Toshikazu Kubo

A functional polymorphism in the 5′ UTR of GDF5 is associated with susceptibility to osteoarthritis

Osteoarthritis (MIM 165720), characterized by degeneration of articular cartilage, is the most common form of human arthritis and a major concern for aging societies worldwide. Epidemiological and genetic studies have shown that osteoarthritis is a polygenic disease. Here, we report that the gene encoding growth differentiation factor 5 (GDF5) is associated with osteoarthritis in Asian populations. A SNP in the 5′ UTR of GDF5 (+104T/C; rs143383) showed significant association (P = 1.8 × 10−13) with hip osteoarthritis in two independent Japanese populations. This association was replicated for knee osteoarthritis in Japanese (P = 0.0021) and Han Chinese (P = 0.00028) populations. This SNP, located in the GDF5 core promoter, exerts allelic differences on transcriptional activity in chondrogenic cells, with the susceptibility allele showing reduced activity. Our findings implicate GDF5 as a susceptibility gene for osteoarthritis and suggest that decreased GDF5 expression is involved in the pathogenesis of osteoarthritis.

Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture.

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs...

Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.

Circulation‐derived cells play a crucial role in the healing processes of tissue. In early phases of tendon healing processes, circulation‐derived cells temporarily exist in the wounded area to initiate the healing process and decrease in number with time. We assumed that a delay of time‐dependent decrease in circulation‐derived cells could improve the healing of tendons. In this study, we injected platelet‐rich plasma (PRP) containing various kinds of growth factors into the wounded area of the patellar tendon, and compared the effects on activation of circulation‐derived cells and enhancement of tendon healing with a control group (no PRP injection). To follow the circulation‐derived cells, we used a green fluorescent protein (GFP) chimeric rat expressing GFP in the circulating cells and bone marrow cells. In the PRP group, the numbers of GFP‐positive cells and heat‐shock protein (HSP47; collagen‐specific molecular chaperone)‐positive cells were significantly higher than in the control group at 3 and 7 days after injury. At the same time, the immunoreactivity for types I and III collagen was higher in the PRP group than in the control group at early phase of tendon healing. These findings suggest that locally injected PRP is useful as an activator of circulation‐derived cells for enhancement of the initial tendon healing process. J. Cell. Physiol. 215: 837–845, 2008.

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection.

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.

A functional SNP in CILP , encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T → C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-β1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-β1–mediated induction of cartilage matrix genes through direct interaction with TGF-β1 and inhibition of TGF-β1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-β1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-β signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-β.

Initial MRI findings of non-traumatic osteonecrosis of the femoral head in renal allograft recipients

Fifty-one renal allograft recipients (15-62 years old, mean: 37 years) were monitored for 2.5-6.5 years (average: 4.3 years) after surgery by using magnetic resonance imaging (MRI) to find (i) initial signs of osteonecrosis of the femoral head (ONF), (ii) the presence of bone marrow edema as an initial sign of ONF, (iii) any changes of MRI patterns, and (iv) the relationship between these MRI findings and prognosis. MRI was performed preoperatively (baseline), and whenever possible during the 6-9th week, 12-16th week, 12th month, and yearly thereafter. T1- and T2-weighted images were obtained by using a spin echo technique. Abnormalities were first detected on MRI of 23 femoral heads in 13 patients between 6 weeks and 12 months. All lesions first showed a low intensity band on T1-weighted images and a high intensity band on T2-weighted images. No symptoms or diffuse patterns, such as bone marrow edema, preceded the appearance of the band pattern. After the 12th month, no new abnormal findings on MRI were detected. The lesions were classified into Type A, B, or C, according to the location. 12 of the 16 Type C femoral head lesions, which extend beyond the medial two thirds of the weight-bearing portion of the acetabulum, became symptomatic 7-14 months after transplantation and then progressed to collapse. Bone marrow edema appeared with radiological collapse and symptoms. With the exception of five lesions in three patients who failed to be MR imaged until 12 months postoperatively, all lesions were first detected on MRI within 16 weeks after transplantation. We therefore postulate that the ischemic event that causes ONF will have occurred within 12 weeks after transplantation, considering the time lag of reparative reaction to the dead bone.

Effect of acetabular cup position and orientation in cemented total hip arthroplasty.

Long-term clinical results of total hip arthroplasty for patients with developmental acetabular dysplasia of the hip have been reported, but placement of the femoral head center or cup orientation remains controversial, especially with a severe anterolateral shallow acetabulum or dislocated femoral head. Results of 41 Müller and 34 Harris Design 2 cemented total hip arthroplasties were evaluated for developmental dysplasia of the hip. The femoral head center and acetabular cup inclination angle were measured from the interteardrop line. Linear wear and wear direction were measured using the Livermore technique. The best position of the femoral head center was less than 35 mm vertically from the interteardrop line and 25 mm laterally from the teardrop. Femoral head center analysis showed that hips with the cup in a lateral and superior cup position all were revised, but a superior and medial position combined with a cup inclination angle less than 40° did not require revision. Hips with a cup inclination angle more than 45° had superior and lateral penetration patterns of the polyethylene. However, hips with an inclination angle less than 35° and medial placement had medial head penetration patterns. With these all-polyethylene monolithic cemented cups, regardless of the femoral head diameter or cup thickness, better long-term results occurred with a cup inclination angle of 40° or less and medial position of the cup.

Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

A Functional Polymorphism in COL11A1, Which Encodes the α1 Chain of Type XI Collagen, Is Associated with Susceptibility to Lumbar Disc Herniation

Lumbar disc herniation (LDH), degeneration and herniation of the nucleus pulposus of the intervertebral disc (IVD) of the lumbar spine, is one of the most common musculoskeletal diseases. Its etiology and pathogenesis, however, remain unclear. Type XI collagen is important for cartilage collagen formation and for organization of the extracellular matrix. We identified an association between one of the type XI collagen genes, COL11A1, and LDH in Japanese populations. COL11A1, which encodes the alpha 1 chain of type XI collagen, was highly expressed in IVD, but its expression was decreased in the IVD of patients with LDH. The expression level was inversely correlated with the severity of disc degeneration. A single-nucleotide polymorphism (c.4603C-->T [rs1676486]) had the most significant association with LDH (P=3.3 x 10(-6)), and the transcript containing the disease-associated allele was decreased because of its decreased stability. These observations indicate that type XI collagen is critical for IVD metabolism and that its decrease is related to LDH.

Hyaluronan suppressed nitric oxide production in the meniscus and synovium of rabbit osteoarthritis model.

Nitric oxide (NO) plays an important role in cartilage degeneration, and NO donors induce meniscus degeneration and synovium inflammation. This study evaluated the effect of intraarticular injections of hyaluronan (HA) on NO production in meniscus and synovium using an experimental osteoarthritis (OA) model. Thirty‐six New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT), and were divided into three groups. Four weeks after ACLT, the HA group started to receive intraarticular HA injections once a week for 5 weeks; the vehicle group started to receive the carrier of HA; and the no injection group, no treatment. All ACLT knees were harvested at the 9th week. Meniscus and synovium sections were examined by immunohistochemistry for nitrotyrosine. The pieces of these two tissues were cultured for 24 h. Culture supernatants were analyzed for nitrite concentration. The amount of NO produced by the meniscus was much larger than that produced by the synovium. NO productions in the meniscus and synovium of the HA group were significantly lower than those of the other groups. The results suggest that the inhibition of NO production in meniscus and synovium might be a part of the mechanism of the therapeutic effect of HA on OA.