Holger Köhler

Biomaterial-induced sarcoma: A novel model to study preneoplastic change.

In the study of carcinogenesis most interest has focused on carcinomas, as they represent the majority of human cancers. The recognition of the adenoma-carcinoma sequence both in humans and in animal experimental models has given the field of basic oncology the opportunity to elucidate individual mechanisms in the multistep development of carcinoma. The relative scarcity of human sarcomas coupled with the lack of adequate animal models has hampered understanding of the molecular genetic steps involved. We present an experimental model in the rat in which a high incidence of malignant mesenchymal tumors arise around a subcutaneously implanted biomaterial. Nine commercially available biomaterials were implanted in a total of 490 rats of the Fischer strain for 2 years. On average, macroscopic tumors were found in 25.8% of implantation sites over a period from 26 to 110 weeks after implantation. The most frequent tumors were malignant fibrous histiocytomas and pleomorphic sarcomas, although fibrosarcomas, leiomyosarcomas, and angiosarcomas readily developed, the latter especially around polyurethane implants. Of particular interest are the results of a detailed histological study of the capsules around the implanted biomaterials without tumors. Here a spectrum of change from focal proliferative lesions through preneoplastic proliferation to incipient sarcoma could be observed. A parallel immunohistochemical study of peri-implant capsules showed that proliferating cell nuclear antigen was of particular help in identifying these atypical proliferative lesions. To our knowledge this is the first description of a sarcoma model in which preneoplastic lesions can be readily identified and also reproducibly induced. This model provides the molecular biologist with defined stages in the development of mesenchymal malignancy, with which the multistage tumorigenesis hypothesis can be tested, analogous to the well-known adenoma-carcinoma sequence.

Comparative Studies on Vascular Endothelium in vitro

Recent studies have presented evidence that the processes of hypoxaemia and reperfusion are involved in several pathogenetic mechanisms of atherosclerotic lesions. The ability of hypoxaemia to activate circulating white blood cells (WBCs) and enhance WBC-endothelial cell (EC) interactions is suspected to be a major factor in deleterious processes in the blood vessel wall. Various groups have suggested that cell adhesion molecules (CAMs), such as ICAM-1, VCAM-1 and E-selectin and their leukocyte ligands are involved in intercellular activities of the relevant cell types. We studied the effects of different oxygen tensions, simulating normoxic conditions, hypoxia and hyperoxia in vitro with the help of an umbilical vein EC model in order to determine the effects of oxygenation on CAM presentation on vascular ECs with and without further cytokine and endotoxin (lipopolysaccharides; LPS) stimulation. Semi-quantitative analysis of ICAM-1, E-selectin and VCAM-1 was performed using cell enzyme immunoassay techniques. The presentation of ICAM-1, E-selectin and VCAM-1 remained on the whole unaffected by both hypoxia and hyperoxic conditioning after both 7 and 24 h. Stimulation of ICAM-1 by cytokines and LPS was only marginally influenced by the oxygen tension. Cytokine induction of E-selectin was not affected after 7 h and was even reduced under hypoxia, compared to the control culture after 24 h, while stimulation was increased by hyperoxia. VCAM-1 was reduced in both the hypoxic and hyperoxic culture, while being maximally stimulated by cytokines and LPS after 7 h. In general, an effect of hypoxia was not found without any further stimulation. Moreover, evidence is presented that reoxygenation might be the more important aspect in the mechanisms of ischaemia/reperfusion.

Effects of Cytokines on the Expression of Cell Adhesion Molecules by Cultured Human Omental Mesothelial Cells

