Yoshiki Hira

Anti-colitis and -adhesion effects of daikenchuto via endogenous adrenomedullin enhancement in Crohn's disease mouse model

BACKGROUND AND AIMS Adrenomedullin (ADM) is a member of the calcitonin family of regulatory peptides, and is reported to have anti-inflammatory effects in animal models of Crohns disease (CD). We investigated the therapeutic effects of daikenchuto (DKT), an extracted Japanese herbal medicine, on the regulation of endogenous ADM in the gastrointestinal tract in a CD mouse model. METHODS Colitis was induced in mice by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS); afterwards, DKT was given orally. Colonic damage was assessed on day 3 by macroscopic and microscopic observation, enzyme immunoassays of proinflammatory cytokines in the colonic mucosa, and serum amyloid A (SAA), a hepatic acute-phase protein. To determine the involvement of ADM, an ADM antagonist was instilled intrarectally before DKT administration. The effect of DKT on ADM production by intestinal epithelial cells was evaluated by enzyme immunoassay and real-time PCR. RESULTS DKT significantly attenuated mucosal damage and colonic inflammatory adhesions, and inhibited elevations of SAA in plasma and the proinflammatory cytokines TNFα and IFNγ in the colon. Small and large intestinal epithelial cells produced higher levels of ADM after DKT stimulation. A DKT-treated IEC-6 cell line also showed enhanced ADM production at protein and mRNA levels. Abolition of this effect by pretreatment with an ADM antagonist shows that DKT appears to exert its anti-colitis effect via up-regulation of endogenous ADM in the intestinal tract. CONCLUSION DKT exerts beneficial effects in a CD mouse model through endogenous release and production of ADM. Endogenous ADM may be a therapeutic target for CD.

Daikenchuto (TU-100) ameliorates colon microvascular dysfunction via endogenous adrenomedullin in Crohn’s disease rat model

BackgroundDaikenchuto (TU-100), a traditional Japanese medicine, has been reported to up-regulate the adrenomedullin (ADM)/calcitonin gene-related peptide (CGRP) system, which is involved in intestinal vasodilatation. The microvascular dysfunction of the intestine in Crohn’s disease (CD), due to down-regulation of the ADM/CGRP system, is etiologically related to the recurrence of CD. Therefore, we investigated the vasodilatory effect of TU-100 in a CD rat model.MethodsColitis was induced by the rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. Laser Doppler blood flowmetry was used to measure colonic blood flow. ADM, CGRP, and their receptors in the ischemic colon were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme immunoassays. Additionally, we determined whether the intestinal epithelial cell line IEC-6 released ADM in response to TU-100.ResultsTU-100 increased blood flow in ischemic segments of the colon but not in hyperemic segments. Pretreatment with an antibody to ADM abolished the vasodilatory effect of TU-100. CGRP levels and βCGRP mRNA expression were decreased in the ischemic colon, while protein and mRNA levels of ADM were unchanged. Hydroxy α-sanshool, the main constituent of TU-100, was the most active component in improving blood flow. Additionally, both TU-100 and hydroxy α-sanshool enhanced the release of ADM from IEC-6 cells.ConclusionsIn the ischemic colon, endogenous βCGRP, but not ADM, was decreased. Thus, it was concluded that TU-100 ameliorated microvascular dysfunction by the up-regulation of endogenous ADM in the CD rat model. TU-100 may be a possible therapeutic agent for gastrointestinal ischemia-related diseases including CD.

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.

Peptidergic peripheral nervous systems in the mammalian pineal gland.

The distribution and density of tyrosine hydroxylase (TH) and neuropeptide Y (NPY)‐immunoreactive, sympathetic fibers and calcitonin gene‐related peptide (CGRP)‐, substance P (SP)‐, and vasoactive intestinal polypeptide (VIP)‐immunoreactive, non‐sympathetic fibers in the pineal gland, the effects of superior cervical ganglionectomy (SCGX) on these fibers, and the location of their terminals in the pineal gland were compared between rodents and non‐rodents. A dense network of TH/NPY‐positive fibers is present all over the pineal gland. A less dense network of CGRP/SP‐ or VIP‐positive fibers occurs in the whole pineal gland of non‐rodents, but these fibers are usually confined to the superficial pineal gland in rodents. After SCGX, some TH/NPY‐fibers remain only in the deep pineal gland in rodents, whereas considerable numbers of these fibers persist throughout the gland in non‐rodents. Thus, the remaining fibers, probably originating from the brain, may be more numerous in non‐rodents. Since CGRP‐, SP‐ or VIP‐immunoreactive fibers in the pineal capsule can be traced to those in the gland, and since these fibers are ensheathed by Schwann cells, it is concluded that these fibers belong to the peripheral nervous system. However, the existence of SP‐positive central fibers cannot be denied in some species. In the superficial pineal gland of rodents, sympathetic terminals are mostly localized in perivascular spaces, whereas the parenchymal innervation by sympathetic fibers in the pineal gland is more dense in non‐rodents than in rodents. Synapses between sympathetic nerve terminals and pinealocytes occur occasionally in non‐rodents, but only rarely in the superficial pineal gland of rodents. The occurrence of the synapses may depend on the frequency of intraparenchymal sympathetic terminals. Microsc. Res. Tech. 46:265–280, 1999.

