αvß5 integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes.

OBJECTIVE: When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating. METHODS: Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process. RESULTS: In monolayer-cultured chondrocytes, suppression of αv or ß5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of αvß5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by αvß5 integrin. In articular chondrocytes, the affinity of αvß5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced αvß5 integrin activation and the subsequent increase in ERK signaling. CONCLUSION: Our findings indicate that αvß5 integrin may be involved in the change that occurs in monolayer-cultured chondrocytes after plating.

Involvement of a disintegrin and metalloproteinase 10 and 17 in shedding of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is initially synthesized as a membrane-bound protein and converted into a soluble form by proteolytic cleavage. Although a disintegrin and metalloproteinase 17 (ADAM17) is considered to be the primary sheddase for TNF-alpha, it is not known whether ADAM17 is solely responsible for that process in any type of cells. To identify the TNF-alpha sheddase(s) in varieties of cells, we performed experiments using a unique screening system and observed that ADAM9, ADAM10, ADAM17, and ADAM19 were capable of cleaving TNF-alpha. We then performed RNA interference experiments and confirmed that ADAM10 and ADAM17 were in fact involved in TNF-alpha shedding in 293A cells. In mouse macrophages, ADAM17 was confirmed to be the primary sheddase, but the involvement of ADAM10 was also demonstrated. In NIH3T3 cells, ADAM10 could be more important in the shedding than ADAM17. In mouse vascular endothelial cell line UVfemale2, ADAM10 and ADAM17 were equally involved in TNF-alpha shedding, whereas ADAM17 was a major sheddase in human osteoarthritic chondrocytes. From these observations and others, we concluded that both ADAM10 and ADAM17 can be a TNF-alpha sheddase and that their significance could be determined by their expression levels and the abundance of tissue inhibitor of metalloproteinases.

Acute pericardial diverticulum caused by pericarditis treated with drainage of pericardial effusion.

A 50-year-old woman was admitted with congestive heart failure due to cardiac tamponade, which was caused by acute pericarditis with pericardial effusion. Although images of contrast computed tomography (CT) obtained two weeks prior to admission had shown no abnormality, CT on admission showed a mediastinal tumor communicating with the pericardial cavity. It had rapidly appeared in a few weeks. We diagnosed it as acute pericardial diverticulum caused by acute pericarditis. They improved after treatment with antibiotic therapy and pericardial drainage. .

Effect of GDF-5 on ligament healing.

The effects of growth and differentiation factor-5 (GDF-5) on ligament healing were studied using a gap injury model of the medial collateral ligament in rat knee joints. The administration of GDF-5 once at the time of surgery significantly improved the mechanical properties of the femur-ligament-tibia complex. At 3 weeks after surgery, 30 microg of GDF-5 improved the ultimate tensile strength of the complex by 41%, and the stiffness by 60%, compared with the vehicle control (p < 0.05 for both; Fisher's PLSD test). The observation with a transmission electron microscopy revealed that GDF-5 increased the diameter of collagen fibrils in the repair tissue, which was considered to be a possible mechanism for the positive result in the biomechanical testing. Quantitative PCR and in situ hybridization revealed enhanced type I procollagen expression by GDF-5, and the PCR analysis also revealed that the GDF-5 treatment reduced the expression of type III procollagen relative to type I procollagen. The PCR analysis further showed that the expression of decorin and fibromodulin was relatively reduced against type I procollagen by the growth factor, which was considered to be responsible for the increase of collagen fibril diameter in the repair tissue. No adverse effects were observed, and the use of GDF-5 was considered a promising approach to facilitate ligament healing.

Progressing small vessel pontine infarction includes different etiologies.

BACKGROUND: The aim of this study was to investigate the clinical features of progressing stroke of pontine infarction as small vessel disease. METHODS: Enrolled 38 acute pontine infarctions were confirmed by magnetic resonance imaging and magnetic resonance angiography at the first and seventh days. Ten patients (26.3%) presented progression (NIH Stroke Scale ≥2 increase within 72 h). RESULTS: Progressing patients showed no relation to the size and the distribution of lesion. Expansion of ischemic lesion showed correlation with basilar artery atherosclerosis. Stable lesion related to delayed worsening. CONCLUSION: These findings suggest that progressing stroke may be caused by not only the worsening of blood flow of ischemic lesion but also delayed neuronal death.