Cultured mesothelial cells (HOMES) are very responsive to the proinflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha). E-selectin, ICAM-1 and VCAM-1 are known to play an important role, because they are presented by diverse cell types, for example endothelial cells (ECs), and interact with co-responding ligands on white blood cell membranes. In this study, the expression of ICAM-1, VCAM-1, E-selectin as well as PECAM-1 on cultured HOMES was studied over 5, 24, 48 and 72 h exposure to IL-1 beta, interferon-gamma and TNF-alpha. In previous studies we have shown that IL-1 beta and TNF-alpha increase the expression of ICAM-1, E-selectin and VCAM-1 on the cytoplasmatic membranes of HUVECs, HSVECs and HAFECs (ECs from human umbilical vein, saphenous vein and femoral artery, respectively). Using a comparative quantitative cell enzyme immunoassay, we found that expression of the adhesion molecules ICAM-1 and VCAM-1 was significantly increased on HOMES in a dose- and time-dependent manner, compared to nonstimulated cells. Thus, ICAM-1 increased dramatically after 5 h incubation with TNF-alpha. Values of about 450% of the control level were measured. VCAM-1 was similarly stimulated after 24 h incubation with the same cytokine, although its level of expression was significantly lower than that of ICAM-1. In contrast to findings in the literature, VCAM-1 was not found to be expressed constitutively. E-selectin was neither constitutively expressed nor markedly inducible on HOMES. Only weak expression was found after 24 h incubation with high-dose IL-1 beta. PECAM-1 was expressed constitutively, as became evident in antibody dilution studies. These data indicate that HOMES respond to inflammatory stimuli, in some ways in a similar fashion to vascular endothelial cells, but also show a specific pattern of antigen presentation. The results are important for a better understanding of inflammatory processes in serous cavities. The data are also relevant for the improvement of antithrombogenous surfaces of the lumina of vascular prostheses by cell seeding.

From cytotoxicity to biocompatibility testing in vitro: cell adhesion molecule expression defines a new set of parameters

Determination of potential cytotoxicity is a central issue in current biocompatibility testing standards such as ISO and ASTM. Most of these tests do not assess biocompatibility of a biomaterial with regard to cell function. This study was aimed at screening a number of potential parameters that could be included in assessment of cell functional aspects of biocompatibility. Human umbilical vein endothelial cells (HUVEC) were seeded directly on titanium, NiCr alloy, CoCr alloy, PMMA, PE, PU, PVC, and silicone, or were exposed to the material extracts. Cytotoxicity was assessed for these materials through MTT conversion, crystal violet protein determination and Ki67 expression. In addition, expression of the cell adhesion molecules E-selectin, cadherin-5 and PECAM, as well as of the adhesion-associated proteins fibronectin and vinculin (focal adhesions), was determined by immunocytochemistry and western blotting. Cytotoxicity was not detected with the material extracts. Cells were able to adhere to bare metals, but not polymers. Fibronectin preadsorption resulted in adhesion and spreading also on the polymers. Cells were able to establish cell–cell contacts and focal adhesions. Western blotting, in combination with differential detergent extraction, indicated that linkage of cell–cell adhesion markers to the cytoskeleton may be used as an additional parameter relevant to cell function.

Physiology and cell biology of the endothelium: a dynamic interface for cell communication.

This manuscript presents a brief overview of the physiology and cell biology of the endothelium, which is the basis for understanding the role of endothelial cells in pathological processes as diverse as atherosclerosis, tumour intravasation and multiple organ failure. Following consideration of general aspects of endothelial function in regulating haemostasis, vascular tone and growth, special emphasis will be placed on endothelial regulation of the inflammatory response, which centres on the microcirculation. A particular role in inflammation is played by cell adhesion molecules (CAM), expressed both on endothelial and blood cells. Cell and molecular biological methods to investigate the expression of CAM in endothelial cells in vitro will be presented, as well as novel data, indicating that cytokine-induced up-regulation of CAM in the endothelium may involve signal transduction pathways other than those culminating in the activation of NF-kappa B. Finally, the phenomenon of angiogenesis will be briefly reviewed as a characteristic of endothelial cell activity of central importance to both physiology and pathology and new experimental data presented from an in vitro model to study the ability of individual endothelial cells to form vessel-like structures. In comparative studies to investigate the roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, the dominant role of VEGF in the formation of capillary networks could be unequivocally demonstrated.

