VEGF (vascular endothelial growth factor) is not only one of the most important angiogenesis factors, but is involved also in inflammatory processes. Recent studies have shown that VEGF as well as its receptor VEGFR-2 are expressed on osteoarthritic chondrocytes, but not on normal adult chondrocytes. Since mechanical overload is one of the causative factors for osteoarthritis, we studied its effect on VEGF expression on bovine cartilage disks that were compressed once with a strain of 50% and a strain rate of 1/second. Under these conditions, control disks (without pressure) were completely negative for VEGF expression as evidenced by immunocytochemical stainings as well as by enzyme-linked immunosorbent assay (ELISA) measurements. In contrast, 4 days after mechanical overload, the cartilage disks were positive in both detection methods. In addition, after mechanical overload chondrocytes were strongly immunopositive for hypoxia-inducible factor-1alpha (HIF-1alpha), the limiting protein of the dimeric transcription factor HIF-1 that is known to induce VEGF expression. Furthermore, the matrix metalloproteases MMP-1, MMP-3, and MMP-13, could be easily detected in pressure-treated disks by immunohistochemistry whereas staining in controls was low or undetectable. The tissue inhibitors of metalloproteinases (TIMP-1 and -2) could be detected in controls but not in samples treated with mechanical overload. To prove that increased MMP or decreased TIMP expression could be a result of the autocrine action of VEGF on chondrocytes, we repeated the experiments in the presence of a specific inhibitor for the kinase activity of the VEGFR-2. This inhibitor was effective to reduce mechanically induced MMP-1, -3, and -13 immunostaining and to restore TIMP expression. Taking together, these findings indicate that VEGF is induced in chondrocytes by mechanical overload and mediates destructive processes in osteoarthritis as an autocrine factor.
Objective. To study the influence of tissue maturation and antioxidants on apoptosis in bovine articular cartilage induced by injurious compression.Methods. Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5-23 months and placed in culture. Cartilage disks were preincubated overnight with the cellpermeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0-12.5 M) or ␣-tocopherol (0-50 M) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium.Results. Injurious compression induced significantly more apoptosis in cartilage disks from newborn calves (22% of cells) than in cartilage from more mature cows (2-6%). In cartilage from 22-month-old animals, the SOD mimetic reduced the percentage of apoptotic cells induced by injury in a dose-dependent manner (complete inhibition with 2.5 M), while ␣-tocopherol had no effect. Neither antioxidant altered protein biosynthesis or cellular ultrastructure.Conclusion. Our data suggest that the apoptotic response of articular cartilage to mechanical injury is affected by maturation and is mediated in part by reactive oxygen species. The antioxidative status of the tissue might be important for the prevention of mechanically induced cell death in articular cartilage.
Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.
IntroductionLittle is known about factors that induce meniscus damage. Since joint inflammation appears to be a causative factor for meniscal destruction, we investigated the influence of tumor necrosis factor (TNFα) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model.MethodsMeniscal explant disks (3 mm diameter × 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFα-treatment GAG release (DMMB assay), biosynthetic activity (sulfate incorporation), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR, zymography), and immunostaining of the aggrecan fragment NITEGE were determined.ResultsTNFα induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFα increased matrix metalloproteinase (MMP)-3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 mRNA expression, whereas collagen type I was decreased, and aggrecan, collagen type II as well as MMP-1, -2, -13 and ADAMTS-5 were variably affected. Zymography also showed a TNFα-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFα-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFα-dependent GAG release and NITEGE production, whereas inhibition of TNFα-dependent NO generation with the NO-synthetase inhibitor L-NMMA failed to inhibit GAG release and NITEGE production.ConclusionsOur study shows that aggrecanase activity (a) is responsible for early TNFα-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFα-dependent source for MMP-3. However, the TNFα-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.
14053 Background: Endothelial progenitor cells (EPCs) may participate in tumor angiogenesis by providing cellular supply. This is a study that compares the effects of two conventional combination chemotherapy schedules to low-dose metronomic trofosfamide (an oral derivate of cyclophosphamide), using the number of circulating EPCs and vascular endothelial growth factor (VEGF) plasma levels in cancer patients. Methods: We measured circulating EPC and VEGF levels in 24 patients that received conventional chemotherapy for either breast cancer in an adjuvant setting or malignant lymphoma, and in 18 patients receiving metronomic chemotherapy with or without celecoxib for advanced cancer. Blood samples were obtained three times: before starting chemotherapy, 10 and 21 days after starting chemotherapy. Peripheral blood EPC levels were determined by fluorescence flow cytometry and defined by CD34- and VEGF-R2-positivity. VEGF plasma concentration was determined by ELISA. Results: The number of circulating EPCs showed a two-fold increase 21 days after conventional chemotherapy but a significant decrease under metronomic chemotherapy. VEGF-plasma-concentrations remained stable in patients under metronomic chemotherapy but significantly increased under conventional chemotherapy.This increase in VEGF plasma levels occurred even in patients that received chemotherapy in an adjuvant setting in supposed absence of tumor. Conclusions: Low-dose metronomic trofosfamide significantly decreased circulating EPCs while conventional chemotherapy increased both the number of circulating EPC and VEGF-concentrations. The increase of VEGF even in an adjuvant setting of chemotherapy without the presence of a tumor may be caused by chemotherapy-induced endothelial cell destruction. Metronomic scheduling of certain cytotoxic drugs may thus prevent tumor progression by inhibiting both tumor cell proliferation and angiogenesis. No significant financial relationships to disclose.
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