Keisuke Kosaki

Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA Stabilization

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase-polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from approximately 23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.

Osteogenesis imperfecta type V: Clinical and radiographic manifestations in mutation confirmed patients

Osteogenesis imperfecta (OI) type V is a specific OI phenotype with interosseous membrane calcification of the forearm and hyperplastic callus formation as typical features. The causative gene mutation for OI type V has been recently discovered. The purpose of this report is to review the clinical and radiographic characteristics of mutation confirmed OI type V in detail. Sixteen (nine familial and seven sporadic) patients were enrolled in the study. Blue sclera and dentinogenesis imperfecta were not evident in any patient. However, hypodontia in the permanent teeth, ectopic eruption, and short roots in molars were additionally observed in 11 patients. Of the radiographic abnormalities, cortical thickening and bony excrescence of interosseous margin of the ulna was the most common finding, followed by overgrowth of the olecranon and/or coronoid process of the ulna. Slender ribs and sloping of the posterior ribs with or without fractures were also a consistent finding. Hyperplastic callus was detected in 75% of patients and was commonly encountered at the femur. Heterotopic ossification in the muscles and tendon insertion sites were noted in four patients, which resulted in bony ankylosis or contracture of joints. The current study confirms common clinical and radiographic findings of OI type V and reports additional phenotypic information. These observations provide clues to recognize OI type V more promptly and guide to direct targeted molecular study.

Pathological characterization of pachydermia in pachydermoperiostosis

Pachydermoperiostosis is a rare hereditary disease, which presents with the cutaneous manifestations of pachydermia and cutis verticis gyrata. Histological findings in pachydermia frequently include dermal edema, mucin deposition, elastic fiber degeneration, dermal fibrosis and adnexal hyperplasia. However, the severity of these findings varies between clinical reports, and a systematic multiple‐case clinicopathological correlative analysis has not been performed to date. In the present study, we reviewed the skin biopsy specimens obtained from the pachydermia of six pachydermoperiostosis patients. The severity of the characteristic histological features was semiquantitatively evaluated and correlated with the grade of pachydermia. Dermal edema, mucin deposition and elastic fiber degeneration were observed in all cases. Patients with severe pachydermia had sebaceous gland hyperplasia and fibrosis. These results suggest that the triad of mucin deposition, dermal edema and elastic fiber degeneration are found from very early stage pachydermia, and could be considered diagnostic findings. To ensure an earlier diagnosis of pachydermoperiostosis, a biopsy should be taken when a patient has grade 1 pachydermia to determine the presence of this histological triad.

Spondylar dysplasia in type X collagenopathy

Background. The type X collagen gene (COL10A1) is currently known as the disease-causing gene of metaphyseal dysplasia type Schmid (MDS), whereas a mutation of COL10A1 has been reported to cosegregate with a disease phenotype of mild spondylometaphyseal dysplasia (SMD) in a Japanese family.¶Objective. To elucidate whether or not spondylar dysplasia is common in patients with mutations of COL10A1.¶Materials and methods. We re-evaluated the radiological manifestations in six patients with mutations of COL10A1, who had been previously reported as having MDS.¶Results. Two of six patients showed mild platyspondyly in infancy and early childhood. In both patients, the spondylar dysplasia tended to normalize with age, but mild alterations of the vertebral bodies persisted, even into late childhood. The other radiological manifestations of both patients were identical to those of MDS.¶Conclusion. Our observation suggests that mild spondylar dysplasia may not be uncommon in MDS.

SOX9 dimerization domain mutation mimicking type 2 collagen disorder phenotype

The classification of bone dysplasia has relied on a clinical/radiographic interpretation and the identification of specific genetic alterations. The clinical presentation of the SOX9 mutation and type 2 collagen disorders overlap with the Pierre-Robin sequence and talipes equinovarus, but the former is often accompanied by the bent long bones. In its milder form, the SOX9 mutation is not necessarily associated with the bent long bones. Here, we report a patient with the Pierre-Robin sequence and talipes equinovarus who did not exhibit either bent long bones or scapular hypoplasia; thus, this patient was instead classified as having a type 2 collagen disorder. Despite this phenotypic presentation, the proposita was found to have a de novo SOX9 mutation. The peculiar location of the mutation within the dimerization domain might account for the relatively mild phenotypic effect of the SOX9 mutation to a degree that is compatible with a clinical diagnosis of type 2 collagen disorder, except for a developmental delay. We concluded that mutations in SOX9 can mimic a type 2 collagen disorder-like phenotype.

Two distinctive mechanisms leading to disruption of the SHOX transcription unit in a single family

Kosuke Izumi, Masao Nakano, Keisuke Kosaki, Rika Kosaki, Noboru Hosogai, Hideo Matsumoto, Tomonobu Hasegawa, Takao Takahashi, and Kenjiro Kosaki* Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan Department of Obstetrics and Gynecology, Saiseikai Kanagawaken Hospital, Yokohama, Japan Department of Orthopedic Surgery, Tokyo Metropolitan Kita Medical & Rehabilitaion Center for the Handicapped, Tokyo, Japan Department of Clinical Genetics and Molecular Medicine, National Center for Child Health and Development, Tokyo, Japan Research and Development Division, Mitsubishi Chemical Medicine Corporation, Tokyo, Japan

Measurements of the microcirculation in a rather deep portion in the parenchymal organ by using laser vital-microscope

To observe the microcirculation in a rather deep portionin the parenchymal organ, we have designed a new in vivo microscope by dual slit lazer beams illumination, in which the fluorescent lighte mitted from the tracer by these beams in the crossing area alone can be picked up. This microscope was appliedto observe the tomographic image of microvasculature and to analyze the macromolecular permeability of single capillary in the anesthetized rabbit tenuisimus muscle. The obtained images make a high vertical zone selectivity of tomographic image from muscle surface to 200 ,ƒÊm in depth. Furthermore, the quick macromolecular leakage can be observed in the single venular capillary region. It was concluded that present method is useful to quantify the microvasculature and capillary permeabilityunder in vivo study.