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Rolf Mentlein

Publications and source records attributed to Rolf Mentlein.

At least 19 recordsLinked to original sources
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

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↗
PubMed2004

Mechanical overload induces VEGF in cartilage discs via hypoxia-inducible factor.

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.

Animals↗
PubMed2004

Cell-surface peptidases.

The cell surface has various functions: communicating with other cells, integrating into the tissue, and interacting with the extracellular matrix. Proteases play a key role in these processes. This review focuses on cell-surface peptidases (ectopeptidases, oligopeptidases) that are involved in the inactivation or activation of extracellular regulatory peptides, hormones, paracrine peptides, cytokines, and neuropeptides. The nomenclature of cell-surface peptidases is explained in relation to other proteases, and information is provided on membrane anchoring, catalytic sites, regulation, and, in particular, on their physiological and pharmacological importance. Furthermore, nonenzymatic (binding) functions and participation in intracellular signal transduction of cell surfaces peptidases are described. An overview on the different cell-surface peptidases is given, and their divergent functions are explained in detail. An example of actual pharmacological importance, dipeptidyl-peptidase IV (CD26), is discussed.

Animals↗
PubMed2004

Expression of VEGFR-1 and VEGFR-2 in degenerative Achilles tendons.

Evidence suggests neovascularization is involved in the etiology of spontaneous Achilles tendon rupture. Therefore we investigated expression of the receptors of one of the most important angiogenic factors, the vascular endothelial growth factor in normal and pathologic human Achilles tendons by immunohistochemical and molecular biology methods. Light microscopy showed degenerative changes in all spontaneously ruptured tendons with a higher vascular density compared with controls of healthy tendons from a cadaver. The vascular endothelial growth factor receptors-1 and 2 could be immunostained in endothelial cells of ruptured and fetal tendons, but not in normal tendons. Reverse transcription-polymerase chain reaction showed messenger ribonucleic acid of both receptors are expressed in ruptured and fetal tendons but not in normal tendons. These results prove the reexpression of the major receptors for the angiogenic peptide vascular endothelial growth factor, the vascular endothelial growth factor receptor-1, and the vascular endothelial growth factor receptor-2 in ruptured and fetal tendons and support our theory that high vascular density is associated with the pathogenesis of tendon degenerations. Inhibiting angiogenesis could be a new approach in the therapy of degenerative Achilles tendon disease.

Achilles Tendon↗
PubMed2003

Angiogenesis factors in gliomas: a new key to tumour therapy?

Angiogenesis, the formation of new blood vessels, is required for the growth and expansion of tumours. Gliomas, the most common brain tumours, are particularly highly vascularized and, therefore, serve as a model to elucidate the process of tumour angiogenesis and to investigate new anti-angiogenic therapies. This review describes the role of angiogenic factors in glioma angiogenesis and new strategies to inhibit glioma growth by application of anti-angiogenic substances. We focus on vascular endothelial growth factor (VEGF), but also examine the role of angiopoietin and pleiotropic factors such as platelet-derived growth factor (PDGF), pleiotrophin and transforming growth factor-beta (TGF-beta). Strategies to inhibit glioma growth by reducing the action of angiogenic factors, by the application of anti-angiogenic substances such as angiostatin or endostatin, or inactivation of endothelial cells, are discussed. These new anti-angiogenic therapies appear to have a high potential not only for the treatment of gliomas, but also of other tumours.

Angiogenesis Inhibitors↗
PubMed2003

The splice variants 120 and 164 of the angiogenic peptide vascular endothelial cell growth factor (VEGF) are expressed during Achilles tendon healing.

