Biomedical subjects

Rolf Mentlein

Publications and source records attributed to Rolf Mentlein.

At least 19 recordsLinked to original sources

PubMed2006

Differential expression of matrix metalloproteinases in brain- and bone-seeking clones of metastatic MDA-MB-231 breast cancer cells.

Matrix Metalloproteinases (MMPs) play a crucial role in breast cancer metastasis. We examined the mRNA and protein expression of several MMPs in brain- and bone-seeking clones of MDA-MB-231 breast cancer cells, their transcriptional regulation and their functional role in the metastatic process. MMP mRNA expression was examined using real-time reverse transcription polymerase chain reaction. Protein expression was examined using enzyme linked immunosorbent essay (ELISA). The inducibility of mRNA and protein expression was tested with TPA (phorbol 12-myristate 13-acetate; 50 microM); epidermal growth factor and transforming growth factor beta (20 ng/ml both). Migration and invasion assays were performed with the QCM 96-Well Migration/Invasion Assay (8 microm; Chemicon) over 24 h with or without specific MMPs inhibitors (MMP Inhibitor I Mix (5 microM); MMP-2/MMP-9 Inhibitor III (50 microM); EMD Biosciences). We found significantly higher mRNA expression of MMP-1 and -9 in brain-seeking 231-clones in comparison to -bone and -parental cells. In contrast, the mRNA expression of MMP-3 and -14 was comparable in all cells lines examined and MMP-13 expression was lower in both selective metastatic lines. MMP-2 and -8 were not expressed. ELISA revealed a higher amount of total as well as active MMP-1 and -9 in brain-seeking cells. TPA stimulation showed that MMP-1 and -9 transcription was inducible on the mRNA and protein level in 231-parental but not in 231-brain or -bone. 231-brain showed the highest migration and invasive capacity which could be decreased by the application of MMP-1 and/or MMP-9 inhibitor. Our results indicate functional importance of MMP-1 and -9 overexpression in brain metastasis in an in vitro model.

Bone Neoplasms↗

PubMed2006

Matrix-degrading proteases ADAMTS4 and ADAMTS5 (disintegrins and metalloproteinases with thrombospondin motifs 4 and 5) are expressed in human glioblastomas.

Brain tumors, in particular glioblastomas, have a high morbidity and mortality, mainly due to their invasive nature. A prerequisite for this invasiveness is cell migration based on increased expression of proteases digesting the extracellular matrix. Brevican, an important extracellular proteoglycan that is upregulated in glioblastomas, can be degraded by certain proteases. We demonstrate that in human glioblastomas secretory proteases like ADAMTS4 and ADAMTS5 (aggrecanases 1 and 2; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs) are expressed on the mRNA and protein levels in considerable amounts. Real-time RT-PCR shows a higher levels of ADAMTS4 and 5 expressions in glioblastomas in situ, compared to cultured human glioblastoma cells. The upregulation of these proteases in vivo by cytokines may explain this difference. In vitro, transforming growth factor-beta induces ADAMTS4, but less ADAMTS5, and interleukin-1beta ADAMTS5, but not ADAMTS4. As demonstrated by immunohistochemistry and confocal microscopy in situ, ADAMTS5 expression is confined to proliferating glioblastoma cells of surgical tumor sections and with lower intensity to astroglial cells in normal brain sections, as opposed to brevican. In vitro, glioblastoma-derived ADAMTS5 degrades recombinant human brevican to several smaller fragments. Our results show that ADAMTS4 and 5 are upregulated on proliferating glioblastoma cells and these proteases may contribute to their invasive potential.

ADAM Proteins↗

PubMed2006

Flavonoids and vitamin E reduce the release of the angiogenic peptide vascular endothelial growth factor from human tumor cells.

Neoangiogenesis is required for tumor development and progression. Many solid tumors induce vascular proliferation by production of angiogenic factors, prominently vascular endothelial growth factor (VEGF). Because nutrition is a causative factor for tumor prevention and promotion, we determined whether secondary plant constituents, i.e., flavonoids, tocopherols, curcumin, and other substances regulate VEGF in human tumor cells in vitro. VEGF release (concurrent with synthesis) from MDA human breast cancer cells and, for comparison, U-343 and U-118 glioma cells was measured by ELISA. Of 21 compounds tested, 9 showed significant inhibitory activity at 0.1 micromol/L in MDA human breast cancer cells. The rank order of inhibitory potency was naringin > rutin > alpha-tocopheryl succinate (alpha-TOS) > lovastatin > apigenin > genistein > alpha-tocopherol >or= kaempferol > gamma-tocopherol; chrysin and curcumin were inactive except at a concentration of 100 micromol/L. Glioma cells were similarly sensitive, with U343 more than U118, especially for alpha-TOS and tocopherols. Among the tocopherol derivatives, alpha-TOS (0.1 micromol/L) was the most effective in reducing VEGF release. Overall, the glycosylated flavonoids (i.e., naringin, a constituent of citrus fruits, and rutin, a constituent of cranberries) induced the greatest response to treatment at the lowest concentration in MDA human breast cancer cells. Inhibition of VEGF release by flavonoids, tocopherols, and lovastatin in these models of neoplastic cells suggests a novel mechanism for mammary cancer prevention.

