Publications by authors named "Takaya Abe"

166 Publications

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice.

Sci Transl Med 2021 Mar;13(587)

Department of Developmental Neurobiology, Graduate School of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.
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http://dx.doi.org/10.1126/scitranslmed.aay7896DOI Listing
March 2021

Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

Sci Rep 2021 Mar 12;11(1):5857. Epub 2021 Mar 12.

Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo, 162-8480, Japan.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.
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http://dx.doi.org/10.1038/s41598-021-85416-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954811PMC
March 2021

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription.

J Biol Chem 2020 12;295(51):17632-17645

Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan; Graduate School of Medical Sciences, Nara Medical University, Kashihara, Nara, Japan. Electronic address:

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.
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http://dx.doi.org/10.1074/jbc.RA120.015003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762959PMC
December 2020

Arf1 and Arf6 Synergistically Maintain Survival of T Cells during Activation.

J Immunol 2021 Jan 11;206(2):366-375. Epub 2020 Dec 11.

Department of Cell Signaling, Institute of Biomedical Science, Kansai Medical University, Hirakata, Osaka 573-1010, Japan;

ADP-ribosylation factor (Arf) family consisting of six family members, Arf1-Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage-specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.
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http://dx.doi.org/10.4049/jimmunol.2000971DOI Listing
January 2021

Stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution.

Nat Metab 2020 12 7;2(12):1382-1390. Epub 2020 Dec 7.

Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.
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http://dx.doi.org/10.1038/s42255-020-00318-yDOI Listing
December 2020

Formation of quantum dots in GaN/AlGaN FETs.

Sci Rep 2020 Sep 22;10(1):15421. Epub 2020 Sep 22.

ROHM Co., Ltd, 21 Saiinnmizosakicho, Ukyo-ku, Kyoto, 615-8585, Japan.

GaN and the heterostructures are attractive in condensed matter science and applications for electronic devices. We measure the electron transport in GaN/AlGaN field-effect transistors (FETs) at cryogenic temperature. We observe formation of quantum dots in the conduction channel near the depletion of the 2-dimensional electron gas (2DEG). Multiple quantum dots are formed in the disordered potential induced by impurities in the FET conduction channel. We also measure the gate insulator dependence of the transport properties. These results can be utilized for the development of quantum dot devices utilizing GaN/AlGaN heterostructures and evaluation of the impurities in GaN/AlGaN FET channels.
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http://dx.doi.org/10.1038/s41598-020-72269-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508848PMC
September 2020

Amyloidogenic processing of amyloid β protein precursor (APP) is enhanced in the brains of alcadein α-deficient mice.

J Biol Chem 2020 07 27;295(28):9650-9662. Epub 2020 May 27.

Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic β-amyloid (Aβ) peptide generated from proteolytic cleavage of β-amyloid protein precursor (APP). Except for familial AD arising from mutations in the and presenilin () genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aβ generation , and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic β-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aβ in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aβ-dependent pathogenicity in AD.
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http://dx.doi.org/10.1074/jbc.RA119.012386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363152PMC
July 2020

Prc1-rich kinetochores are required for error-free acentrosomal spindle bipolarization during meiosis I in mouse oocytes.

Nat Commun 2020 05 27;11(1):2652. Epub 2020 May 27.

Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, 650-0047, Japan.

Acentrosomal meiosis in oocytes represents a gametogenic challenge, requiring spindle bipolarization without predefined bipolar cues. While much is known about the structures that promote acentrosomal microtubule nucleation, less is known about the structures that mediate spindle bipolarization in mammalian oocytes. Here, we show that in mouse oocytes, kinetochores are required for spindle bipolarization in meiosis I. This process is promoted by oocyte-specific, microtubule-independent enrichment of the antiparallel microtubule crosslinker Prc1 at kinetochores via the Ndc80 complex. In contrast, in meiosis II, cytoplasm that contains upregulated factors including Prc1 supports kinetochore-independent pathways for spindle bipolarization. The kinetochore-dependent mode of spindle bipolarization is required for meiosis I to prevent chromosome segregation errors. Human oocytes, where spindle bipolarization is reportedly error prone, exhibit no detectable kinetochore enrichment of Prc1. This study reveals an oocyte-specific function of kinetochores in acentrosomal spindle bipolarization in mice, and provides insights into the error-prone nature of human oocytes.
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http://dx.doi.org/10.1038/s41467-020-16488-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253481PMC
May 2020

Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes.