BACKGROUND: The Achilles tendon is one of the most common sites of tendon injury and rupture. One of the early events of wound healing is angiogenesis, in which neovascularization prompts delivery of inflammatory cells and fibroblasts to the wound site. Angiogenesis is controlled by a variety of mitogenic, chemotactic, or inhibitory peptides and lipid factors that act on invading endothelial and smooth muscle cells. One of the most important angiogenic factors is the vascular endothelial cell growth factor (VEGF), a glycosylated protein of 46-48 kDa composed of two disulfide-linked subunits. METHODS: We therefore investigated the expression of VEGF during healing of artificial lesions of the Achilles tendon in a sheep model by immunohistochemical, biochemical, molecular, and cell biology methods. Two groups were created, the Achilles tendon was tenotomized, and the animals were killed at 3 and 24 weeks. Each group consisted of 6 specimens. Six animals which did not undergo surgery served as controls. RESULTS: VEGF could be immunostained in tenocytes of ruptured but not in normal adult tendons. At microvessels, the receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR) could also be visualized. High VEGF levels in ruptured and negligible levels in normal Achilles tendons could be confirmed and quantified by enzyme-linked immunoassay (ELISA). The highest VEGF concentrations were found in ruptured tendons, whereas the VEGF content in healthy adult tendons was negligible. Interestingly, the VEGF concentration of the original tendon stump was higher after 3 weeks than that of the newly regenerated tendon tissue. However, this difference was not significant (p>0.05). Reverse transcription-polymerase chain reaction (RT-PCR) showed that the splice variants VEGF(120 )and VEGF(164) are expressed at 3 weeks and 24 weeks, respectively. CONCLUSION: These results prove the presence of the splice variants VEGF(120 )and VEGF(164) in ruptured tendons during the healing process. The demonstrated up-regulation of VEGF in intrinsic tenocytes suggests a role for VEGF in mechanisms of angiogenesis and Achilles tendon repair. Further research is needed to evaluate if VEGF might be a possible tool to enhance the process of tendon healing.

Animals↗
PubMed2003

Hypoxia and PDGF have a synergistic effect that increases the expression of the angiogenetic peptide vascular endothelial growth factor in Achilles tendon fibroblasts.

BACKGROUND: Angiogenesis, the formation of new blood vessels from pre-existing vessels, is an essential step in the process of tendon healing and tendon graft remodelling, e.g. after anterior cruciate ligament reconstruction. To address the question of whether platelet-derived growth factor (PDGF) could influence vascular endothelial growth factor (VEGF) expression in tenocytes, we challenged primary cultures of rat tenocytes with different stimulators (hypoxia, PDGF) and measured their VEGF secretion. METHODS: To measure the VEGF secretion and the effect of PDGF, we used cell culture and stimulation of rat tenocytes (2-5 days old), immunohistochemistry as well as enzyme-linked immunosorbent assay (ELISA). RESULTS: Tenocytes cultivated under normal oxygen pressure released measurable amounts of VEGF into their culture supernatants. Stimulation with PDGF, a cytokine that activates mitogen-activated protein kinases, raised VEGF secretion 2-fold. Hypoxic conditions alone (5% O(2)) raised VEGF secretion only 2-fold. However, the combination of cytokines and hypoxia increased VEGF production 5-fold, apparently a synergistic effect. CONCLUSION: These data are useful to further our understanding of angiogenesis in tendon healing and tendon graft remodelling after ACL reconstruction.

Achilles Tendon↗
PubMed2003

Expression of pleiotrophin, an embryonic growth and differentiation factor, in rheumatoid arthritis.

OBJECTIVE: Pleiotrophin (PTN), a 15.3-kd heparin-binding peptide, is expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. Since developmentally regulated factors often reappear during disease, we sought to determine whether there was PTN expression in the synovial membranes of patients with rheumatoid arthritis (RA). METHODS: PTN messenger RNA expression was assayed by quantitative reverse transcriptase-polymerase chain reaction. The protein was localized by immunohistochemistry and quantified by enzyme-linked immunosorbent assay (ELISA). Effects of PTN on cell proliferation in vitro were determined by DNA measurements. RESULTS: PTN expression in normal adult synovial membranes and cartilage was barely detectable. However, PTN was strongly up-regulated in synovial tissues from patients with RA. In contrast, samples from patients with pyogenic arthritis had moderate PTN levels, and those from patients with osteoarthritis had only a slight increase in PTN, as measured by ELISA. In RA patients, PTN was localized primarily in synoviocytes but was also found in endothelial cells of blood vessels. In cultured mouse fibroblasts used as a model, PTN expression was up-regulated by tumor necrosis factor alpha and was more weakly up-regulated by epidermal growth factor. Recombinant PTN stimulated the proliferation of cultured human synoviocytes and the monocyte cell line THP-1, but not human dermal fibroblasts, in which PTN increased the synthesis of vascular endothelial growth factor. CONCLUSION: In addition to certain types of cancer, the embryonic growth and differentiation factor PTN is expressed in adults with inflammatory diseases, in particular, RA. Proinflammatory cytokines enhance the expression of PTN. Thus, we propose that PTN is a further paracrine angiogenesis and growth factor for synovial cells in RA.