Differential expression of cell adhesion molecules in inflamed appendix: correlation with clinical stage

The diagnosis of ‘early inflamed’, ‘recurrent’ or ‘sub‐acute’ appendicitis is often difficult and accompanied by controversies between clinical data, histological findings, and their interpretation. The expression of the intercellular cell adhesion molecule‐1 (ICAM‐1), the vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin has been studied in 61 appendicectomy specimens for possible use as a diagnostic tool. This study demonstrates a different expression of CAM by endothelial (EC) and mesothelial cells (MC) in the various stages of appendicitis, with early E‐selectin and ICAM‐1 expression in EC, followed by VCAM‐1 in EC and MC. Appendices from patients with prolonged clinical symptoms defined by clinicians as ‘chronic’ appendicitis showed VCAM‐1 expression and occasionally weak expression of E‐selectin in EC. In several cases, discrepancies were found between the pre‐operative ‘clinical’ diagnosis, the histomorphological findings, and the immunohistological results. In this context, the expression of E‐selectin and VCAM‐1 in comparison with the histological features has potential significance in the diagnosis of ‘early acute’, ‘sub‐acute’ or ‘recurrent’ appendicitis. In addition, a correlation was demonstrated between the histological stages of appendicitis and the kinetics of CAM expression. The study also indicates that the time course of E‐selectin expression in vivo is longer than is suggested from in vitro data. Copyright

Increased Adhesion and Activation of Polymorphonuclear Neutrophil Granulocytes to Endothelial Cells under Heavy Metal Exposure in vitro

Heavy metals have been implicated in the mechanisms of endothelial damage. Influences of heavy metal ions on diverse cell types have been studied using a variety of in vitro and in vivo methods. Polymorphonuclear neutrophil granulocytes (PMNs) have physiological and pathological functions, including the modulation of adhesion to and destruction of endothelial cells (ECs). PMNs were studied during interaction with human umbilical vein ECs under exposure to zinc, nickel and cobalt using an in vitro model. We studied adhesion processes with the help of a computer-controlled image-analyzing system and examined the activation of PMNs by quantification of leukotriene B4 (LTB4) release. The biphasic effects of the evaluated heavy metals on PMN-EC adhesion, with stimulation at very high and very low molar concentrations, were observed. The release of LTB4 by PMNs increased during exposure to very low metal concentrations. The initiation of these important pathogenetic mechanisms of inflammation at very low metal ion concentrations, which give no morphological changes, must be regarded as potentially significant with respect to the toxic effects of heavy metals.

PECAM-1 Expression in Human Mesothelial Cells: An in vitro Study

Mesothelial cells are actively involved in inflammatory processes by expressing a set of cell adhesion molecules (CAMs). Transmigration of leukocytes into inflamed tissues requires a chemotactic stimulus and engagement of platelet-endothelial cell adhesion molecule-1 (PECAM-1). To investigate the kinetics involved in peritonitis, pure cultures of mesothelial cells are necessary. In previous studies, we have found that human mesothelial cells (HOMES) show a weak constitutive expression of PECAM-1, which cannot be further stimulated by cytokines. It is known that all serous cavities and body fluids contain numerous macrophages which strongly express this adhesion molecule. To identify the cells responsible for the expression of PECAM-1, mesothelial cells freshly obtained from omental tissue were isolated using PECAM-1-conjugated magnetic beads by cell sorting. For these studies, the negative as well as the positive fraction of isolated cells were used. As a control, freshly isolated monocytes were studied. Cell cultures were characterized by light and electron microscopy, as well as immunocytochemistry. The negative cell fraction was cultivated and stimulated for different times with tumor necrosis factor-alpha (30 and 300 U/ml), interleukin-1 beta (10 and 100 U/ml) and interferon-gamma (500 U/ml) and PECAM-1 expression was analyzed by a comparative quantitative cell enzyme immunoassay (EIA). The positive cell fraction was treated in the same manner. Both fractions of isolated cells showed strong positivity for cytokeratins 8, 18, 7 and 19, as well as vimentin. CD68, a monocyte marker, was not detected on mesothelial cells. In addition, EIA analysis confirmed the constitutive expression of PECAM-1 obtained from previous studies. This expression on HOMES was not inducible, irrespective of the type and concentration of cytokine studied. These data confirm PECAM-1 expression on mesothelial cells obtained from human omental tissue and suggest a critical role in transmigration of leukocytes during peritoneal inflammation.

Methodological Approaches to Biocompatibility and Haemocompatibility Testing of Biomaterials

Die ISO beschreibt Standards zur In-vitro-Testung von Materialien fur medizinische oder zahnmedizinische Applikationen hinsichtlich deren Zytotoxizitat und Hamokompatibilitat. Auf der Basis dieser ISO