Cell Rep 2020 05;31(7):107653

Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima Minami-machi, Chuou-ku, Kobe 650-0047, Japan. Electronic address:

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.
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http://dx.doi.org/10.1016/j.celrep.2020.107653DOI Listing
May 2020

GCN2 regulates pancreatic β cell mass by sensing intracellular amino acid levels.

JCI Insight 2020 05 7;5(9). Epub 2020 May 7.

Division of Diabetes and Endocrinology, Department of Internal Medicine, and.

EIF2AK4, which encodes the amino acid deficiency-sensing protein GCN2, has been implicated as a susceptibility gene for type 2 diabetes in the Japanese population. However, the mechanism by which GCN2 affects glucose homeostasis is unclear. Here, we show that insulin secretion is reduced in individuals harboring the risk allele of EIF2AK4 and that maintenance of GCN2-deficient mice on a high-fat diet results in a loss of pancreatic β cell mass. Our data suggest that GCN2 senses amino acid deficiency in β cells and limits signaling by mechanistic target of rapamycin complex 1 to prevent β cell failure during the consumption of a high-fat diet.
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http://dx.doi.org/10.1172/jci.insight.128820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253016PMC
May 2020

Evaluation of a new measurement method of indoxyl sulfate in hemodialysis patients.

Ther Apher Dial 2021 Feb 30;25(1):44-49. Epub 2020 Apr 30.

Department of Biopharmaceutics, Kumamoto University, Graduate School of Pharmaceutical Sciences, School of Pharmacy, Kumamoto, Japan.

Indoxyl sulfate (IS) is related to the development of cardiovascular disease and total mortality in dialysis patients. High-performance liquid chromatography (HPLC) is the conventional measurement approach. However, the HPLC method is difficult to perform in real time. Recently, the IS Assay Kit "NIPRO", which enables the measuring of total IS by the enzyme method, was developed. This new reagent allows the easy and quick measurement of many samples using the automatic biochemical analyzer. Moreover, it was reported that it demonstrated satisfactory analytical performance. If this enzyme method is useful for measuring IS in hemodialysis, we can expect that the mechanism in which the IS effects adversely on a body as uremic toxins will be clarified. However, the enzyme method is more easily influenced by other coexisting substances. In this study, we have assessed on how the uremic toxins and anticoagulation effect on this new reagent and evaluate whether it can be put into practice effectively for hemodialysis patients. For the enzyme method, accuracy, simultaneous repeatability, linearity, limit of detection, influence of coexisting materials, and correlation with the HPLC method were examined. Accuracy and simultaneous repeatability were satisfactory, and linearity was good. The limit of detection was acceptable, and there was no influence of coexisting materials. With regard to the correlation, the regression equation was y = 0.947X + 7.987 and the correlation coefficient (r) was 0.972. This new reagent showed sufficient fundamental performance and had a good correlation with the conventional HPLC method for assessing the plasma of dialysis patients.
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http://dx.doi.org/10.1111/1744-9987.13500DOI Listing
February 2021

Exophilin-5 regulates allergic airway inflammation by controlling IL-33-mediated Th2 responses.

J Clin Invest 2020 07;130(7):3919-3935

Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.

A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma.
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http://dx.doi.org/10.1172/JCI127839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324203PMC
July 2020

The large GTPase Rab44 regulates granule exocytosis in mast cells and IgE-mediated anaphylaxis.

Cell Mol Immunol 2020 12 1;17(12):1287-1289. Epub 2020 Apr 1.

Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 8528588, Japan.

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http://dx.doi.org/10.1038/s41423-020-0413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784977PMC
December 2020

The CCR4-NOT complex maintains liver homeostasis through mRNA deadenylation.

Life Sci Alliance 2020 05 1;3(5). Epub 2020 Apr 1.

Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of , encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation.
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http://dx.doi.org/10.26508/lsa.201900494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119370PMC
May 2020

CDK1 dependent phosphorylation of hTERT contributes to cancer progression.

Nat Commun 2020 03 25;11(1):1557. Epub 2020 Mar 25.