Angiogenesis Inhibitors↗

PubMed2006

Cannabinoid receptors in human astroglial tumors.

In animal models, cannabinoids are reported to inhibit the growth of tumors, including gliomas. These effects have been claimed to be mediated via cannabinoid receptors 1 and 2 (CB1, CB2). To elucidate a possible relevance for treatment of human gliomas, we investigated receptor subtype expression in surgical material of solid human astrocytomas, gliomas and cultivated glioma cells by quantitative reverse transcriptase polymerase chain reaction, western blot and immunohistochemistry and assayed their functionality. In normal brain, cultivated glioma cells and solid tumors, CB1 mRNA was expressed to a much greater extent than CB2, which in some samples was even undetectable. Expression of both receptor subtypes was unrelated to malignancy, varied between patients, and was not significantly increased in relation to normal brain tissues. In normal brain, CB1 protein was localized on astroglial and other cell types; in gliomas, it was found on astroglial/glioma cells. CB2 protein was detected on microglial cells/macrophages but rarely on astroglial cells. Functionally, CB1 receptor agonists reduced elevated cyclic AMP levels and slightly reduced proliferation of glioma cells in vitro, but did not induce apoptosis. We conclude that cannabinoid therapy of human gliomas targets not only receptors on tumor, but also on other cell types. Therefore, complex and potential side-effects should be considered carefully.

Apoptosis↗

PubMed2005

Human beta-defensin 3 mediates tissue remodeling processes in articular cartilage by increasing levels of metalloproteinases and reducing levels of their endogenous inhibitors.

OBJECTIVE: Beta-defensins are broad-spectrum antimicrobial peptides (APs) that are components of innate immunity. Recent investigations showed the induction of beta-defensins in synovial membranes of osteoarthritic (OA) joints and suggested that they have functions other than the ability to kill microbes. As a result of these findings, we undertook this study to investigate the production of human beta-defensin 3 (HBD-3) in OA cartilage and to determine its influence on chondrocyte function. METHODS: Healthy and OA cartilage were assessed for HBD-3 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. HBD-3 expression in C28/I2 chondrocytes after administration of tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) was determined by real-time RT-PCR and immunodot blot. Enzyme-linked immunosorbent assay experiments were used to study the effects of HBD-3 in cultured articular chondrocytes and in healthy and OA cartilage discs. Immunohistochemical analyses were performed to study the expression of mouse beta-defensins (MBDs) in OA cartilage of STR/Ort mice. RESULTS: HBD-3 was induced in OA cartilage without bacterial challenge. Cytokines involved in the pathogenesis of OA, namely, TNFalpha and IL-1, were strong inducers of HBD-3 in cultured chondrocytes. Application of the recombinant HBD-3 protein to cultured chondrocytes and cartilage discs resulted in increased production of cartilage-degrading matrix metalloproteinases and in down-regulation of their endogenous regulators, tissue inhibitors of metalloproteinases 1 and 2. Furthermore, STR/Ort mice, which are genetically predisposed to develop OA-like lesions in the knee joint, demonstrated an increased expression of MBDs 3 and 4 in cartilage compared with that in healthy animals. CONCLUSION: These findings widen our knowledge of the functional spectrum of APs and demonstrate that HBD-3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissue-remodeling processes in articular cartilage. Moreover, our data suggest that HBD-3 is an additional factor in the pathogenesis of OA.

Adult↗

PubMed2005

Expression and regulation of antimicrobial peptides in articular joints.

