Signaling Pathways Crucial for Craniofacial Development Revealed by Endothelin-A Receptor-Deficient Mice

AbstractMost of the bone and cartilage in the craniofacial region is derived from cephalic neural crest cells, which undergo three primary developmental events: migration from the rhombomeric neuroectoderm to the pharyngeal arches, proliferation as the ectomesenchyme within the arches, and differentiation into terminal structures. Interactions between the ectomesenchymal cells and surrounding cells are required in these processes, in which defects can lead to craniofacial malformation. We have previously shown that the G-protein-coupled endothelin-A receptor (ETA) is expressed in the neural crest-derived ectomesenchyme, whereas the cognate ligand for ETA, endothelin-1 (ET-1), is expressed in arch epithelium and the paraxial mesoderm-derived arch core; absence of either ETA or ET-1 results in numerous craniofacial defects. In this study we have attempted to define the point at which cephalic neural crest development is disrupted in ETA-deficient embryos. We find that, while neural crest cell migration in the head of ETA−/− embryos appears normal, expression of a number of transcription factors in the arch ectomesenchymal cells is either absent or significantly reduced. These ETA-dependent factors include the transcription factorsgoosecoid, Dlx-2, Dlx-3, dHAND, eHAND, and Barx1, but not MHox, Hoxa-2, CRABP1, or Ufd1. In addition, the size of the arches in E10.5 to E11.5 ETA−/− embryos is smaller and an increase in ectomesenchymal apoptosis is observed. Thus, ETA signaling in ectomesenchymal cells appears to coordinate specific aspects of arch development by inducing expression of transcription factors in the postmigratory ectomesenchyme. Absence of these signals results in retarded arch growth, defects in proper differentiation, and, in some mesenchymal cells, apoptosis. In particular, this developmental pathway appears distinct from the pathway that includesUFD1L, implicated as a causative gene in CATCH 22 patients, and suggests parallel complementary pathways mediating craniofacial development

Glycemic control in type 2 diabetes

Type 2 diabetes is a typical lifestyle disease. We aimed to identify the factors affecting glycemic control in 64 outpatients with type 2 diabetes over a 2-year period. We defined poor glycemic control using a change in glycosylated hemoglobin (ΔHbA1c) of ≥ 0.5% over 2 years and/or HbA1c ≥ 7.5% at the end of the study period. We used a questionnaire to collect information on oral health behavior and lifestyle, including eating and smoking habits, and analyzed the relationships between indices of diabetes control and responses to the questionnaire. The mean (SD) HbA1c of the participants was 6.87% (0.77%) at a baseline, and 6.93% (0.69%) after 2 years. Twenty-three participants (36.0%) had poor glycemic control. ΔHbA1c and the change in body mass index (ΔBMI) correlated (Spearman’s rank correlation, r = 0.350, p < 0.01). The HbA1c at baseline was associated with eating slowly / chewing well, and ΔBMI was associated with perceived oral symptoms. Binominal logistic regression analysis revealed that poor glycemic control was associated with ΔBMI and a smoking habit (odds ratio : 1.62, 95% confidence interval : 1.08–2.42 ; and 4.01, 1.12–14.36, respectively). These findings imply that weight gain and a smoking habit are associated with poor glycemic control in patients with type 2 diabetes

Identification of the matricellular protein Fibulin-5 as a target molecule of glucokinase-mediated calcineurin/NFAT signaling in pancreatic islets

Glucokinase-mediated glucose signaling induces insulin secretion, proliferation, and apoptosis in pancreatic β-cells. However, the precise molecular mechanisms underlying these processes are not clearly understood. Here, we demonstrated that glucokinase activation using a glucokinase activator (GKA) significantly upregulated the expression of Fibulin-5 (Fbln5), a matricellular protein involved in matrix-cell signaling, in isolated mouse islets. The islet Fbln5 expression was induced by ambient glucose in a time- and dose-dependent manner and further enhanced by high-fat diet or the deletion of insulin receptor substrate 2 (IRS-2), whereas the GKA-induced increase in Fbln5 expression was diminished in Irs-2-deficient islets. GKA-induced Fbln5 upregulation in the islets was blunted by a glucokinase inhibitor, KATP channel opener, Ca2+ channel blocker and calcineurin inhibitor, while it was augmented by harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1 A inhibitor. Although deletion of Fbln5 in mice had no significant effects on the glucose tolerance or β-cell functions, adenovirus-mediated Fbln5 overexpression increased glucose-stimulated insulin secretion in INS-1 rat insulinoma cells. Since the islet Fbln5 expression is regulated through a glucokinase/KATP channel/calcineurin/nuclear factor of activated T cells (NFAT) pathway crucial for the maintenance of β-cell functions, further investigation of Fbln5 functions in the islets is warranted

