Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Prevention of amyloid beta-induced memory impairment by fluvastatin, associated with the decrease in amyloid beta accumulation and oxidative stress in amyloid beta injection mouse model

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized by amyloid beta (Abeta)-containing plaques and neurofibrillary tangles, and synaptic and neuronal loss, along with progressive cognitive impairment. Although growing evidence suggests the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) on AD, this notion is still controversial. To evaluate the efficacy of statins for Abeta-induced cognitive impairment, we employed an Abeta injection model. Using this model, the present study demonstrated that pretreatment with fluvastatin, but not post-treatment just after Abeta exposure, prevented Abeta-induced memory impairment. We also observed that fluvastatin significantly decreased Abeta accumulation and oxidative stress after Abeta injection. Mice treated with simvastatin, but not fluvastatin, did not demonstrate the prevention of Abeta-induced memory impairment, and showed no significant decrease in oxidative stress. More importantly, fluvastatin significantly prevented the loss of neurons in the basal forebrain induced by Abeta. Overall, the present study demonstrated that fluvastatin significantly prevented memory impairment induced by Abeta. The beneficial effects of fluvastatin might be explained by the preservation of neurons through a significant decrease in Abeta accumulation and oxidative stress. In clinical practice, the timing of the start of fluvastatin treatment might be critical in achieving a beneficial effect on cognitive function.     

Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow, the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. A subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared with normoxia. AT cells released vascular endothelial growth factor (VEGF) protein and expressed CD31 and kinase insert domain receptor/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells before in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.     

Metabolic cardiovascular disease risk factors and their clustering in subclinical hypothyroidism

Objective A possible association between subclinical hypothyroidism and cardiovascular disease (CVD) has been reported. Monitoring of atomic‐bomb survivors for late effects of radiation exposure at the Radiation Effects Research Foundation has provided the opportunity to examine associations between subclinical hypothyroidism and metabolic CVD risk factors. The objective of the study was to evaluate associations between subclinical hypothyroidism and metabolic CVD risk factors, and a cluster of these factors. Design and participants This was a cross‐sectional study of 3549 subjects (mean age 70 years; 1221 men and 2328 women) between 2000 and 2003 comprising 306 subjects with subclinical hypothyroidism and 3243 control euthyroid subjects in Japan. Measurements We investigated associations between subclinical hypothyroidism and metabolic CVD risk factors such as hypertension, diabetes mellitus, dyslipidaemia and hyperuricaemia, and a cluster of these factors. Results Subclinical hypothyroidism was not significantly associated with either hypertension, diabetes mellitus or hyperuricaemia defined by taking into account the use of medications in both men and women, but in men it was associated with dyslipidaemia (P = 0·02). We observed a significantly increased odds ratio (OR) for the presence of three or more metabolic CVD risk factors in men with subclinical hypothyroidism after adjusting for age, body mass index (BMI), and smoking status [OR: 1·83, 95% confidence interval (CI): 1·13–2·94, P = 0·01]. The significant associations remained after an additional adjustment for atomic‐bomb radiation dose. Conclusions There appears to be a significant increase in a cluster of metabolic CVD risk factors among people with subclinical hypothyroidism.     

Regenerative potential of basic fibroblast growth factor contained in biodegradable gelatin hydrogel microspheres applied following vocal fold injury: Early effect on tissue repair in a rabbit model

IntroductionPostoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging.ObjectiveThe goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor.MethodsVideoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement.ResultsThe presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection.ConclusionA prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.     

Primrose syndrome associated with unclassified immunodeficiency and a novel ZBTB20 mutation

Primrose syndrome is a congenital malformation syndrome characterized by intellectual disability, developmental delay, progressive muscle wasting, and ear lobe calcification. Mutations in the ZBTB20 gene have been established as being accountable for this syndrome. In this study, a novel de novo ZBTB20 mutation, NM_001164342.2:c.1945C>T (p.Leu649Phe), has been identified through whole exome sequencing (WES) in a female patient presenting a typical Primrose phenotype. Because the present patient exhibited recurrent otitis media, detailed immunological examinations were performed in this study and subnormal immunoglobulin levels were firstly identified in a Primrose patient. Anatomical anomaly of the inner ear has never been reported in this patient and WES data did not include any relevant variants causally linked with the immunologic defect. Thus, there is a possibility of a relation between an unclassified immunodeficiency with selective IgG2 deficiency and Primrose syndrome and this may be the reason of recurrent otitis media frequently observed in Primrose patients. Because subnormal levels of IgG2 in this patient might be caused by an unrelated and still uncharacterized genetic cause, further studies are required to prove the causal link between aberrant ZBTB20 function and immunodeficiency.     

The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly.

Holoprosencephaly (HPE) is a common developmental anomaly of the human forebrain and midface where the cerebral hemispheres fail to separate into distinct left and right halves. We have previously reported haploinsufficiency for Sonic Hedgehog ( SHH ) as a cause for HPE. We have now performed mutational analysis of the complete coding region and intron-exon junctions of the SHH gene in 344 unrelated affected individuals. Herein, we describe 13 additional unrelated affected individuals with SHH mutations, including nonsense and missense mutations, deletions and an insertion. These mutations occur throughout the extent of the gene. No specific genotype-phenotype association is evident based on the correlation of the type or position of the mutations. In conjunction with our previous studies, we have identified a total of 23 mutations in 344 unrelated cases of HPE. They account for 14 cases of familial HPE and nine cases of sporadic HPE. Mutations in SHH were detected in 10 of 27 (37%) families showing autosomal dominant transmission of the HPE spectrum, based on structural anomalies. Interestingly, three of the patients with an SHH mutation also had abnormalities in another gene that is expressed during forebrain development. We suggest that the interactions of multiple gene products and/or environmental elements may determine the final phenotypic outcome for a given individual and that variations among these factors may cause the wide variability in the clinical features seen in HPE.