve analysis, SD of systolic BP predicted the cardiovascular end point (AUC 0.63, best cutoff value 14.2 mm Hg, p = 0.003). Kaplan-Meier analysis demonstrated a significantly higher incidence of the cardiovascular end point in patients with SD of systolic BP ≥14.2 mm Hg compared to their counterpart (p = 0.003). A multivariable analysis showed SD of systolic BP as an independent predictor for the cardiovascular end point. When assessed with coefficient variation, BP variability was similarly related to eGFR change and clinical outcomes. CONCLUSION: Greater in-hospital BP variability was associated with renal function decline and cardiovascular events in patients with stable CAD.

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.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

Chemical and thermal sensitivity of medaka TRPA1 analyzed in heterologous expression system.

TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water.