Keizo Furukawa

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD‐C6, from Runx2‐deficient mouse embryos. RD‐C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) or transduction of BMP‐2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD‐C6 cells. BMP‐2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD‐C6 cells with or without rhBMP‐2 treatment demonstrated that BMP‐2 upregulated 66 genes including 13 transcription‐related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD‐C6 cells, we transplanted RD‐C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP‐2. RD‐C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP‐2 induces Runx2‐deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2‐independent pathway, but it failed to induce these cells to differentiate into bone‐forming osteoblasts and mature chondrocytes. J. Cell. Physiol. 211: 728–735, 2007.

Primary human bone marrow adipocytes support TNF-α-induced osteoclast differentiation and function through RANKL expression

PURPOSE In previous reports, it was demonstrated that bone marrow adipocytes were related to steroid osteoporosis through osteoclastogenesis induced by Receptor Activator of Nuclear factor κ-B Ligand (RANKL) expression. The purpose of this study was to evaluate the effect of Tumor necrosis factor-alpha (TNF-α) on RANKL expression in bone marrow adipocytes, and osteoclast differentiation supported by human bone marrow adipocytes. METHODS RANKL, osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) mRNA expression in bone marrow adipocytes and their regulation by TNF-α treatment were measured by real-time RT-PCR. Co-cultures of bone marrow adipocytes and osteoclast precursors were performed with or without TNF-α, and osteoclast differentiation was evaluated morphologically and functionally. RESULTS RANKL expression and an increase in the RANKL/OPG ratio in bone marrow adipocytes were stimulated by TNF-α treatment. In co-culture of bone marrow adipocytes and osteoclast precursors with TNF-α, the number of TRAP-positive multinuclear cells and resorption cavity formations of calcium phosphate film were increased. Osteoclast differentiation was suppressed by anti-RANKL antibody treatment. In co-culture with non-cell-contact conditions, no TRAP-positive cells or resorption cavity formations were observed. CONCLUSIONS TNF-α increased RANKL expression in primary human bone marrow adipocytes. TNF-α induced the ability of bone marrow adipocytes to promote osteoclast differentiation and activity in a manner directly related to RANKL expression.

Pulmonary diffusing capacity, serum angiotensin-converting enzyme activity and the angiotensin-converting enzyme gene in Japanese non-insulin-dependent diabetes mellitus patients

We investigated the independent change in pulmonary diffusing capacity (DLCO) as one manifestation of pulmonary microangiopathy and to analyze the correlation between DLCO and serum ACE. We also examined the association between DLCO and the ACE genes. We examined pulmonary functions, especially %DLCO/VA (DLCO corrected by alveolar volume, percent predicted) in 54 NIDDM patients and 34 age-matched normal control subjects. Subjects were subdivided according to the degree of retinopathy. Serum ACE level was assayed by a colorimetric method in 54 patients and an insertion/deletion polymorphism in the ACE gene was amplified using the polymerase chain reaction in 52 of the 54 patients. There was a significant reduction of %DLCO/VA (percent predicted P < 0.05) in diabetic patients. In the proliferative retinopathy (PDR) group. %DLCO/VA was significantly (P < 0.05) lower than in the no diabetic retinopathy (NDR) and simple diabetic retinopathy (SDR) groups. Although the levels of serum ACE were within normal ranges in all diabetic groups, %DLCO/VA was negatively correlated with serum ACE values (r = 0.49, P < 0.0002, y = -1.4x + 109.3). Differences among DD, ID and II type of the ACE gene, with respect to the incidence of abnormal values of each clinical parameter, were not significant. DLCO was significantly reduced in patients with PDR and the serum ACE was significantly related to impaired DLCO. Our study suggests the existence of microangiopathic involvement of pulmonary vessels in NIDDM patients.

Functional domains of paired-like homeoprotein Cart1 and the relationship between dimerization and transcription activity.