Articular joint infection is a surprisingly rare event considering the frequency of joint arthrocentesis and other invasive procedures applied to limb joints. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides synovial membrane and articular cartilage with resistance to infection. We subsequently began a systematic analysis of in vitro and in vivo antimicrobially active proteins in healthy articular joints and in disease states such as pyogenic arthritis, rheumatoid arthritis, and osteoarthritis. An anatomical approach with systematic characterization combined with antimicrobial testing revealed expression and production of human antibiotic peptides and proteins. In this review, we focus on the most prominent antimicrobial proteins in articular joints, which we have identified as lysozyme, lactoferrin, secretory phospholipase A2, RNase 7, CAP37, the cathelicidin LL37, and especially the human beta-defensin-2 and -3 (HBD-2/-3). Activation pathways and possible antimicrobial functions are discussed and the involvement in non-antimicrobial processes such as tissue remodelling is also considered.

Cartilage, Articular↗

PubMed2005

The influence of biomechanical parameters on the expression of VEGF and endostatin in the bone and joint system.

Many degenerative processes in the skeletal system are induced by mechanical overload. Osteoarthritis and spontaneous tendon ruptures are two examples of mechanically influenced diseases. Incubator-housed compression apparatuses and cyclic strain chambers are adequate models to investigate the cellular processes. Recent studies have shown that growth factors are involved in the transduction pathways of mechanical overload leading to tissue degradation. Vascular endothelial growth factor (VEGF) is a dimerized, 45 kDa peptide that normally attracts endothelial cells in wound healing. VEGF can be detected in the superficial zone of the tibial plateau in osteoarthritic (OA) patients with degenerative changes but not in healthy articular cartilage. Blood vessels are only rarely observed in OA cartilage suggesting that there are other roles for VEGF in cartilage. VEGF is also detectable in ruptured but not in normal tendons. The mechanically induced expression of VEGF in avascular tissues like articular cartilage or fibrocartilage of contact areas from gliding tendons initiates degenerative processes. Chondrocytes from OA cartilage also express the VEGF receptor 2. In vitro assays have shown that VEGF binds the VEGFR-2 leading to a phosphorylation of MAP kinases (ERK1/2) with subsequent transcription factor accumulation (activator protein 1 = AP-1). One of the antagonists of VEGF is endostatin. Endostatin, a fragment of collagen type XVIII, is expressed in avascular tissues and has the potency to decrease VEGF induced effects (ERK1/2 phosphorylation). The increase in matrix metalloproteinase (MMP) production and the decrease in tissue inhibitor metalloproteinase (TIMP) synthesis is a result of the signal transduction cascade activation. MMPs participate in the degradation processes of osteoarthritis whereas TIMPs are inhibitors of the MMPs. Taken together mechanically induced VEGF is involved in the destruction and endostatin in the maintenance of avascular tissues of the bone and joint system.

Biomechanical Phenomena↗

PubMed2005

Differential expression of vascular endothelial growth factor implies the limbal origin of pterygia.

OBJECTIVE: To study the distribution of isoforms of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 in pterygia and to compare it with that in healthy conjunctivas. DESIGN: Nonrandomized comparative (cadaver controlled) study with histopathologic correlations. METHODS: Tissue specimens from 75 patients treated for primary pterygia were analyzed using immunohistochemical studies as well as different molecular biological examinations. Healthy conjunctivas from 33 patients treated for cataracts as well as specimens from the conjunctiva, limbus, and lens of both eyes of 12 body donors served as controls. TESTING: Surgical specimens of pterygia and normal conjunctiva specimens were processed with paraffin, sectioned, stained using specific antibodies against VEGF and its receptors, and examined by light microscopy. The other part of both groups of specimens as well as specimens from body donors were prepared and analyzed by means of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, enzyme-linked immunosorbent assay, and Western blots. MAIN OUTCOME PARAMETERS: Vascular endothelial growth factor and VEGFR1 and VEGFR2 were analyzed to indentify the splice variants of VEGF as well as the distribution and amount of VEGF and both receptors in pterygia and the control tissues. RESULTS: In analysis of specimens from pterygium patients as well as normal conjunctivas, VEGF121 and VEGF165 were identified as the only VEGF splice forms expressed. In addition to VEGF, VEGFR1 and VEGFR2 were detected in pterygia and conjunctivas and immunostained within the epithelium of pterygia and conjunctivas and on intrapterygial and intraconjunctival endothelial cells. Levels of VEGFR1 and VEGFR2 mRNA were lower in pterygia than in conjunctivas but similar in limbal and pterygium samples. Vascular endothelial growth factor levels were higher in pterygia than in conjunctivas, but were similar in the limbus and pterygia. CONCLUSIONS: The results reveal similar behaviors in limbal and pterygium epithelial cells in terms of VEGF and VEGFR expression, with the presumption that pterygia arise from limbal epithelial cells and that human conjunctivas are not a suitable control for the analysis of pterygia. Moreover, the results suggest that VEGF might play an active role in the physiology of conjunctival epithelial cells.