Division of Cancer Stem Cell, National Cancer Center Research Institute, Tokyo, 104-0045, Japan.

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.
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http://dx.doi.org/10.1038/s41467-020-15289-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096428PMC
March 2020

Dmrt genes participate in the development of Cajal-Retzius cells derived from the cortical hem in the telencephalon.

Dev Dyn 2020 06 12;249(6):698-710. Epub 2020 Feb 12.

Department of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

Background: During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood.

Results: We found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced.

Conclusion: Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development.
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http://dx.doi.org/10.1002/dvdy.156DOI Listing
June 2020

The Chiral Looping of the Embryonic Heart Is Formed by the Combination of Three Axial Asymmetries.

Biophys J 2020 02 18;118(3):742-752. Epub 2019 Dec 18.

Laboratories for Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Chūō-ku, Kobe, Hyogo, Japan; Division of Embryology, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi, Japan.

In mammals and birds, embryonic development of the heart involves conversion of a straight tubular structure into a three-dimensional helical loop, which is a chiral structure. We investigated theoretically the mechanism of helical loop formation of the mouse embryonic heart, especially focusing on determination of left-/right-handedness of the helical loop. In geometrical terms, chirality is the result of the combination of three axial asymmetries in three-dimensional space. We hypothesized the following correspondences between axial asymmetries and morphogenesis (bending and displacement): the dorsal-ventral asymmetry by ventral bending of a straight tube of the initial heart and the left-right and anterior-posterior asymmetries, the left-right asymmetry by rightward displacement of the heart tube, which is confined to the anterior region of the tube. Morphogenesis of chiral looping of the embryonic heart is a large-scaled event of the multicellular system in which substantial physical force operates dynamically. Using computer simulations with a cell-based physico-mechanical model and experiments with mouse embryos, we confirmed the hypothesis. We conclude that rightward displacement of the tube determines the left-handed screw of the loop. The process of helix loop formation consists of three steps: 1) the left-right biasing system involving Nodal-related signals that leads to left-right asymmetry in the embryonic body; 2) the rightward displacement of the tube; and finally 3) the left-handed helical looping. Step 1 is already established. Step 3 is elucidated by our study, which highlights the need for step 2 to be clarified; namely, we explore how the left-right asymmetry in the embryonic body leads to the rightward displacement of the heart tube.
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http://dx.doi.org/10.1016/j.bpj.2019.11.3397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002914PMC
February 2020

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability.

RNA Biol 2020 03 10;17(3):403-416. Epub 2020 Jan 10.

Laboratory for Immunogenetics, Riken Center of Integrative Medical Sciences, Yokohama, Japan.

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in -dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in -dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in -dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in -dKO MEFs than in -dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in -dKO MEFs than in -dKO MEFs. Importantly, -dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.
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http://dx.doi.org/10.1080/15476286.2019.1709747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999631PMC
March 2020

Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development.

Nat Cell Biol 2020 01 23;22(1):26-37. Epub 2019 Dec 23.

Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch-R-Ras-integrin β1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.
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http://dx.doi.org/10.1038/s41556-019-0436-9DOI Listing
January 2020

Behavioral analysis in mice deficient for GAREM2 (Grb2-associated regulator of Erk/MAPK subtype2) that is a subtype of highly expressing in the brain.

Mol Brain 2019 11 12;12(1):94. Epub 2019 Nov 12.

The Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 5562 Nanatsuka, Shobara, Hiroshima, 727-0023, Japan.

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.
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http://dx.doi.org/10.1186/s13041-019-0512-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852768PMC
November 2019

Complete response to nivolumab for metastatic renal cell carcinoma on hemodialysis patient; a case report.

Urol Case Rep 2020 Jan 15;28:101048. Epub 2019 Oct 15.

Department of Urology, Iwate Medical University, Japan.

A 73-year-old-male patient with hypertension and grade 3B chronic kidney disease underwent radical nephrectomy in the year 2000. As the renal function gradually worsened, the patient was started on hemodialysis in 2003. Eleven years after nephrectomy, the metastases were pointed out. The patient received sunitinib as first-line therapy. Thereafter, as the metastatic lesions became larger, we initiated nivolumab therapy (3 mg/m2 every 2 weeks). After 52 weeks, the metastatic lesions continued showing complete response (CR). No adverse effects were observed during nivolumab treatment.
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http://dx.doi.org/10.1016/j.eucr.2019.101048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833355PMC
January 2020

Semaphorin 6A-Plexin A2/A4 Interactions with Radial Glia Regulate Migration Termination of Superficial Layer Cortical Neurons.

iScience 2019 Nov 21;21:359-374. Epub 2019 Oct 21.

Brain Function Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.

Precise regulation of neuronal migration termination is crucial for the establishment of brain cytoarchitectures. However, little is known about how neurons terminate migration. Here we focused on interactions between migrating cortical neurons and their substrates, radial glial (RG) cells, and analyzed the role of Plexin A2 and A4 (PlxnA2/A4) receptors and their repulsive ligand, Semaphorin 6A (Sema6A), for this process. In both PlxnA2/A4 double-knockout and Sema6A mutant mice, the outermost cortical plate neurons ectopically invade layer 1 at a stage when they should reach their destinations. PlxnA2/A4 proteins are abundantly expressed on their leading processes, whereas Sema6A mRNA is enriched in RG cell somata. Cell-targeted gene expression and conditional knockouts indicate critical roles for these molecules. We hypothesize that the timely appearance of repulsive signaling mediated by Sema6A-PlxnA2/A4 weakens migrating neuron-RG cell interactions, leading to migration termination.
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http://dx.doi.org/10.1016/j.isci.2019.10.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889767PMC
November 2019

Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

Proc Natl Acad Sci U S A 2019 11 6;116(47):23653-23661. Epub 2019 Nov 6.

Department of Molecular Immunology, Institute of Industrial Science, The University of Tokyo, 153-8505 Tokyo, Japan;

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.
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http://dx.doi.org/10.1073/pnas.1915326116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876158PMC
November 2019

A Novel Birthdate-Labeling Method Reveals Segregated Parallel Projections of Mitral and External Tufted Cells in the Main Olfactory System.

eNeuro 2019 Nov/Dec;6(6). Epub 2019 Nov 20.

Brain Function Laboratory, National Institute of Genetics.

A fundamental strategy in sensory coding is parallel processing, whereby unique, distinct features of sensation are computed and projected to the central target in the form of submodal maps. It remains unclear, however, whether such parallel processing strategy is employed in the main olfactory system, which codes the complex hierarchical odor and behavioral scenes. A potential scheme is that distinct subsets of projection neurons in the olfactory bulb (OB) form parallel projections to the targets. Taking advantage of the observation that the distinct projection neurons develop at different times, we developed a Cre-loxP-based method that allows for birthdate-specific labeling of cell bodies and their axon projections in mice. This birthdate tag analysis revealed that the mitral cells (MCs) born in an early developmental stage and the external tufted cells (TCs) born a few days later form segregated parallel projections. Specifically, the latter subset converges the axons onto only two small specific targets, one of which, located at the anterolateral edge of the olfactory tubercle (OT), excludes widespread MC projections. This target is made up of neurons that express dopamine D1 but not D2 receptor and corresponds to the most anterolateral isolation of the CAP compartments (aiCAP) that were defined previously. This finding of segregated projections suggests that olfactory sensing does indeed involve parallel processing of functionally distinct submodalities. Importantly, the birthdate tag method used here may pave the way for deciphering the functional meaning of these individual projection pathways in the future.
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http://dx.doi.org/10.1523/ENEURO.0234-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868177PMC
June 2020

Holocrine Secretion Occurs outside the Tight Junction Barrier in Multicellular Glands: Lessons from Claudin-1-Deficient Mice.

J Invest Dermatol 2020 02 22;140(2):298-308.e5. Epub 2019 Aug 22.

Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; Keio-Maruho Laboratory of Skin Barriology, Keio University School of Medicine, Tokyo, Japan. Electronic address:

Holocrine secretion is a specific mode of secretion involving secretion of entire cytoplasmic materials with remnants of dead cells, as observed in multicellular exocrine glands of reptiles, birds, and mammals. Here, we found that sebaceous glands in mice, representative of multicellular exocrine glands of mammals, exhibit a form of polarized stratified epithelium equipped with tight junctions (TJs), and found that holocrine secretion occurred outside the TJ barriers. Sebaceous glands share characteristics of stratified epithelia with interfollicular epidermis, including basal-layer-restricted cell proliferation, TJ barrier formation at a specific single layer of cells with apico-basolateral plasma membrane polarity, and cell death outside the TJ barrier. Knockout of claudin-1, a transmembrane adhesive protein in TJs, in mice caused leakage of the TJ barrier in sebaceous glands and incomplete degradation of the plasma membrane and nuclei during holocrine secretion. Claudin-1 knockout resulted in the accumulation of incompletely degenerated sebocytes in sebaceous ducts, suggesting that the TJ barrier was necessary for differentiation of holocrine secretion. The redefinition of sebaceous glands as TJ-forming stratified epithelia provides an important framework to understand the molecular mechanism of holocrine secretion.
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http://dx.doi.org/10.1016/j.jid.2019.06.150DOI Listing
February 2020

Cm/Eu induced structural, mechanistic and functional implications for calmodulin.

Phys Chem Chem Phys 2019 Oct 16;21(38):21213-21222. Epub 2019 Aug 16.

Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, 01328, Germany. and Tokyo Tech World Research Hub Initiative (WRHI), Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, 152-8550, Japan.

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An/Ln ions. As will be demonstrated herein, our data confirm the higher affinity of Cm and Eu compared to Ca to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu and Cm to carry one or two additional waters compared to Ca-CaM, while also causing the structure of Cm/Eu-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.
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http://dx.doi.org/10.1039/c9cp03750kDOI Listing
October 2019

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

JCI Insight 2019 06 20;4(12). Epub 2019 Jun 20.

Department of Otolaryngology-Head and Neck Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
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http://dx.doi.org/10.1172/jci.insight.128561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629139PMC
June 2019

Fhod1, an actin-organizing formin family protein, is dispensable for cardiac development and function in mice.

Cytoskeleton (Hoboken) 2019 02 29;76(2):219-229. Epub 2019 Apr 29.

Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.

The formin family proteins have the ability to regulate actin filament assembly, thereby functioning in diverse cytoskeletal processes. Fhod3, a cardiac member of the family, plays a crucial role in development and functional maintenance of the heart. Although Fhod1, a protein closely-related to Fhod3, has been reported to be expressed in cardiomyocytes, the role of Fhod1 in the heart has still remained elusive. To know the physiological role of Fhod1 in the heart, we disrupted the Fhod1 gene in mice by replacement of exon 1 with a lacZ reporter gene. Histological lacZ staining unexpectedly revealed no detectable expression of Fhod1 in the heart, in contrast to intensive staining in the lung, a Fhod1-containing organ. Consistent with this, expression level of the Fhod1 protein in the heart was below the lower limit of detection of the present immunoblot analysis with three independent anti-Fhod1 antibodies. Homozygous Fhod1-null mice did not show any defects in gross and histological appearance of the heart or upregulate fetal cardiac genes that are induced under stress conditions. Furthermore, Fhod1 ablation did not elicit compensatory increase in expression of other formins. Thus, Fhod1 appears to be dispensable for normal development and function of the mouse heart, even if a marginal amount of Fhod1 is expressed in the heart.
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http://dx.doi.org/10.1002/cm.21523DOI Listing
February 2019

Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence.

Blood 2019 05 5;133(22):2385-2400. Epub 2019 Apr 5.

Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.

Inactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first null (knockout [KO]) mice and show that they are viable and fertile with no gross abnormalities. However, by 6 months, they exhibit increased peripheral blood cell counts, splenomegaly, and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sublethal γ irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and COP9 signalosome subunit 5 (CSN5)-mediated p53 ubiquitination, and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a posttranslational regulator of wild-type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.
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http://dx.doi.org/10.1182/blood.2019000530DOI Listing
May 2019

Adipolin/CTRP12 protects against pathological vascular remodelling through suppression of smooth muscle cell growth and macrophage inflammatory response.

Cardiovasc Res 2020 01;116(1):237-249

Molecular Cardiovascular Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya 466-8550, Japan.

Aims: Secreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling.

Methods And Results: Adipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-β (TGF-β) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-β receptor II (TGF-βRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-βRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-β1 concentration in media from cultured VSMCs and macrophages.

Conclusion: These data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.
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http://dx.doi.org/10.1093/cvr/cvz074DOI Listing
January 2020