Embracing Diversity, Equity, and Inclusion in the Scientific Community-Viewpoints of the Diversity, Equity, and Inclusion Committee of the North American Vascular Biology Organization

Recent increased visibility on racial issues in the United States elicited public outcry and a collective call for action. The social justice movement has facilitated energetic discussions about race, sexual orientation, and various issues of diversity, equity, and inclusion. This article discusses issues faced by people of color that we as scientists can address, as well as challenges faced by women and internationally trained scientists in the scientific community that need immediate attention. Moreover, we highlight various ways to resolve such issues at both institutional and individual levels. Silence and incremental solutions are no longer acceptable to achieving lasting social justice and ensure prosperous societies that work for all

包丁技術習得に関する研究―上達の客観的指標に関する検討―

　本研究では、調理頻度や得意意識の異なる者を対象者に包丁動作分析を行い、客観的な評価方法の検討を行うことを目的とした。和洋女子大学教職員33名（男性9名、女性24名）を対象にし、長さと太さを調整したきゅうりを用いた輪切りの薄切りを実施した。包丁には「携帯型電気生理計測装置　超小型無線生体モニターマイクロDAQターミナル」を取り付け、動作解析を行った。分析項目は、20秒あたりの包丁の1ストローク所要時間、包丁動作（包丁サイクル・振り下ろし・振り上げ、前・後方向）の加速度とした。切断物に関しては薄切りの枚数、厚みを分析した。加えて、調理頻度や得意意識をアンケート調査した。20秒間のきゅうりの薄切りの枚数は、対象者の調理頻度より、本人の自己評価である得意意識の方が関連することが示され、対象を「得意群」、「中間群」、「不得意群」に分類し分析した。輪切り枚数、厚みは、「得意群」、「不得意群」間に有意差があり、包丁サイクルは「得意群」の方が「不得意群」より1サイクルの時間が有意に短く、包丁の振り下ろし・振り上げの最大加速度が有意に大きいことが認められた。　以上の結果から、輪切り枚数、厚み、包丁サイクル、振り下ろし・振り上げの最大加速度の指標を用いることで、包丁技術の上達の経緯を客観的な数値を用いて評価することが可能になると考えられた

Novel ELN mutation in a Japanese family with a severe form of supravalvular aortic stenosis

BackgroundSupravalvular aortic stenosis (SVAS) is one of the congenital cardiovascular diseases characterized by stenosis of the aorta. The stenotic lesions occur anywhere above the aortic valve in the aortic tree as well as pulmonary arteries and eventually leads to circulatory failure. The disease gene has been identified on the elastin gene (ELN) and two types of SVAS have been categorized; a familial type and an isolated type with the de novo mutation.MethodsFluorescent In situ hybridization (FISH) analysis and gene sequencing were performed in a two‐generation family in which severe form of SVAS was diagnosed.ResultsNone of the patients tested showed microdeletion of ELN, LIMK1, and D7S613. A novel nonsense mutation of ELN (c.160G>T (p.(Gly54*)), RNA not analyzed) was found in exon 3 in three members; two of them died suddenly due to rapid progression of SVAS with possible arrhythmia in early infancy. A point mutation in the 5’ untranslated region, which was previously suggested to be associated with SVAS, did not co‐segregate with the SVAS phenotype and found to be SNPs.ConclusionOur report shows a broad spectrum of clinical features in family members sharing the identical mutations, suggesting a potential contribution of modifier gene(s) or interactions with environmental factors

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations

Fibulin-5, an integrin-binding matricellular protein: its function in development and disease

Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including α5β1, αvβ3, and αvβ5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF165-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the α5β1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of tumor-derived versus host microenvironment-derived fibulin-5 remains to be evaluated

Decreased mitochondrial respiration in aneurysmal aortas of Fibulin-4 mutant mice is linked to PGC1A regulation

Aim Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation. Methods and results In Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I–V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFβ reduced PGC1α levels, indicating involvement of TGFβ signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator. Conclusion Our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation