Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis.

Cancer cells gain a growth advantage through the so-called Warburg effect by shifting glucose metabolism from oxidative phosphorylation to aerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) has been suggested to function in metabolic reprogramming; however, the underlying mechanism has not been fully elucidated. We found that the aberrant expression of wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer, decreased α-ketoglutarate levels and increased the stability and transactivation activity of HIF-1α in cancer cells. The silencing of IDH3α significantly delayed tumor growth by suppressing the HIF-1-mediated Warburg effect and angiogenesis. IDH3α expression was associated with the poor postoperative overall survival of lung and breast cancer patients. These results justify the exploitation of IDH3 as a novel target for the diagnosis and treatment of cancers.

Involvement of decreased hypoxia-inducible factor 1 activity and resultant G1-S cell cycle transition in radioresistance of perinecrotic tumor cells.

Cancer patients often suffer from local tumor recurrence after radiation therapy. Some intracellular and extracellular factors, such as activity of hypoxia-inducible factor 1 (HIF-1), cell cycle status and oxygen availability, have been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. But when, where, and how these factors affect one another and induce cellular radioresistance is largely unknown. Here, we analyzed mechanistic and spatio-temporal relationships among them in highly heterogeneous tumor microenvironments. Experiments in vitro demonstrated that a decrease in the glucose concentration reduced the transcriptional activity of HIF-1 and expression of a downstream gene for the cell cycle regulator p27(Kip1) even under hypoxic conditions. Then, the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased, significantly. Immunohistochemical analyses showed that cancer cells in perinecrotic hypoxic regions, which should be under low-glucose conditions, expressed little HIF-1α, and therefore, were mainly in S phase and less damaged by radiation treatment. Continuous administration of glucagon, which increases the blood glucose concentration and so improves glucose availability in perinecrotic hypoxic regions, induced HIF-1α expression and increased radiation-induced DNA damage. Taken all together, these results indicate that cancer cells in perinecrotic regions, which would be under low-glucose and hypoxic conditions, obtain radioresistance by decreasing the level of both HIF-1 activity and p27(Kip1) expression, and adjusting their cell cycle to the radioresistant S phase.

Effects of the systemic administration of alendronate on bone formation in a porous hydroxyapatite/collagen composite and resorption by osteoclasts in a bone defect model in rabbits.

Several bisphosphonates are now available for the treatment of osteoporosis. Porous hydroxyapatite/collagen (HA/Col) composite is an osteoconductive bone substitute which is resorbed by osteoclasts. The effects of the bisphosphonate alendronate on the formation of bone in porous HA/Col and its resorption by osteoclasts were evaluated using a rabbit model. Porous HA/Col cylinders measuring 6 mm in diameter and 8 mm in length, with a pore size of 100 µm to 500 µm and 95% porosity, were inserted into a defect produced in the lateral femoral condyles of 72 rabbits. The rabbits were divided into four groups based on the protocol of alendronate administration: the control group did not receive any alendronate, the pre group had alendronate treatment for three weeks prior to the implantation of the HA/Col, the post group had alendronate treatment following implantation until euthanasia, and the pre+post group had continuous alendronate treatment from three weeks prior to surgery until euthanasia. All rabbits were injected intravenously with either saline or alendronate (7.5 µg/kg) once a week. Each group had 18 rabbits, six in each group being killed at three, six and 12 weeks post-operatively. Alendronate administration suppressed the resorption of the implants. Additionally, the mineral densities of newly formed bone in the alendronate-treated groups were lower than those in the control group at 12 weeks post-operatively. Interestingly, the number of osteoclasts attached to the implant correlated with the extent of bone formation at three weeks. In conclusion, the systemic administration of alendronate in our rabbit model at a dose-for-weight equivalent to the clinical dose used in the treatment of osteoporosis in Japan affected the mineral density and remodelling of bone tissue in implanted porous HA/Col composites.

Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy.

Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tumour radioresistance; therefore, it is recognised as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimised their combination. Hypoxic tumour cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1alpha and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1alpha accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumour hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumour hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumour growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

Treatment of distal radius fractures with a wrist-bridging external fixation: the value of alternating electric current stimulation.

The radiological and functional outcomes of two groups of comminuted intraarticular fractures of the distal radius that were treated with wrist-bridging external fixation, with or without an alternating electric current stimulation (EF + ES or EF group, respectively), were compared. The radial length, radial inclination and palmar tilt were measured on radiographs taken immediately after removal of wrist external fixator and also after bone union had occurred. Furthermore, active range of motion was compared at the final consultation. Bridging callus was observed earlier postoperatively in the EF + ES group than in the EF group. The radial length and palmar tilt were significantly larger, and the loss of radial length and radial inclination significantly smaller, in the EF + ES group. We believe that callus maturation is enhanced by alternating electric current stimulation, which enables the early removal of external fixator.

Cementless total hip replacement after previous intertrochanteric valgus osteotomy for advanced osteoarthritis.

We present a series of 30 uncemented total hip replacements performed between June 1985 and January 2002 with a mean follow-up of seven years (5 to 20) in 27 patients who had previously undergone a valgus intertrochanteric osteotomy. No further osteotomy was undertaken to enable hip replacement. We used a number of uncemented modular or monoblock femoral components, acetabular components and bearings. The patients were followed up clinically and radiologically. We report 100% survival of the femoral component. One acetabular component was revised at five years post-implantation for aseptic loosening. We noted cortical hypertrophy around the tip of the monoblock stems in six patients. We believe that modular femoral components should be used when undertaking total hip replacement in patients who have previously undergone valgus femoral osteotomy.

ED-71, a novel vitamin D analog, promotes bone formation and angiogenesis and inhibits bone resorption after bone marrow ablation.

ED-71, a novel analog of 1alpha,25-(OH)2 D3, increases bone mass to a greater extent than alfacalcidol, an 1alpha,25-(OH)2 D3 prodrug. In this study, we used a murine bone marrow ablation model to compare the effect of ED-71 on bone formation and resorption in vivo with that of 1alpha,25-(OH)2 D3. We discovered that bone matrix remodeling occurring within the first week after bone marrow ablation was enhanced by a single injection of ED-71, but not by 1alpha,25-(OH)2 D3. This enhancement was associated with an increase in bone surface. Trabecular bone resorption occurring from 1 to 2 weeks after the procedure was suppressed by a single injection of ED-71, but not 1alpha,25-(OH)2 D3, with treated mice exhibiting a reduction in osteoclast numbers, despite increases in osteoblast surface. As seen with the single injection, daily administration of ED-71 also enhanced bone modeling. Bone marrow osteoblast differentiation was also augmented by ED-71 pretreatment. Furthermore, ED-71 treatment immediately after bone marrow ablation enhanced angiogenesis within the bone marrow cavity via enhancement of VEGF(120) expression. In this paper, we clearly demonstrate that ED-71 is an orally administered small molecular weight compound with an anabolic effect on bone metabolism.

Plasminogen activator inhibitor-1 aids nerve growth factor-induced differentiation and survival of pheochromocytoma cells by activating both the extracellular signal-regulated kinase and c-Jun pathways.

Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator-plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway.

Establishment and characterization of a cell line from a malignant fibrous histiocytoma of bone developing in a patient with multiple fibrous dysplasia.

PURPOSE AND METHODS: In order to provide material for genetic analysis of fibrous dysplasia (FD), a cell line designated GBS-1 was established from a secondary bone malignant fibrous histiocytoma (MFH) developing in a rib of a 44-year-old male polyostotic FD patient. RESULTS: The GBS-1 cells are characterized by a pleomorphic spindle cell morphology with abundant mucus production. On transplantation to nude mouse subcutis the cell line forms myxoid-spindle cell sarcomas with giant cells, the myxoid product being positive for periodic acid-Schiff (PAS) and alcian blue (Al-B) stains and completely digested by hyaluronidase, mimicking the original tumor. Chromosome and genetic analyses revealed multiple structural and numerical abnormalities of chromosomes with a large number of unidentifiable chromosomes and p53 mutation in exon 7 with LOH in the counterpart. CONCLUSIONS: Since cell lines for FD have hitherto not been available, the GBS-1 cells should prove useful for genetic analyses of FD and also MFH of bone origin.

Effects of neurectomy and tenotomy on the bone mineral density and strength of tibiae.

The right hindlimbs of 5 or 6-week old Wistar male rats were sciatic/femoral neurectomized, tenotomized or sham operated. The rats were sacrificed 2 weeks after the surgery and the tibiae were removed. pQCT measurement was performed on total, cortical, and trabecular bone separately at different regions. Reduction of the bone mineral density by unloading was observed more significantly at metaphysis than at diaphysis due to histological heterogeneity between metaphysis and diaphysis; metaphysis is rich in trabecular bone and diaphysis is abundant in cortical bone. Trabecular bone might be more sensitive to unloading because the reduction rate of volumetric bone mineral density in trabecular bone was approximately 10 times and 3 times larger than that of cortical bone in both neurectomy and tenotomy rats, respectively, Unloading also reduced the cross-sectional area and stress strain index at metaphysis.

The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene.

It has been suggested that matrix metalloproteinase-3 (MMP-3, stromelysin-1) has an important role in the degeneration of intervertebral discs (IVDs). A human MMP-3 promoter 5A/6A polymorphism was reported to be involved in the regulation of MMP-3 gene expression. We suggest that IVD degeneration is associated with 5A/6A polymorphism. We studied 54 young and 49 elderly Japanese subjects. Degeneration of the lumbar discs was graded using MRI in the younger group and by radiography in the elderly. 5A/6A polymorphism was determined by polymerase-chain reaction-based assays. We found that the 5A5A and 5A6A genotype in the elderly was associated with a significantly larger number of degenerative IVDs than the 6A6A (p < 0.05), but there was no significant difference in the young. In the elderly, the IVD degenerative scores were also distributed more highly in the 5A5A and 5A6A genotypes (p = 0.0029). Our findings indicate that the 5A allele is a possible risk factor for the acceleration of degenerative changes in the lumbar disc in the elderly.

Osteopontin facilitates angiogenesis, accumulation of osteoclasts, and resorption in ectopic bone.

Osteoclastic bone resorption requires a number of complex steps that are under the control of local regulatory molecules. Osteopontin is expressed in osteoclasts and is also present in bone matrix; however, its biological function has not been fully understood. To elucidate the role of osteopontin in the process of osteoclastic bone resorption, we conducted ectopic bone implantation experiments using wild-type and osteopontin knockout mouse. In the wild-type group, bone discs from calvariae implanted ectopically in muscle were resorbed, and their mass was reduced by 25% within 4 weeks. In contrast, the mass of the bone discs from calvariae of osteopontin knockout mice was reduced by only 5% when implanted in osteopontin knockout mice. Histological analyses indicated that the number of osteoclasts associated with the implanted bones was reduced in the osteopontin knockout mice. As osteopontin deficiency does not suppress osteoclastogenesis per se, we further examined vascularization immunohistologically and found that the number of vessels containing CD31-positive endothelial cells around the bone discs implanted in muscle was reduced in the osteopontin knockout mice. Furthermore, sc implantation assays indicated that the length and branching points of the newly formed vasculatures associated with the bone discs were also reduced in the absence of osteopontin. In this assay, tartrate-resistant acid phosphatase-positive area of the bone discs was also reduced in the osteopontin knockout mice, indicating further the link between the osteopontin-dependent vascularization and osteoclast accumulation. The bone resorption defect could be rescued by topical administration of recombinant osteopontin to the bones implanted in muscle. These observations indicate that osteopontin is required for efficient vascularization by the hemangiogenic endothelial cells and subsequent osteoclastic resorption of bones.

A new continuous-flow cell separation method based on cell density: principle, apparatus, and preliminary application to separation of human buffy coat.

With the recent progress in transfusion medicine, separation and isolation of cells in a large quantity is becoming increasingly important. At present, the continuous cell separation method in a preparative scale is limited to apheresis and elutriation: the former is mainly used for collection of platelet and buffy coat from the whole blood, while the latter separates cells virtually according to their size. Here we introduce a continuous flow method that separates cells entirely based on cell density. The method is gentle and capable of processing a large number of cells. The potential capability of the method was demonstrated on separation of lymphocytes and granulocytes from human buffy coat. Lymphocytes were enriched to 90% in the fraction at density = 1.065 and granulocytes are isolated in fractions at density = 1.075-1.080 while red cells were completely retained at the periphery of the channel. CD34 cells were distributed around 1.065 and coeluted with lymphocytes, suggesting that further enrichment requires focusing the density gradient around 1.070. The method could process 10(9) nucleated cells in 2 hours. Our preliminary results suggest that the present method is an effective and efficient means to separate blood cells.

Prevention of pulmonary embolism by a foot sole pump.

We carried out a prospective, randomised study of 62 patients to determine the efficacy of a foot sole pump (the A-V Impulse System) for the prevention of pulmonary embolism (PE) after hip surgery. PE was assessed by pulmonary perfusion scintigraphy before and after operation. We defined a PE as any new scintigraphic defect which was larger than a bronchopulmonary segment. The incidence of PE was 55% in the control group and 21% in the treatment group. The foot sole pump significantly reduced the incidence of PE (p = 0.008) and we encountered no side-effects from its use.

Association of plasma adrenomedullin with carotid atherosclerosis in chronic ischemic stroke.

Adrenomedullin is a potent vasodilator peptide exerting anti-atherosclerotic actions in vitro. We investigated the impact of the severity of atherosclerosis on plasma mature-adrenomedullin (m-AM) levels in 38 patients with chronic ischemic stroke. The variables of carotid artery atherosclerosis assessed using ultrasound measurement, blood pressure, and risk factors were related to m-AM levels. Severe atherosclerosis was associated with a further elevation of the increased m-AM level in patients with high systolic blood pressure. Even in patients with fewer risk factors, the presence of severe atherosclerosis was associated with an increased m-AM level. Thus, atherosclerosis elevates m-AM independent of the blood pressure level or presence of risk factors.

Long-term results of valgus-extension femoral osteotomy for advanced osteoarthritis of the hip.

We treated 31 hips in 30 patients with advanced osteoarthritis of the hip secondary to acetabular dysplasia, by valgus-extension femoral osteotomy. The mean follow-up was 12.7 years (10 to 17). Acetabuloplasty was added in ten severely dysplastic hips. In 28 hips, radiological widening of the joint space was seen three years after operation, but in 12 had narrowed again by ten years. Survivorship analysis showed that the rate of survival was 82% using the pain score as the index of failure, and 72% based on radiological findings at ten years. Better long-term results were obtained in hips which had an acetabular head index greater than 70% or a roof osteophyte more than 5 mm in length three years after operation. Acetabuloplasty should be added for the hip which is severely dysplastic and with a poorly developed roof osteophyte.

Prevention of fibrous layer formation between bone and adhesive bone cement: in vivo evaluation of bone impregnation with 4-META/MMA-TBB cement.

We have studied a new adhesive bone cement, that consists of 4-methacryloyloxyethyl trimellitate anhydride (4-META) and methylmethacrylate (MMA) as monomers, tri-n-butyl borane (TBB) as the initiator, and polymethylmethacrylate powder (4-META/MMA-TBB cement). This cement has shown remarkable adhesive properties to bone in vitro. In this study, we assessed the interface in vivo periodically. The femora of rabbits were fenestrated and filled with either the 4-META/MMA-TBB cement or a conventional polymethylmethacrylate cement. The animals were killed after 1, 4, 12, and 24 weeks to analyze the interface by optical microscopy and transmission electron microscopy. Optical microscopic examinations showed that the cured 4-META/MMA-TBB adhesive cement bonded to bone directly for 24 weeks, whereas a fibrous tissue layer was observed between the bone and cured conventional cement at 12 weeks after the operation. The transmission electron microscopy views of 4-META/MMA-TBB cement bonded to bone demonstrated a unique "hybridized bone" with the cement in the subsurface of the substrate in every case. The formation of the hybridized bone indicates the bonding mechanism of the adhesive cement to bone, which prevents the fibrosis intervention between bone and cement. These results suggest that the biomechanical and adhesive properties of 4-META/MMA-TBB cement make it a useful bone-bonding agent in orthopedic surgery.

Matrix metalloproteinase-3-dependent generation of a macrophage chemoattractant in a model of herniated disc resorption.

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption.

Herniated disc (HD), one of the major causes of low back pain, is often resolved spontaneously without surgical intervention. Resorption is associated with a marked increase in infiltrating macrophages, and the matrix metalloproteinases (MMP) MMP-3 and MMP-7 have been implicated in this phenomenon. We developed a murine organ culture model in which intact intervertebral discs were cocultured with peritoneal macrophages to investigate the role of MMPs in HD resorption. Using macrophages isolated from MMP-null mice, we report that macrophage-produced MMP-7 was required for proteoglycan degradation, loss of wet weight, and macrophage infiltration of cocultured discs. The inability of MMP-7-deficient macrophages to infiltrate discs could not be attributed to a defect in macrophage migration. MMP-7 was required for the release of the cytokine TNF-alpha from peritoneal macrophages. The generation of soluble TNF-alpha was essential for the induction of MMP-3 in disc cocultures, which in turn is required for the generation of a macrophage chemoattractant and subsequent macrophage infiltration. TNF-alpha release from macrophages was necessary but insufficient for disc resorption, which required macrophage infiltration. We conclude that there is extensive communication between macrophages and chondrocytes in HD resorption and that an essential component of this communication is the requirement for MMPs to release soluble bioactive factors.