Browsing by Author "Pufe, T."

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 fibrochondrccytes 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 inununohistochemical 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. (C) 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Background: After clinical lung transplantation, the amount of vascular endothelial growth factor (VEGF) was found to be decreased in the bronchoalveolar lavage from lungs with acute lung injury. Since Type II pneumocytes are a major site of VEGF synthesis, VEGF depression may be an indicator of pulmonary epithelial damage after ischemia and reperfusion. Methods: Using an established rat lung model, we investigated the relationship between VEGF protein expression, oxygenation capacity and structural integrity after extracorporeal ischemia and reperfusion (ischemia 6 hours at 10degreesC, reperfusion 50 minutes) and preservation with either low-potassium dextran solution (Perfadex 40 kD, n = 8) or Celsior (n = 6). Untreated, non-ischemic lungs served as controls (n = 5 per group). Perfusate oxygenation was recorded during reperfusion. An enzyme-linked immunoassay (ELISA) for VEGF protein and reverse transcription-polymerase chain reaction (RT-PCR) for mRNA splice variants were determined on tissue collected from the left lungs, whereas the right lungs were fixed by vascular perfusion for VEGF immunohistochemistry as well as structural analysis by light and electron microscopy. Tissue collection by systematic uniform random sampling was representative for the whole organ and allowed for quantification of structures by stereological means. Results: After ischemia and reperfusion, the 3 major VEGF isoforms, VEGF(120), VEGF(164) and VEGF(188), were present. VEGF protein expression was reduced, which correlated significantly with perfusate oxygenation (r = 0.736; p = 0.002) at the end of reperfusion. It was inversely related to Type 11 cell volume (r = 0.600; p = 0.047). VEGF protein was localized by immunohistochemistry in Type 11 pneumocytes, alveolar macrophages as well as bronchial epithelium, and staining intensity of Type II cells was reduced after ischemia and reperfusion. Alveolar edema did not occur but significant interstitial edema accumulated around vessels and in the blood-gas barrier, which showed a higher degree of epithelial damage after preservation with Celsior compared with the other groups. Conclusions: Depression in VEGF protein expression can be considered an indicator for increased alveolar epithelial damage. Preservation with low-potassium, dextran solution resulted in improved oxygenation and tissue integrity compared with Celsior.