Animals↗
PubMed2003

The angiogenic peptide vascular endothelial growth factor (VEGF) is expressed in chronic sacral pressure ulcers.

Chronic decubital lesions have a limited potential to heal. Evidence suggests that a lack of local revascularization is involved in this aetiology. The present study investigated the expression of one of the most important angiogenic factors, the vascular endothelial growth factor (VEGF), in different regions of sacral chronic decubital lesions and in normal skin by immunohistochemical, biochemical, molecular, and cell biology methods. To elucidate some of the factors responsible for the induction of VEGF in chronic skin ulcers, cultured fibroblasts were exposed to hypoxia and/or growth factors. In the central part (zone I) of chronic ulcers and in normal skin, immunostaining for VEGF remained largely negative. However, VEGF could be immunostained in cells in the granulation tissue adjacent to central necrosis (zone II). VEGF receptor 2 (VEGFR-2; KDR) could also be identified in microvessels. High VEGF levels were present in homogenates from granulation tissue by enzyme-linked immunosorbent assay (ELISA) and western blot experiments: low concentrations were found in areas of central necrosis and negligible amounts were present in normal skin. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only the splice variants VEGF(121) and VEGF(165) were expressed. In cultured fibroblasts, hypoxia or platelet-derived growth factor (PDGF) raised VEGF production. The angiogenic peptide VEGF is present in all zones of chronic decubital ulcers. Its strong expression within the adjacent granulation tissue supports the view that there is no deficiency of VEGF. VEGF may be involved in the healing process of chronic skin lesions, but it seems that loss of another factor may be responsible for the poor healing response.

Aged↗
PubMed2003

CD26 expression determines lung metastasis in mutant F344 rats: involvement of NK cell function and soluble CD26.

The association between CD26 expression, tumor cell adhesion, metastasis, and natural killer (NK) cell function was investigated in a CD26 mutant Fischer 344 (F344/DuCrj) substrain from Japanese breeders (F344JAP) in comparison with wild-type F344 substrains from US (F344/Crl) and Hannover (HAN; F344/Ztm) breeders. F344JAP rats lack the dipeptidyl peptidase IV activity of CD26 and show a reduced cell surface expression of the mutated CD26 glycoprotein. In vivo adhesion of vital dye-labeled MADB106 tumor cells, tumor colonization, CD26 enzymatic activity, and CD26 immunoreactivity in lungs and soluble CD26-like protein expression in serum were markedly reduced in F344JAP rats. These findings demonstrate that CD26 protein expression exerts a key role in lung metastasis. In addition, NK cell cytotoxicity against MADB106 cells was diminished in the mutant F344 substrain, suggesting that CD26 enzymatic activity sustains NK cytotoxicity. Interestingly, tumor cells lacked CD26 immunoreactivity in vitro, but displayed CD26 immunoreactivity in situ after in vivo inoculation as well as after incubation with rat serum, indicating that soluble CD26-like protein assembles in tumor cells during in vivo passage, which may interact with the process of tumor adhesion and metastasis. Overall, these findings indicate that altered expression and function of a single enzyme-the CD26 protein--can drastically change the outcome of metastatic disease.

Adenocarcinoma↗
PubMed2003

The role of vascular endothelial growth factor in glucocorticoid-induced bone loss: evaluation in a minipig model.

Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis. To our knowledge there are no previous studies investigating the role of VEGF in osteoporosis. We hypothesized that VEGF expression in bone would be reduced under glucocorticoid (GC) treatment and tested this in a minipig model. As part of a larger study, 17 primiparous sows (Göttingen minipig) were allocated to two experimental groups when they were 15 months old: a control group (n = 9) and a group receiving GC treatment for 15 months (n = 8). All animals were fed a semisynthetic diet until they were sacrificed. The GC group received prednisolone orally at a daily dose of 1 mg/kg body wt for 8 weeks and thereafter 0.5 mg/kg body wt. VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. VEGF and its receptors (VEGFR) were localized by immunohistochemistry. Expression of VEGF-mRNA was analyzed by reverse transcription-polymerase chain reaction. VEGF protein was quantified in supernatants of cultivated osteoblasts by ELISA. Spinal bone mineral density was assessed in vivo by quantitative computed tomography. Expression of cyclooxygenase-2 (COX-2) protein was investigated by immunohistochemistry. High VEGF concentrations were measured in normal lumbar vertebrae whereas VEGF concentrations were 60% lower (P < 0.0001) in GC-treated minipigs. VEGF levels were associated (r = 0.7) with rates of spinal trabecular bone loss, which differed significantly (P < 0.0013) between controls (-0.47 +/- 2.2% SEM) and GC-treated minipigs (-12.8 +/- 2.3% SEM). Osteoblasts were immunopositive for VEGF. VEGF receptors VEGFR-2 (KDR, flk-1) and VEGFR-1 (flt-1) could be immunostained on osteoclasts and osteoblasts. VEGF-mRNA and protein were detectable in the lumbar vertebrae of all animals. The expression of COX-2 protein was decreased in GC-treated animals. VEGF is produced in osteoblasts and its concentration is decreased in GC-treated animals as well as in osteoblasts exposed to GC. Since reductions in VEGF concentrations correlate with parallel measurement of bone mineral density in GC-treated minipigs we hypothesize that VEGF may be an important modulating factor for bone remodeling, specifically in GC-induced osteoporosis. GC inhibit COX-2 and hence prostaglandin E2 (PGE2) production. Since PGE2 is able to increase VEGF synthesis, this may be the link between GC and VEGF decrease.

Adipocytes↗
PubMed2003

Mechanical factors influence the expression of endostatin--an inhibitor of angiogenesis--in tendons.

Avascular zones of tendons are predisposed for degenerative changes and spontaneous rupture. Therefore, we analyzed the expression of the endogenous angiogenesis inhibiting factor endostatin in human fetal and adult tendons by immunohistochemical and biochemical methods. Moreover, to elucidate factors involved in the regulation of vascularity, we exposed primary cultures of rat tendon cells to intermittent hydrostatic pressure (0.2 MPa, 0.1 Hz for 24 h), and measured the endostatin content by ELISA and the effect of the conditioned medium to the proliferation of human umbilical vein endothelial cells (HUVEC). In fetal tendons high endostatin levels could be quantified by ELISA whereas low levels were found in adult tissue. In fetal tendons endostatin could also be immunostained in endothelial cells but mainly in fibroblasts. In adult Achilles tendons endostatin immunostaining was restricted to endothelial cells. In the tibialis posterior tendon--as an example for "wrap around"--endostatin immunostaining remained positive in the fibrocartilage adjacent to the medial malleolus. Fibrochondrocytes of the type II collagen positive fibrocartilage were intensively stained with the endostatin antibody. Factor VIII immunostaining showed that this region was largely avascular. Monolayer cultures of tendon cells released measurable amounts of endostatin into their culture supernatants. Application of intermittent hydrostatic pressure increased endostatin expression significantly. The conditioned media of tendon fibroblasts cultivated under intermittent hydrostatic pressure inhibited the proliferation of HUVEC in a dose dependent way. The spatial expression of endostatin in adult gliding tendons suggests that mechanical factors are involved in the regulation of this anti-angiogenic factor. In accordance, tendon cells exposed to intermittent hydrostatic pressure inhibit endothelial cell proliferation via humoral factors and produce endostatin. These findings support the view that the development and maintenance of avascular zones in tendons might be caused by a mechanically induced upregulation of anti-angiogenic factors.

Achilles Tendon↗