Shinji Imai

Heparin-binding proteins HB-GAM (pleiotrophin) and amphoterin in the regulation of cell motility ☆

Fractionation of proteins from perinatal rat brain was monitored using a neurite outgrowth assay. Two neurite-promoting proteins, HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin) and amphoterin, were isolated, cloned and produced by baculovirus expression for structural and functional studies. HB-GAM is highly expressed in embryonic and early post-natal fiber pathways of the nervous system, and it enhances axonal growth/guidance by binding to N-syndecan (syndecan-3) at the neuron surface. N-syndecan in turn communicates with the cytoskeleton through the cortactin/src-kinase pathway to enhance neurite extension. In addition to N-syndecan, the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) is implicated in the receptor mechanism of HB-GAM. HB-GAM is also prominently expressed in developing and regenerating bone as a matrix-bound cue for migration of osteoblasts/osteoblast precursors to the site of bone deposition. HB-GAM is suggested to regulate motility in osteoblasts through a similar mechanism as in neurons. Structural studies using heteronuclear NMR reveal two similar protein domains in HB-GAM, both consisting of three anti-parallel beta-strands. Search of sequence databases shows that the beta structures of HB-GAM and of the similar domains of MK (midkine) correspond to the thrombospondin type I (TSR) sequence motif. We suggest that the TSR sequence motif, found in several neurite outgrowth-promoting and other cell surface and matrix-binding proteins, defines a beta structure similar to those found in HB-GAM and MK. In general, amphoterin is highly expressed in immature and transformed cells. We suggest a model, according to which amphoterin is an autocrine/paracrine regulator of invasive migration. Amphoterin binds to RAGE (receptor of advanced glycation end products), an immunoglubulin superfamily member related to N-CAM (neural cell adhesion molecule), that communicates with the GTPases Cdc42 and Rac to regulate cell motility. In addition, ligation of RAGE by amphoterin activates NF-kappaB to regulate transcription.

Neuropeptides and the puzzle of bone remodeling: State of the art

Bone metabolism is dependent on cells of the osteoblast and osteoclast lineage. These cells play a major role in the synthesis and degradation of osteoid and in its mineralization and demineralization. Bone cells are under the influence of various systemic and local auto/paracrine factors. One further regulatory element that can play both a sensory/ afferent and a regulatory/efferent role, consists of neuropeptide-containing nerves. In particular, the calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) have been implicated; their distribution in bone and their molecular biology are discussed in some detail. Bone neuropeptides can function as direct bone cell regulators, with additional amplifying indirect effects mediated by vascular endothelial cells, monocyte/macrophages and mast cells and their mediators. Recent experimental and clinical work has implicated bone nerves in processes varying from normal remodelling to fracture healing and non-union. Apart from systemic endocrine influences on bone stock and osteoclast/ osteoblast coupling (activation-resorption-formation cycle) mediated by local auto/paracrine factors, bone nerves/neuropeptides may explain why various inputs/outputs are transformed in a meaningful way to altered mass and quality of bone.

Cytokines in aseptic loosening of total hip replacement

It has been more than 30 years since Charnley, credited with being the father of present-day total hip replacement (THR), in 1961 first reported his clinical experience with THR at his clinic in Wrightington, England? THR has now become well accepted throughout the world as a means of treating patients with end-stage degenerative/inflammatory hip disorders such as rheumatoid arthritis, osteoarthritis, severe traumatic arthritis, and consequences of

Efferent targets of osseous CGRP-immunoreactive nerve fiber before and after bone destruction in adjuvant arthritic rat : An ultramorphological study on their terminal-target relations