Up-regulation of interleukin-6 and vascular endothelial growth factor-A in the synovial fluid of temporomandibular joints affected by synovial chondromatosis.

Our aim was to explore important inflammatory mediators for synovial chondromatosis in the temporomandibular joints (TMJs) by analysing synovial fluid. Samples were collected from 10 patients with unilateral synovial chondromatosis of the TMJ. Control samples were obtained from 11 subjects with no symptoms in the TMJ. Concentrations of aggrecan, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A were measured in the samples of synovial fluid, and the results in the two groups compared. The tissues from the affected TMJ were examined histologically and immunohistochemically. Of the proteins evaluated, the concentrations of aggrecan, IL-6, and VEGF-A were significantly higher in the group with synovial chondromatosis. The immunohistochemical analysis showed that the synovial cells around the osteocartilaginous nodules were vigorously expressing VEGF-A. IL-6 and VEGF-A are thought to have important roles in the pathology of synovial chondromatosis of the TMJ.

α5ß1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers.

INTRODUCTION: Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes. METHODS: All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes. RESULTS: In dedifferentiating chondrocytes, α5ß1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5ß1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls. CONCLUSIONS: The result of this study has shown, for the first time, that α5ß1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.

Cerebrovascular lesions in elderly Japanese patients with Alzheimer's disease.

OBJECTIVE: Cerebrovascular lesions (CVLs) are known to play important roles in the pathophysiology underlying Alzheimer's disease (AD), especially in elderly AD cases. The present study was conducted to elucidate the relationship between the CVLs and vascular risk factors (VRFs) in elderly Japanese patients with AD. SUBJECTS AND METHODS: The CVLs such as lacunar infarcts, old microbleeds (OMBs), white matter lesions (WMLs), and occlusive vascular lesions on MRI were analyzed in relation to the risk factors in 120 Japanese patients with probable AD. Their mean age was 75.6 years. The subjects were divided into two age groups: young-old group (YOG) consisting of 55 cases being younger than 75 years and old-old group (OOG) consisting of 65 cases being 75 years or older. RESULTS: In overall analysis, 10 cases (8.3%) showed brain atrophy without CVLs on MRI, 46 cases (38.3%) showed WMLs in addition to the brain atrophy, 61 cases (50.8%) showed lacunar lesions, and 3 cases (2.5%) were diagnosed as having a superficial siderosis. Lacunar infarcts and OMBs were more frequently observed in OOG than in YOG, and were also more frequently observed in those with 2 or more VRFs than those with less than 2 VRFs (p<0.05). The WMLs were more pronounced in OOG, and in those with more VRFs. CONCLUSION: The CVLs including lacunes, WMLs, and OMBs were present more than 90% of elderly Japanese patients with AD. As the severity of CVLs was associated with VRFs and age, VRFs may modify clinical presentation of elderly AD patients.

The use of laser capture microdissection on adult human articular cartilage for gene expression analysis.

The integrity of articular cartilage is maintained by chondrocytes, the sole type of cell that resides within the tissue. The noncalcified region of articular cartilage can be divided into three zones based on histological features, in which the chondrocyte metabolism is known to differ obviously among the zones. In pathological cartilage, the chondrocyte metabolism may change dramatically, which could play a pivotal role in the progression of the disease. Since such change in metabolism differs obviously from site to site within cartilage, it is crucial to determine the chondrocyte metabolism in respective regions. To this end, we have employed laser-capture microdissection (LCM) to analyze chondrocyte metabolism in various regions of pathological and control cartilage. In this report, we describe our protocol for LCM on adult human cartilage tissue. With this protocol, a specific site of cartilage tissue was successfully obtained by LCM for gene expression analysis.