Comparisons of sizes of pinealocyte nuclei and pinealocytes in young and adult Chinese hamsters (Cricetulus griseus) under different photoperiod conditions

Semiquantitative electron microscopic observations on pinealocytes of the Chinese hamster (Cricetulus griseus) were made to compare the sizes of pinealocyte nuclei and pinealocytes as a whole, their nuclei and cytoplasm together, over a 24‐hr period, between young animals (60‐70 days old) and adult animals (120–130 days old) under LD 12: 12 and between adults under LD 12: 12 and LD 14: 10. Under LD 12: 12, similar 24‐hr rhythms exist in the nuclear area and the area of pinealocytes of young animals, whereas in adults these values exhibit no significant 24‐hr rhythm. In adults under LD 14: 10, there is no significant 24‐hr rhythm in the nuclear area, but the area of pinealocytes shows a statistically significant 24‐hr rhythm. Thus, in the Chinese hamster, the relationships between nuclear area and area of pinealocytes with time of day vary, depending on the age of animals as well as different photoperiodic conditions, although they differ only slightly.

A unique ball-shaped Golgi apparatus in the rat pituitary gonadotrope: its functional implications in relation to the arrangement of the microtubule network.

In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes.

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for γ‐aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar‐King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA‐immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA‐positive fibers, and some of them were followed to the pineal gland. GABA‐positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA‐immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal. J. Comp. Neurol. 430:72–84, 2001.

Nerve growth factor stimulates regeneration of perivascular nerve, and induces the maturation of microvessels around the injured artery.

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay. The vasa vasorum around arterial walls was induced to grow using wire-mediated mouse femoral arterial injury. When collagen-coated tube (CCT) was placed beside the injured artery for 7-14 days, microvessels grew two-dimensionally in a thin layer on the CCT (CCT-membrane) in accordance with the development of the vasa vasorum. The perivascular nerve was found at not only arterioles but also capillaries in the CCT-membrane. Biodegradable hydrogels containing VEGF and NGF were applied around the injured artery/CCT. VEGF significantly increased the total length and instability of microvessels within the CCT-membrane. In contrast, NGF induced regeneration of the peripheral nerve around the microvessels and induced the maturation and stabilization of microvessels. In an ex vivo nerve-free angiogenesis assay, although NGF potentially stimulated vascular sprouting from aorta tissues, no effects of NGF on vascular maturation were observed. These data demonstrated that NGF had potent angiogenic effects on the microvessels around the injured artery, and especially induced the maturation/stabilization of microvessels in accordance with the regeneration of perivascular nerves.

Light and electron microscopic immunocytochemical study on the innervation of the pineal gland of the tree shrew (Tupaia glis), with special reference to peptidergic synaptic junctions with pinealocytes

Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the tree shrew (Tupaia glis) were made. Neuropeptide Y (NPY)-immunoreactive fibers, which were abundantly distributed in the gland, disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympathetic fibers. By contrast, tyrosine hydroxylase (TH)-immunoreactive fibers, which were less numerous than NPY-fibers, remained in considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources. Bundles of substance P (SP)- or calcitonin gene-related peptide (CGRP)-immunoreactive fibers, entering the gland at its distal end, were left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers were myelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- and SP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsed frequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directly control the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered the gland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to contain also unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained in ganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals of commissural fibers.

Immortalized multipotent pericytes derived from the vasa vasorum in the injured vasculature. A cellular tool for studies of vascular remodeling and regeneration.

Adventitial microvessels, vasa vasorum in the vessel walls, have an active role in the vascular remodeling, although its mechanisms are still unclear. It has been reported that microvascular pericytes (PCs) possess mesenchymal plasticity. Therefore, microvessels would serve as a systemic reservoir of stem cells and contribute to the tissues remodeling. However, most aspects of the biology of multipotent PCs (mPCs), in particular of pathological microvessels are still obscure because of the lack of appropriate methods to detect and isolate these cells. In order to examine the characteristics of mPCs, we established immortalized cells residing in adventitial capillary growing at the injured vascular walls. We recently developed in vivo angiogenesis to observe adventitial microvessels using collagen-coated tube (CCT), which also can be used as an adventitial microvessel-rich tissue. By using the CCT, CD146- or NG2-positive cells were isolated from the adventitial microvessels in the injured arteries of mice harboring a temperature-sensitive SV40 T-antigen gene. Several capillary-derived endothelial cells (cECs) and PCs (cPCs) cell lines were established. cECs and cPCs maintain a number of key endothelial and PC features. Co-incubation of cPCs with cECs formed capillary-like structure in Matrigel. Three out of six cPC lines, termed capillary mPCs demonstrated both mesenchymal stem cell- and neuronal stem cell-like phenotypes, differentiating effectively into adipocytes, osteoblasts, as well as schwann cells. mPCs differentiated to ECs and PCs, and formed capillary-like structure on their own. Transplanted DsRed-expressing mPCs were resident in the capillary and muscle fibers and promoted angiogenesis and myogenesis in damaged skeletal muscle. Adventitial mPCs possess transdifferentiation potential with unique phenotypes, including the reconstitution of capillary-like structures. Their phenotype would contribute to the pathological angiogenesis associated with vascular remodeling. These cell lines also provide a reproducible cellular tool for high-throughput studies on angiogenesis, vascular remodeling, and regeneration as well.