Publications by authors named "Tomoaki Iwayama"

13 Publications

  • Page 1 of 1

Zbp1-positive cells are osteogenic progenitors in periodontal ligament.

Sci Rep 2021 Apr 6;11(1):7514. Epub 2021 Apr 6.

Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-87016-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024286PMC
April 2021

Hypoxia stimulates collagen hydroxylation in gingival fibroblasts and periodontal ligament cells.

J Periodontol 2021 Mar 4. Epub 2021 Mar 4.

Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.

Background: Cellular responses to hypoxia regulate various biological events, including angiogenesis and extracellular matrix metabolism. Collagen is a major component of the extracellular matrix in periodontal tissues and its coordinated production is essential for tissue homeostasis. In this study, we investigated the effects of hypoxia on collagen production in human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLs).

Methods: HGFs and HPDLs were cultured under either normoxic (20% O ) or hypoxic (1% O ) conditions. Nuclear expression of hypoxia-inducible factor-1α (HIF-1α) was determined by western blotting. Peri-cellular expression of type I collagen was examined by immunocytochemistry analysis. Synthesis of type I collagen was evaluated by measuring the concentration of procollagen type I C-peptide (PIP) in culture supernatant using enzyme-linked immunosorbent assay. Expression of collagen hydroxylase enzymes prolyl 4-hydroxylase alpha polypeptide 1 (P4HA1) and 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was determined by RT-qPCR and western blotting. The roles of these enzymes were analyzed using siRNA transfection.

Results: Cultivation under hypoxic conditions stimulated type I collagen production via HIF-1α in both cell types. Interestingly, hypoxic conditions did not affect collagen 1a1 or 1a2 gene expression but upregulated that of P4HA1 and PLOD2. Additionally, suppressing P4HA1 significantly decreased the levels of hypoxia-induced procollagen type I C-peptide, a product of stable triple helical collagen, in the supernatant. In contrast, PLOD2 suppression decreased cross-linked collagen expression in the pericellular region.

Conclusion: Our results suggest that hypoxia activates collagen synthesis in HGFs and HPDLs by upregulating hydroxylases P4HA1 and PLOD2 in an HIF-1α-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/JPER.20-0670DOI Listing
March 2021

Mice lacking PLAP-1/asporin counteracts high fat diet-induced metabolic disorder and alveolar bone loss by controlling adipose tissue expansion.

Sci Rep 2021 Mar 2;11(1):4970. Epub 2021 Mar 2.

Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.

Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-84512-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925592PMC
March 2021

Nanoscale observation of PM2.5 incorporated into mammalian cells using scanning electron-assisted dielectric microscope.

Sci Rep 2021 Jan 8;11(1):228. Epub 2021 Jan 8.

Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8566, Japan.

PM2.5 has been correlated with risk factors for various diseases and infections. It promotes tissue injury by direct effects of particle components. However, effects of PM2.5 on cells have not been fully investigated. Recently, we developed a novel imaging technology, scanning electron-assisted dielectric-impedance microscopy (SE-ADM), which enables observation of various biological specimens in aqueous solution. In this study, we successfully observed PM2.5 incorporated into living mammalian cells in culture media. Our system directly revealed the process of PM2.5 aggregation in the cells at a nanometre resolution. Further, we found that the PM2.5 aggregates in the intact cells were surrounded by intracellular membrane-like structures of low-density in the SE-ADM images. Moreover, the PM2.5 aggregates were shown by confocal Raman microscopy to be located inside the cells rather than on the cell surface. We expect our method to be applicable to the observation of various nanoparticles inside cells in culture media.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-80546-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794539PMC
January 2021

Association of periodontal disease with atherosclerosis in 70-year-old Japanese older adults.

Odontology 2021 Apr 4;109(2):506-513. Epub 2020 Nov 4.

Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.

Periodontal disease and arteriosclerotic disease are greatly affected by aging. In this study, the association of conventional risk factors and periodontal disease with atherosclerosis was longitudinally examined in Japanese older adults. Subjects in this study were 490 community-dwelling septuagenarians (69-71 years) randomly recruited from the Basic Resident Registry of urban or rural areas in Japan. At the baseline examination, all subjects underwent socioeconomic and medical interviews; medical examinations, including examinations for carotid atherosclerosis, hypertension, diabetes mellitus, and dyslipidemia; and conventional dental examinations, including a tooth count and measurement of probing pocket depth (PPD). After 3 years, 182 septuagenarians who had no atherosclerosis at the baseline examination were registered and received the same examination as at the baseline. In the re-examination conducted 3 years after the baseline survey, 131 (72.0%) of the 182 participants who had no atherosclerosis at the baseline examination were diagnosed with carotid atherosclerosis. Adjusting and analyzing the mutual relationships of the conventional risk factors for atherosclerosis by multiple logistic regression analysis for the 171 septuagenarians with a full set of data, the proportion of teeth with PPD ≥ 4 mm was independently related to the prevalence of atherosclerosis (odds ratio: 1.029, P < 0.022). This longitudinal study of Japanese older adults suggests that periodontal disease is associated with the onset/progression of atherosclerosis. Maintaining a healthy periodontal condition may be an important factor in preventing the development and progression of atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10266-020-00567-zDOI Listing
April 2021

Osteoblastic lysosome plays a central role in mineralization.