Myocardial infarction in a premenopausal woman with a decreased serum estrogen level due to leuprorelin acetate.

A 45-year-old premenopausal woman was admitted with acute myocardial infarction. Her serum estrogen level was decreased because of leuprorelin acetate administration, 3 months prior to admission for the treatment of uterine myoma. Emergency coronary angiography revealed diffuse narrowing of the distal half of the left anterior descending artery (LAD). The second coronary angiography after anti-anginal medication revealed significant improvement in LAD narrowing, which suggested prolonged coronary vasospasm. She had no coronary risk factors except for a positive family history. This case suggests that a decreased serum estrogen level could cause ischemic heart disease even in premenopausal women.

Regional differences in chondrocyte metabolism in osteoarthritis: a detailed analysis by laser capture microdissection.

OBJECTIVE: To determine the change in metabolic activity of chondrocytes in osteoarthritic (OA) cartilage, considering regional difference and degree of cartilage degeneration. METHODS: OA cartilage was obtained from knee joints with end-stage OA, at both macroscopically intact areas and areas with various degrees of cartilage degeneration. Control cartilage was obtained from age-matched donors. Using laser capture microdissection, cartilage samples were separated into superficial, middle, and deep zones, and gene expression was compared quantitatively in the respective zones between OA and control cartilage. RESULTS: In OA cartilage, gene expression changed markedly with the site. The expression of cartilage matrix genes was highly enhanced in macroscopically intact areas, but the enhancement was less obvious in the degenerated areas, especially in the upper regions. In contrast, in those regions, the expression of type III collagen and fibronectin was most enhanced, suggesting that chondrocytes underwent a phenotypic change there. Within OA cartilage, the expression of cartilage matrix genes was significantly correlated with SOX9 expression, but not with SOX5 or SOX6 expression. In OA cartilage, the strongest correlation was observed between the expression of type III collagen and fibronectin, suggesting the presence of a certain link(s) between their expression. CONCLUSION: The results of this study revealed a comprehensive view of the metabolic change of the chondrocytes in OA cartilage. The change of gene expression profile was most obvious in the upper region of the degenerated cartilage. The altered gene expression at that region may be responsible for the loss of cartilage matrix associated with OA.

Zonal gene expression of chondrocytes in osteoarthritic cartilage.

OBJECTIVE: To determine the chondrocyte metabolism in respective zones of osteoarthritic (OA) cartilage. METHODS: OA cartilage was obtained from macroscopically intact areas of 4 knee joints with end-stage OA. The cartilage was divided into 3 zones, and gene expression profiles were determined in the respective zones by a custom-designed microarray that focused on chondrocyte-related genes. For the genes whose expression was significantly different among the zones, the expression was compared between OA and control cartilage in the respective zones by an analysis using laser capture microdissection and real-time polymerase chain reaction (PCR). For some genes, the correlation of expression was investigated in specific cartilage zones. RESULTS: A total of 198 genes (approximately 40% of those investigated) were found to be expressed at significantly different levels among the zones. Expression of 26 of those genes was evaluated by laser capture microdissection and real-time PCR, which confirmed the validity of microarray analysis. The expression of cartilage matrix genes was mostly enhanced in OA cartilage, at similar levels across the zones but at different magnitudes among the genes. The expression of bone-related genes was induced either in the superficial zone or in the deep zone, and positive correlations were found among their expression in the respective zones. The expression of 5 proteinase genes was most enhanced in the superficial zone, where their expression was correlated, suggesting the presence of a common regulatory mechanism(s) for their expression. CONCLUSION: In OA cartilage, the metabolic activity of chondrocytes differed considerably among zones. Characteristic changes were observed in the superficial and deep zones.

Pro-inflammatory cytokine tumor necrosis factor-alpha induces bone morphogenetic protein-2 in chondrocytes via mRNA stabilization and transcriptional up-regulation.

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-alpha induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3'-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3'-untranslated regions, and transient transfection experiments demonstrated that TNF-alpha increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-alpha modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-kappaB.