Background: Cart1 encodes the paired‐like homeodomain in the central portion of the gene, and plays a crucial role in the developmental lineage of bone and cartilage, especially in head formation. However, its transactivation mechanism is still poorly understood, including the target gene. Here, we report biochemical dissections of Cart1 functional domains and a relationship between dimerization and transcription activity.

P300/CBP acts as a coactivator to cartilage homeoprotein-1 (Cart1), paired-like homeoprotein, through acetylation of the conserved lysine residue adjacent to the homeodomain.

The paired‐like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation.

Serial MRI findings in a relapsing-remitting form of neuro-Behcet's disease A case report

We describe a case of neuro-Behcets disease (NBD) characterized by recurrent attacks of neurologic deficit. T2-weighted images showed a high signal intensity lesion with extensive edema in the right thalamolenticular region, midbrain, and pons as well as the cerebral white matter. After a relapse of the disease, MRI demonstrated a high signal intensity in the left thalamus, internal capsule, and midbrain. These MRI abnormalities showed marked resolution with steroid treatment. We observed sequential MRI findings in a patient with a relapsing-remitting form of NBD who had parenchymal CNS involvement, and we examined the correlation among the MRI findings and clinical features during the clinical course.

Acute Cerebellar Ataxia with an MRI Abnormality: A Sequential Imaging Study

Introduction Acute cerebellar ataxia (ACA), or acute cerebellitis, is a benign disease occurring with or after a nonspecific viral infection and generally has a good prognosis [1, 2]. ACA occurs most commonly in young children, and in the majority of cases, cerebrospinal fluid (CSF) and neuroimaging analysis are normal. Serial MRI findings have been reported in single-case studies in children [3, 4]. We report a sequential study of MRI in adult with ACA.

One Example of Osteochondoroma Generating in the Cervical Vertebrae with Spinal Cord Symptoms

［はじめに］頚椎に発生し脊髄症状を呈した骨軟骨腫の一例を経験したので報告する．［症例］13歳女性．多発性骨軟骨腫の既往があり3度の摘出術（右前腕，右手，両膝）を施行されている．入院2ヶ月前より右上腕から前腕にかけてしびれ，脱力感が出現．入院1ヶ月より箸の保持困難を自覚．歩行時につまずくようになり近医受診．頚部脊柱管を狭窄する椎弓由来の腫瘍を指摘され当科入院となった．腫瘍はC5椎弓より発生，脊柱管内に達し硬膜を圧迫していた為，これによる脊髄症状と診断．腫瘍摘出術を行い，症状の著明な改善を得た．病理診断は骨軟骨腫であった．［考察］骨軟骨腫は長管骨骨幹端部に好発する良性骨腫瘍としてよく知られているが，脊椎発生はまれである．今回我々は，頚椎に発生し脊髄症状を呈した骨軟骨腫の1例を経験したので若干の文献的考察を加えて報告する．

A Case of Multiple Bone Tuberculosis with Bilateral Lower-Leg Paralysis

症例は29歳男性．14ヶ月前より頸背部痛，腰痛が出現．近医外来にて加療したが症状軽快せず，総合病院入院．頸椎から仙椎にかけての多発する溶骨性変化，Th1/2，3/4，7の硬膜外膿瘍および腸骨，肋骨に溶骨性変化を認めた．骨生検にて乾酪性肉芽腫，ラングハンス巨細胞，抗酸菌を認めたことから多発性骨結核と診断され，抗結核薬の内服を開始したが，内服加療開始後よりTh4以下のしびれが出現し，両下肢不全麻痺を呈した．約6ヶ月間の抗結核薬内服と臥位安静加療を行ったが麻痺の十分な改善が得られなかったため当科入院．Th3，4に認めた膿瘍が硬膜管を圧排しており，後方よりインストゥルメンテーションを使用した7椎間におよぶ脊椎固定術，骨移植術を施行し，症状の著明な改善を得た．術後4ヶ月で再発兆候はなく，独歩も可能となった．今回我々は，病変が極めて広範囲である稀な骨結核の1例を経験したので若干の文献的考察を加えて報告する．