Blotting, Western↗

PubMed2005

Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells.

The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT-PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up-regulated by tumour necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin-like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA-induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT-PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6-positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.

Animals↗

PubMed2005

Expression of VEGF and its receptors in different brain tumors.

INTRODUCTION: Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and -2 are considered to play a major in tumor angiogenesis, which is a prerequisite for growth of solid tumors. Glioblastoma multiforme is a prominent example of VEGF-induced tumor vascularization; however, little is known about VEGF and in particular VEGFR expression in other types of brain tumors. METHODS: VEGFR mRNA was quantified by real time RT-PCR in 12 different types of brain tumors and compared to VEGF protein content measured by ELISA. VEGF splice variants were determined by an RT-PCR method. RESULTS: VEGF protein was highest in glioblastoma and metastatic kidney tumors. In all types of tumors the diffusible splice forms VEGF(121) and VEGF(165) were expressed; VEGF(189) was minor in a few tumors. Expression of VEGF receptors did not necessarily correlate with VEGF content. Both were highly expressed in glioblastomas, but in meningiomas VEGF was low and VEGFR high, and in metastatic tumors the reverse. With few exceptions, in particular oligodendrogliomas, VEGFR-1 expression was parallel to VEGFR-2 expression. Interestingly, for the astrocytic gliomas, the expression of VEGFR correlated well to the tumor malignancy, even better than VEGF content. CONCLUSIONS: These results show that VEGF and VEGFR expression in various types of brain tumors differ and are not necessarily parallel.

Blotting, Northern↗

PubMed2005

Therapeutic assessment of glucagon-like peptide-1 agonists compared with dipeptidyl peptidase IV inhibitors as potential antidiabetic drugs.

The most prevalent form of diabetes is non-insulin-dependent or Type 2 diabetes. Innovative strategies to enhance insulin secretion and thereby improve glucose tolerance in patients with this type of diabetes are currently under preclinical and clinical investigation. These therapies include the applications of incretin hormones; gut hormones released postprandially that stimulate insulin secretion in pancreatic beta-cells. Because incretin actions are rapidly terminated by N-terminal cleavage of these peptide hormones by the amino-peptidase dipeptidyl peptidase IV (DPP IV, CD26), the utility of DPP IV inhibitors for the treatment of Type 2 diabetes is also under investigation. This review compares the therapeutic potential and possible side effects of metabolically stable analogues/peptide agonists of the incretin glucagon-like peptide-1 (GLP-1) with the application of DPP IV inhibitors that reduce the rate of endogenous degradation of GLP-1 and other incretins. GLP-1 analogues have been shown to be highly efficacious in the treatment of Type 2 diabetes, with minimal side effects. Of particular importance is the fact that they do not induce hypoglycaemia. However, they are currently available only in an injectable form. In contrast, DPP IV inhibitors have the clear advantage of oral application resulting in better patient compliance. Furthermore, they also potentiate the actions of other incretins normally degraded by the action of DPP IV. However, they possess more potential side effects. Taken together, both approaches offer promising new drugs for the treatment of Type 2 diabetes.

Animals↗

PubMed2004

The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10.

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-gamma and TNF-alpha, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-gamma and TNF-alpha synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

ADAM Proteins↗

PubMed2004

Production of endogenous antibiotics in articular cartilage.

OBJECTIVE: Defensins are broad-spectrum antimicrobial peptides that are components of innate immunity. To date, only epithelial surfaces and blood cells have been shown to produce these cationic peptides in bactericidal concentrations when challenged with microorganisms or inflammatory cytokines. Infections caused by gram-negative pathogens occur only infrequently in association with joint surgery. The present study was undertaken to investigate whether this may be explained by intraarticular production of gram-negative-specialized antimicrobial peptides. METHODS: Healthy articular cartilage and cultured T/C-28a2 chondrocytes were assessed, by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, for expression of various antimicrobial peptides. The expression of human beta-defensin 2 (HBD-2) was studied in cultured chondrocytes after exposure to bacterial supernatants and proinflammatory cytokines and was assayed by real-time RT-PCR and immunoblot analysis. A septic arthritis mouse model was used to investigate the regulation of the murine homolog of HBD-2 in articular cartilage after bacterial inoculation. RESULTS: Healthy articular cartilage and T/C-28a2 chondrocytes were able to produce different antimicrobial peptides. After exposure to gram-negative bacteria and proinflammatory cytokines, expression of cartilage-derived HBD-2 strongly increased. Immunoblot analysis revealed up-regulation of the gram-negative-specialized HBD-2 in microbicidal doses. Immunohistochemistry analysis revealed induction of the murine homolog of HBD-2 in vivo after intraarticular injection of bacteria. CONCLUSION: This study demonstrated a previously unrecognized function of human chondrocytes. In addition to its biomechanical properties, articular cartilage has the ability to produce antimicrobial substances when challenged with microorganisms. The expression of HBD-2 in microbicidal doses suggests that antimicrobial peptides may contribute to host defense mechanisms in articular joints.