Sci Adv 2019 07 3;5(7):eaax0672. Epub 2019 Jul 3.

Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan.

Mineralization is the most fundamental process in vertebrates. It is predominantly mediated by osteoblasts, which secrete mineral precursors, most likely through matrix vesicles (MVs). These vesicular structures are calcium and phosphate rich and contain organic material such as acidic proteins. However, it remains largely unknown how intracellular MVs are transported and secreted. Here, we use scanning electron-assisted dielectric microscopy and super-resolution microscopy for assessing live osteoblasts in mineralizing conditions at a nanolevel resolution. We found that the calcium-containing vesicles were multivesicular bodies containing MVs. They were transported via lysosome and secreted by exocytosis. Thus, we present proof that the lysosome transports amorphous calcium phosphate within mineralizing osteoblasts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aax0672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609213PMC
July 2019

Detrimental effects of duplicate reads and low complexity regions on RNA- and ChIP-seq data.

BMC Bioinformatics 2015 25;16 Suppl 13:S10. Epub 2015 Sep 25.

Background: Adapter trimming and removal of duplicate reads are common practices in next-generation sequencing pipelines. Sequencing reads ambiguously mapped to repetitive and low complexity regions can also be problematic for accurate assessment of the biological signal, yet their impact on sequencing data has not received much attention. We investigate how trimming the adapters, removing duplicates, and filtering out reads overlapping low complexity regions influence the significance of biological signal in RNA- and ChIP-seq experiments.

Methods: We assessed the effect of data processing steps on the alignment statistics and the functional enrichment analysis results of RNA- and ChIP-seq data. We compared differentially processed RNA-seq data with matching microarray data on the same patient samples to determine whether changes in pre-processing improved correlation between the two. We have developed a simple tool to remove low complexity regions, RepeatSoaker, available at https://github.com/mdozmorov/RepeatSoaker, and tested its effect on the alignment statistics and the results of the enrichment analyses.

Results: Both adapter trimming and duplicate removal moderately improved the strength of biological signals in RNA-seq and ChIP-seq data. Aggressive filtering of reads overlapping with low complexity regions, as defined by RepeatMasker, further improved the strength of biological signals, and the correlation between RNA-seq and microarray gene expression data.

Conclusions: Adapter trimming and duplicates removal, coupled with filtering out reads overlapping low complexity regions, is shown to increase the quality and reliability of detecting biological signals in RNA-seq and ChIP-seq data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2105-16-S13-S10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597324PMC
May 2016

Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells.

Biochem Biophys Res Commun 2015 Aug 24;464(1):299-305. Epub 2015 Jun 24.

Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.

Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2015.06.147DOI Listing
August 2015

PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity.

Genes Dev 2015 Jun 27;29(11):1106-19. Epub 2015 May 27.

Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA; Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA;

Fibrosis is a common disease process in which profibrotic cells disturb organ function by secreting disorganized extracellular matrix (ECM). Adipose tissue fibrosis occurs during obesity and is associated with metabolic dysfunction, but how profibrotic cells originate is still being elucidated. Here, we use a developmental model to investigate perivascular cells in white adipose tissue (WAT) and their potential to cause organ fibrosis. We show that a Nestin-Cre transgene targets perivascular cells (adventitial cells and pericyte-like cells) in WAT, and Nestin-GFP specifically labels pericyte-like cells. Activation of PDGFRα signaling in perivascular cells causes them to transition into ECM-synthesizing profibrotic cells. Before this transition occurs, PDGFRα signaling up-regulates mTOR signaling and ribosome biogenesis pathways and perturbs the expression of a network of epigenetically imprinted genes that have been implicated in cell growth and tissue homeostasis. Isolated Nestin-GFP(+) cells differentiate into adipocytes ex vivo and form WAT when transplanted into recipient mice. However, PDGFRα signaling opposes adipogenesis and generates profibrotic cells instead, which leads to fibrotic WAT in transplant experiments. These results identify perivascular cells as fibro/adipogenic progenitors in WAT and show that PDGFRα targets progenitor cell plasticity as a profibrotic mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.260554.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470280PMC
June 2015

Involvement of PDGF in fibrosis and scleroderma: recent insights from animal models and potential therapeutic opportunities.

