Masataka Sunohara

Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress

Glomerular hypertension is a major determinant advancing progression to end‐stage renal failure. Podocytes, which are thought to counteract pressure‐mediated capillary expansion, are increasingly challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowmans capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell‐matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up‐regulated by stretch (approximately threefold). By reverse transcriptase‐polymer chain reaction, up‐regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate‐salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV‐coated membranes accelerated stretch‐induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch‐adapting molecule in podocytes.

Immunocytochemical localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 of the human deciduous molar tooth germ development in the human fetus.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel endothelial development. We used immunohistochemical methods to demonstrate the localization of VEGF and its receptors, showing the specific expression pattern of VEGF and VEGF receptor in the human deciduous tooth from the cap to late bell stages in the human fetus. Immunoreactivity to VEGF and its receptor VEGF receptor-2 (VEGFR-2) was intensely positive in the inner enamel epithelium at the cap stage and ranged from negative to moderately positive in the bell stage. At the late bell stage, VEGF immunoreactivity was mainly positive but weak for VEGFR-2. The intensity of VEGF and VEGFR-2 in odontoblasts increases from cap stage to late bell stage. We postulate that the dissimilar expression of VEGF in inner enamel epithelium, ameloblast and odontoblast during each stage of human tooth development may affect tooth germ formation.

Expression of Nuclear and Mitochondrial Thyroid Hormone Receptors in Postnatal Rat Tongue Muscle

In this quantitative study, a competitive RT-PCR analysis was used to measure the level of the thyroid hormone receptors (TRs) in rat tongue muscle during the development of male Wistar rats aged 0, 5, 10, 15 and 21 postnatal days. There were differences between the expression of TR-α1 mRNA and the mRNAs for TR-β1 and TR-β2 in rat tongue muscle. Using Western blot analysis, a difference in expression between TR-α1 protein (c-ErbAα1 protein) and 43-kD c-ErbAα1 protein (T3-binding 43-kD mitochondrial protein) was detected during the development of the rat tongue muscle. Immunohistochemical examination using electron microscopy showed that TR-α1 was found in the mitochondria and nuclei in contrast to TR-β1 detected in rat tongue muscle. In mitochondrial fractions from rat tongue muscle, the expression of 43-kD c-ErbAα1 protein was increased dramatically at 15 and 21 days, and a similar tendency was seen in cytochrome c proteins using Western blot analysis. We presume that the 43-kD c-ErbAα1 protein plays a role in regulating mitochondrial RNA synthesis during the postnatal development of rat tongue. The mRNA and protein myosin heavy chain isoforms of muscle also had a different expression during development. The slow myosin isoform protein was not found from day 10 in contrast to fast myosin isoforms. It is likely that the expression of TR-α1 mRNA from the rat tongue muscle may be related to a specific phase in muscle phenotype during the development.

Distributions of calcitonin gene-related peptide and substance P in the human maxillary sinus of Japanese cadavers ☆

BACKGROUND Substance P (SP) and calcitonin gene-related peptide (CGRP) are released by the nociceptive sensory nerve and are involved in blood flow, pain and inflammation in the nasal mucosa. The purpose of this study was to assess the distribution of the SP and CGRP nerve fibres related to blood supply within human Schneiderian membrane of the maxillary sinus (MS). MATERIAL AND METHODS In this study, the MS from Japanese cadavers was examined by whole-mount immunohistochemistry. Human male cadavers (ranging in age from 80 to 90 years) were used in this study. RESULTS SP- and CGRP-positive fibres were found around large vessels of the medialis superior alveolar branches and also within the floor region of the MS. The floor region of the MS was composed of complex branches of these fibres. CONCLUSION Our results give useful information for surgical sinus floor elevation in this region of the MS. These anatomical features may assist in the execution of a successful surgical procedure.

Distribution of LYVE-1 and CD31 in postnatal rat masseter muscle.

During the development of blood vascular systems in the masseter muscle, one functional property of the blood supply via capillaries is altered by the change in feeding pattern from suckling to mastication. The lymphatic vessel hyaluronan receptor-1 (LYVE-1) is a marker of lymphatic endothelial cells. The PECAM (CD31) is also an important marker of vascular endothelial cells and lymphatic cells. The mechanisms by which circulating lymphatic endothelial cells from blood vessels in masseter muscle form a network of lymphatic capillaries and vessels functioning in jaw muscle movement remain unknown. In our results, LYVE-1- and CD31- positive reactions were located in almost identical regions at the stages examined using double immunofluorescence staining. However, the level of protein for LYVE-1 and CD31 differed between superficial and deep regions in postnatal rat masseter muscle using Western blotting analysis. The different distribution of LYVE-1 and CD31 antibody reactions was found in the deep region in contrast to that of the superficial area in 3-7-week-old rat masseter muscles. Concomitant with the increased level of protein for CD31 in the deep region, many small vessels branch in this region during development in rat masseter muscle. Therefore, different levels of protein and immunohistochemical reactions for CD31- and LYVE-1-positive cells may reflect alterations in the functional properties of the blood supply and collection via capillaries due to the changes in feeding pattern.