Adult↗

PubMed2004

Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes.

VEGF (vascular endothelial growth factor), an important angiogenesis factor, appears also to be involved in inflammatory processes. Recent studies have shown that VEGF and its receptors (VEGFR) are expressed on osteoarthritic, but not on normal adult, chondrocytes. To elucidate possible functions of VEGF in osteoarthritic cartilage, the effects of VEGF were studied on immortalized human chondrocytes. Activated matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, interleukin (IL)-1beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha) were measured in culture supernatants by enzyme-linked immunosorbent assays, nitric oxide with the Griess reagent, and cell proliferation by [3H]thymidine incorporation. VEGFR-2 mRNA was quantified by real-time reverse transcription-polymerase chain reaction and the protein was identified by immuno-gold electron microscopy. Intracellular signal transduction effects were determined by western blots and electrophoretic mobility shift assays. The chondrocyte cell lines C28/I2, C20/A4, and T/C28a2/a4 expressed functionally active VEGFR-2. VEGF stimulation induced receptor phosphorylation, activation of the mitogen-activated protein kinases ERK 1/2, and long-lasting activation of the transcription factor AP-1 (activator protein-1). VEGF increased secreted MMP-1, MMP-3, and especially MMP-13, which could be effectively reduced by an inhibitor of VEGFR-2 kinase activity. Interestingly, VEGF diminished the expression of TIMP-1 and especially TIMP-2. Under hypoxic conditions, as occur in cartilage, the reduction in TIMP levels was even greater. Furthermore, VEGF induced IL-1beta, IL-6, TNF-alpha, and nitric oxide expression to a small extent and stimulated the proliferation of immortalized chondrocytes. These findings indicate that VEGF is an autocrine stimulator of immortalized chondrocytes that mediates mainly destructive processes in osteoarthritis.

Autocrine Communication↗

PubMed2004

VEGF expression in adult permanent thyroid cartilage: implications for lack of cartilage ossification.

Vascular endothelial growth factor (VEGF) has been shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis, but it is not expressed in normal adult articular cartilage. Thyroid cartilage undergoes only partial ossification beginning at the age of about 20. Because it never completely ossifies, we investigated a possible role of VEGF and its receptors (VEGFRs) as well as the angiogenetic inhibitor endostatin in this permanent cartilage. In analysis of cartilage samples from all specimens evaluated, VEGF121 and VEGF165 were identified as the only VEGF splice forms expressed. In addition to VEGF, VEGFR-2 (kinase domain region/fetal liver kinase 1), but not VEGFR-1 (fms-like tyrosine kinase 1), was detectable by RT-PCR in cartilage. However, VEGFR-2 expression was only detectable up to the age of 19 years. Deposition of VEGF and VEGFR was confirmed by immunohistochemistry. VEGF concentrations measured by ELISA in thyroid cartilage increased with age in males but decreased in females. Endostatin concentrations measured by ELISA in thyroid cartilage were three times lower than in articular cartilage and showed no change with age, either in females or males. VEGF was immunostained within the intra- and pericellular matrices of some but not all chondrocytes. Thus, apart from its production in hypertrophic chondrocytes of growth plates, VEGF is also produced in single chondrocytes of thyroid cartilage. The data allow us to speculate that thyroid cartilage persists in an embryological state until it has reached its final size. After reaching its final size at the end of the second decade, VEGFR-2 is downregulated and ossification starts in the posterior part of the thyroid cartilage, proceeding ventrally. Both proteins, VEGF121 and VEGF165, should contribute to this process. VEGF concentration is high and changes in an age-related and sex-specific manner. Therefore, we postulate that VEGF is at least one of the key factors that is important for the lifelong ossification in thyroid cartilage.

Base Sequence↗

PubMed2004

Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles.