Curr Rheumatol Rep 2013 Feb;15(2):304

Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

Fibrosis is the principal characteristic of the autoimmune disease known as scleroderma or systemic sclerosis (SSc). Studies published within the last three years suggest central involvement of platelet-derived growth factors (PDGFs) in SSc-associated fibrosis. PDGFs may also be involved in SSc-associated autoimmunity and vasculopathy. The PDGF signaling pathway is well understood and PDGF receptors are expressed on collagen-secreting fibroblasts and on mesenchymal stem and/or progenitor cells that may affect SSc in profound and unexpected ways. Although much work remains before we fully understand how PDGFs are involved in SSc, there is much interest in using PDGF inhibitors as a therapeutic approach to SSc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11926-012-0304-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570472PMC
February 2013

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

J Cell Physiol 2011 Mar;226(3):809-21

Division of Oral Biology and Disease Control, Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.22406DOI Listing
March 2011

Reduction of N-glycolylneuraminic acid xenoantigen on human adipose tissue-derived stromal cells/mesenchymal stem cells leads to safer and more useful cell sources for various stem cell therapies.

Tissue Eng Part A 2010 Apr;16(4):1143-55

Department of Somatic Stem Cell Therapy, Foundation for Biomedical Research and Innovation , Kobe, Hyogo, Japan.

Adipose tissue is an attractive source for somatic stem cell therapy. Currently, human adipose tissue-derived stromal cells/mesenchymal stem cells (hADSCs/MSCs) are cultured with fetal bovine serum (FBS). Recently, however, not only human embryonic stem cell lines cultured on mouse feeder cells but also bone marrow-derived human MSCs cultured with FBS were reported to express N-glycolylneuraminic acid (Neu5Gc) xenoantigen. Human serum contains high titers of natural preformed antibodies against Neu5Gc. We studied the presence of Neu5Gc on hADSCs/MSCs cultured with FBS and human immune response mediated by Neu5Gc. Our data indicated that hADSCs/MSCs cultured with FBS expressed Neu5Gc and that human natural preformed antibodies could bind to hADSCs/MSCs. However, hADSCs/MSCs express complement regulatory proteins such as CD46, CD55, and CD59 and are largely resistant to complement-mediated cytotoxicity. hADSCs/MSCs cultured with FBS could be injured by antibody-dependent cell-mediated cytotoxicity mechanism. Further, human monocyte-derived macrophages could phagocytose hADSCs/MSCs cultured with FBS and this phagocytic activity was increased in the presence of human serum. Culturing hADSCs/MSCs with heat-inactivated human serum for a week could markedly reduce Neu5Gc on hADSCs/MSCs and prevent immune responses mediated by Neu5Gc, such as binding of human natural preformed antibodies, antibody-dependent cell-mediated cytotoxicity, and phagocytosis. Adipogenic and osteogenic differentiation potentials of hADSCs/MSCs cultured with heat-inactivated human serum were not less than that of those cultured with FBS. For stem cell therapies based on hADSCs/MSCs, hADSCs/MSCs that presented Neu5Gc on their cell surfaces after exposure to FBS should be cleaned up to be rescued from xenogeneic rejection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2009.0386DOI Listing
April 2010

Basic fibroblast growth factor regulates expression of heparan sulfate in human periodontal ligament cells.

Matrix Biol 2008 Apr 26;27(3):232-41. Epub 2007 Oct 26.

Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.

Heparan sulfate (HS) proteoglycan is a widely distributed biological molecule that mediates a variety of physiological responses in development, cell growth, cell migration, and wound healing. We examined the effects of basic fibroblast growth factor-2 (FGF-2), which is known to modulate extracellular matrix (ECM) production of various cell types, on the production of HS proteoglycan by human periodontal ligament (HPDL) cells. We also examined the effects of FGF-2 on the expression of syndecans, a major family of membrane-bound HS proteoglycans. Treatment of HPDL cells with FGF-2 for 72 h resulted in a pronounced increase in the level of HS in the culture supernatant in a dose-dependent manner. However, reverse transcription-polymerase chain reaction data (RT-PCR) revealed that FGF-2 marginally reduced the gene expression of syndecan-1, -2, and -4, and did not alter the level of syndecan-3 mRNA. Furthermore, FGF-2 did not have an effect on the mRNA expression of enzymes associated with HS biosynthesis. Interestingly, FACS analysis revealed that the syndecan family displayed diverse alterations in response to FGF-2. FGF-2 barely altered the expression of syndecan-1, but decreased the expression of syndecan-2 and -4 on HPDL cells. Moreover, dot blot analysis showed that FGF-2 did not alter the level of syndecan-1 and -2, but enhanced the level of syndecan-4 in culture supernatants of FGF-2-stimulated HPDL cells. These results suggest that the FGF-2-activated increase in the level of HS in conditioned medium may be a result of shedding of syndecan-4 from the HPDL cell surface. Taken together, FGF-2 may differentially regulate the expression of HS proteoglycans in a HS-proteoglycan-subtype-dependent manner. The diversity of the expression patterns of HS proteoglycans may be associated with the FGF-2-induced biological functions of HPDL cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2007.10.005DOI Listing
April 2008