Tenomodulin regulated the compartments of embryonic and early postnatal mouse masseter muscle.

The masseter muscle (MM) is a complex tendinous laminar structure during development; however, the stage of the laminar structure formation is unknown. Tenomodulin (TeM) is a useful marker of tendons and has an anti-angiogenic cysteine-rich C-terminal domain. Therefore, we analyzed mRNA of TeM and angiogenesis markers (CD31 and vascular endothelial growth factor (VEGF)) and performed in situ hybridization for the TeM genes in MM from on embryonic day 12.5 (E12.5) to postnatal day 5 (P5). The TeM expression is at first detectable in the middle region of the mesenchymal connective tissue in the MM at E 12.5. The expression domains of the TeM during development typically include the middle region of the MM, particularly surrounding the vascular regions. The level of TeM mRNA in the MM increased from E12.5 to E17.5 and decreased after birth. In contrast, the levels of CD31 and VEGF mRNAs were almost constant from E12.5 to E18.5 and then low from birth onward. Therefore, the development of the laminar tendinous structure in the middle region between superficial and deeper regions of the MM first occurs during the process of tendon formation at embryonic day 12.5. In our study of MM development, the laminar structure regulating TeM also prevents vascular invasion during the formation of compartment of the MM. The tendon may relate to the components of muscle mass of MM.

Quantitative Analysis of Extracellular Matrix Proteins in Hypertrophic Layers of the Mandibular Condyle and Temporal Bone during Human Fetal Development

A computer image analysis of immunostained extracellular matrix (ECM) proteins (collagen types I, II, III and V, fibronectin and tenascin) in hypertrophic layers in the mandibular condyle and temporal bone of human fetuses, which ranged in gestational age from 12 to 32 weeks, was performed. The percentage of cells positive for proliferating cell nuclear antigen (PCNA) increased in almost the same manner in each region. The level of PCNA was markedly elevated at 16 weeks. The percentage of PCNA-positive cells was low in temporal bone and in the bone-forming layer of the mandibular condyle at 24 weeks. Specific concentration patterns of proteins in the ECM were found at each stage of development. The extent of accumulation of fibrillar collagen and of fibronectin differed, while those of other proteins in the ECM, such as tenascin, osteocalcin and osteonectin, were similar at the two sites.

The Endomysium of Human Embryonic Hyoid Muscle during Development

A computer analysis was performed on the endomysium of the hyoid muscle in human fetuses, which ranged in gestational week from 12 to 32 weeks. During development of the human hyoid muscle a level of intramuscular connective tissue as reticular fibers displays a specific change in morphology. We examined quantitates and distributions of endomysium in human hyoid muscles from 12 to 32 weeks. The endomysium of the hyoid muscles formed complex structures, and volume rates increased from 20 weeks gestation. On the other hand, the cross sectional area (CSA) of muscle fibers had almost the same profile. The reticular fibers increased during development from 16 to 28 weeks gestation.

CBCT imaging of the alveolar bone structure in maxilla of elderly donor cadavers and PCA analysis

There is an important bone matrix with remodelling between dentate and edentulous samples of the human maxilla for bone metabolism. Cone beam computed tomography (CBCT) is useful for structural analysis of bone. The objective of this study was to investigate morphological data of donor cadavers in detail using CBCT imaging and principal component analysis (PCA). We analysed 38 donor cadavers using a CBCT apparatus. The analytical results defined differences in skull measurement parameters and dentate and edentulous levels using PCA. We observed cortical bone, trabecular bone, and the distance from the bottom of the maxillary sinus to the oral mucosa at a right angle to the palatal plane of the first molar region between dentate and edentulous samples of the human maxilla using CBCT imaging. In the dentate sample of the maxilla, component 1 was defined by negative contributions from gender (-0.84) and age (-0.54) to positive contributions such as cortical bone structure (CBS, 0.68) and trabecular bone structure (TBS, 0.50). There was a difference in CBS between dentate and edentulous human maxilla samples. This study of CBCT data provides useful basal information for planning dental implant surgery using PCA.

A morphological study of the foramina of the mandible in the Japanese macaque by cone-beam computed tomography

The mandibular canal (MC) contains vessels and nerves in the mandible of the Japanese macaque (JM). The inferior alveolar nerves and vessels of the mandible insert from the mandibular foramen and then run through the MC, the mental foramen and spinal foramen to the outside of the mandible. However, the detailed morphological properties of multiple canals, such as the accessory canal (AC) of the mandible, are unknown in JMs. The purpose of this study was to describe the multiple canals of JMs and to determine the location and analyse the measurements of the JM mandible. In this study, we also showed the course of the lingual foramen in 17 JMs (male: n = 8; female: n = 9) using cone-beam computed tomography (CBCT). In our results, we classified multiple mental foramina and multiple lingual foramina found on the mandibular body at the premolar or molar region. However, there was no significance between the formation of mandibular properties and the lingual foramen. These multiple foramina contain nerves and blood vessels have a few branched canals; these branches run downward and connect with the inferior mandibular nerve and artery. These morphological features may provide useful information about surgical treatment of the alveolus in a human model.