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Endostatin/collagen XVIII--an inhibitor of angiogenesis--is expressed in cartilage and fibrocartilage.

UNLABELLED: Aim of the study was to get a deeper insight in the mechanisms regulating avascularity of cartilaginious tissues. In the center of our interest was the expression of the anti-angiogenic fragment of collagen XVIII and its potency to inhibit angiogenesis. We observed a strong endostatin/collagen XVIII production in articular and fibrocartilage and an inhibitory potency concerning the VEGF-signalling pathway. INTRODUCTION: Cartilaginous tissue is mainly avascular and shows a limited intrinsic capacity for healing. Aim of this study was to investigate the expression of the antiangiogenic peptide endostatin/collagen XVIII in cartilage and fibrocartilage. RESULTS: In fetal epiphyseal cartilage of humans high endostatin/collagen XVIII levels could be detected by ELISA whereas significantly lower levels were found in articular cartilage of adults. In the fibrocartilaginous tissue of the menisci, there was no significant difference in the endostatin/collagen XVIII concentrations between samples of fetuses and adults. But in the menisci of adults, endostatin/collagen XVIII concentrations were higher in the internal avascular two thirds of the meniscus whereas in the fetal menisci higher endostatin/collagen XVIII concentrations were found in the external third. Endostatin/collagen XVIII immunostaining of rat articular cartilage shows that endostatin/collagen XVIII downregulation starts soon after birth. In fetal cartilage and fibrocartilage of rats and humans, endostatin/collagen XVIII could be immunostained in the extracellular matrix and in the pericellular matrix of endothelial cells, fibrochondrocytes and chondrocytes. In adult cells, weak endostatin/collagen XVIII immunostaining was restricted to the pericellular matrix of fibrochondrocytes and chondrocytes. The detection of endostatin/collagen XVIII could be verified by in situ hybridization. Chondrocytes in vitro released measurable amounts of endostatin/collagen XVIII into culture supernatants. Stimulation of chondrocytes with EGF, as an example of a growth factor, or dexamethasone had no influence on endostatin/collagen XVIII expression. Endostatin inhibited VEGF-induced phosphorylation of MAPK in chondrocytes. CONCLUSIONS: The spatial and temporal expression of endostatin/collagen XVIII in cartilaginous tissue and its potency regarding inactivation of VEGF signalling suggests that this antiangiogenic factor is important not only for the development but also for the maintenance of avascular zones in cartilage and fibrocartilage. EXPERIMENTAL PROCEDURES: We analyzed the spatial and temporal expression of endostatin/collagen XVIII--an endogenous angiogenesis inhibiting factor--in cartilage and fibrocartilage of humans and rats by immunohistochemical and biochemical (ELISA) methods and by in situ hybridization. To elucidate possible factors responsible for the induction or suppression of endostatin/collagen XVIII in cartilaginous tissues, chondrocytes (cell line C28/I2) were exposed to EGF and dexamethason. To study the possible interaction of endostatin/collagen XVIII with angiogenic factors, the immortalized human chondrocytes (C28/I2) have been incubated with VEGF and the phosphorylation of the MAPK Erk 1/2 (extracellular-regulated kinases), a known signal transduction pathway for VEGF has been determined under the influence of endostatin.

Aging↗

PubMed2004

Cyclic strain influences the expression of the vascular endothelial growth factor (VEGF) and the hypoxia inducible factor 1 alpha (HIF-1alpha) in tendon fibroblasts.

Neovascularization is involved in beneficial and detrimental processes of tendon pathology. We investigated the influence of repetitive motion on the expression of the most important angiogenic factor, the vascular endothelial growth factor (VEGF) in the 3T3 NIH fibroblast cell line and in cultures of rat Achilles tendon fibroblasts. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic uniaxial movement. Strain was applied over 24 h varying the frequency (0.5-1 Hz). Fibroblasts (3T3 fibroblasts and rat Achilles tendon cultures) cultivated without the application of cyclic strain released measurable VEGF amounts into their culture supernatants. Cyclic stretching of the cells with a frequency of 1 Hz resulted in an increased expression of VEGF. A low frequency (0.5 Hz) reduced VEGF expression to control levels. RT PCR revealed VEGF 121 and VEGF 165 as the only splice forms that were induced by cyclic stretching. Western blot experiments could further show that cyclic stretching induced activation of the transcription factor HIF-1alpha. These results demonstrate that mechanical factors are involved in the regulation of VEGF expression in tendon tissue.

3T3 Cells↗