We report the ultramorphological characterization of the terminal‐target relation of sensory peptidergic nerve fibers in healthy and diseased osseous tissues. Bone tissue sections were immunoelectronmicroscopically investigated for calcitonin gene‐related peptide (CGRP), a neuropeptide widely distributed in sensory peptidergic fibers. Ultramorphological relation of the osseous CGRP‐immunoreactive (ir) nerve terminals and their target cells was comparatively analyzed using healthy, arthritic, and postarthritic bone specimens from control and adjuvant‐induced arthritic rats. Terminal‐like profiles of the osseous CGRP‐ir axons were evidenced in direct contact with the metaphyseal osteoblasts and osteoclasts of the control animals. Terminal‐like profiles were also noted in the vicinity of the periosteal lining cells. Nonterminal‐like profiles did not make intimate spatial relation to the cells/structures surrounding the nerve. Osseous CGRP‐ir terminals and axons, which are either uncovered or thinly ensheathed by the supportive tissues, were extensively degenerated in adjuvant‐induced infiltration, whereas larger fibers were relatively resistant. Numerous CGRP‐ir axons with distinctive features reinnervated the postarthritic, ossifying periosteum. CGRP‐ir axons appeared to reinnervate the eroded surface of metaphyseal bone and cartilage as early as the recruited osteoblasts resume osteogenesis in the postarthritic metaphysis. The observed terminal‐target relations in the healthy and diseased bone tissues give an ultramorphological basis for the putative trophic, modulatory actions of CGRP innervation of the bone cells.

High levels of expression of collagenase-3 (MMP-13) in pathological conditions associated with a foreign-body reaction

A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA). The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3. Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3. We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR.

Tyrosine hydroxylase-immunoreactive nerve fibres in rat posterior longitudinal ligament

The nerve supply to the posterior longitudinal ligament (PLL) of the lumbar vertebrae has been the focus of considerable interest to gain insight into the pathogenesis of low back pain. The present study aimed to characterize the sympathetic fibres in the PLL by immuno-electronmicroscopy for tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. The posterior central branches of the segmental lumbar arteries received numerous communicating fibres from the sinuvertebral nerve (SVN), but only shortly after their entrance to the spinal canal. The non-vessel-associated branches of the SVN formed transverse bundles, which met fibres from the opposite side in a plexus-like mid-sagittal network. As these fibres approached the midline, they gradually lost their Schwann cell cover. The free and naked fibres contained numerous terminal-like varicosities. The TH-ir and 6-hydroxydopamine (6-OH-DA) sensitive fibres were intermingled with non-TH-ir fibres. The TH-ir sympathetic fibres had no obvious target structures except for the numerous, intermingled, closely related and communicating terminal-like axons in the mid-sagittal network in contact with non-TH-ir fibres. This may represent a neuroanatomical equivalent reflecting modulatory functions, which could participate in the pathogenesis of low back pain.

Expression of tenascin-C in aseptic loosening of total hip replacement

OBJECTIVE To assess if the bonding interlayer between the implant and bone in aseptic loosening of total hip replacement (THR) is qualitatively deteriorated by excessive accumulation of anti-adhesive glycoprotein, tenascin-C. METHODS Alkaline phosphatase-anti-alkaline phosphatase (APAAP) method was used for immunohistochemical staining of tenascin-C in interface tissue and control synovial tissue. RESULTS Tenascin-C was found to be a major component of the extracellular matrix at a hitherto unrecognised site, namely the synovial membrane-like interface tissue between implant and bone in aseptic loosening of THR. The overall tenascin-C staining (median score 4.0) was greatly increased in aseptic loosening compared with synovial membrane (median score 2.0; p<0.001) and fibrous capsule (median score 2.0; p<0.001) from primary THR operations. Topological analysis disclosed that tenascin-C was also found at the critical implant-interface and interface-bone surfaces. CONCLUSION Local tenascin-C expression is increased as a result of a chronic foreign body type reaction associated with excessive cytokine production and tissue injury mediated by microtrauma and neutral endoproteinases. This qualitative and topological deterioration of the bonding interlayer by an increase of anti-adhesive tenascin-C expression may inadvertantly contribute to loosening.