Christoph L. Klein

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: Effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts

OBJECTIVE Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

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.

Spot compliant neuronal networks by structure optimized micro-contact printing.

Neuronal cell growth in vitro can be controlled with micropatterned structures of extracellular matrix proteins such as laminin. This technique is a powerful tool for studying neuronal cell function in order to increase experimental reproducibility and to specifically design innovative experimental setups. In this paper the correlation between the structural dimensions of the ECM pattern and the shape of the resulting cellular network is analyzed. The aim of the present study was to position neuronal cell bodies as precisely as possible and to induce directed cell differentiation. PCC7-MzN cells were cultured on laminin patterns. The line width, node size and gap size in-between cell adhesion sites was varied systematically. Micrographs of the samples were taken and statistically analyzed using Students t-test and linear correlation methods. Precise cell positioning has successfully been performed and evidence for controlled neuronal polarization has been found. With a structure geometry of 4 microm line width, 20 microm node size and 10 microm gap size a nodal compliance of 86% (+/- 10%) has been achieved.

The cell and molecular biological approach to biomaterial research: a perspective

The past two decades have witnessed a revolution in our understanding of chemical processes in living organisms. This is mainly a result of the massive advances in the fields of cell and molecular biology. These techniques are highly relevant to the biomaterials sector, as they offer the scientist the possibility to better understand the mechanisms involved in the interactions between cells and a material surface—a prerequisite for the rational development of medical devices with optimal biocompatibility. The purpose of the present article is to explain the rationale of the cell and molecular biological approach to biomaterial research and to present typical examples from the authors’ laboratory, as well as from the literature, to illustrate its application. Important aspects of interfacial biology, including the underlying biological mechanisms and methodology, are presented. Of the latter the combination of morphological techniques with methods of cell and molecular biology as well as molecular genetics (so-called “combinative techniques”) are particularly useful. The applicability of this approach is illustrated from a study on the pathomechanisms of metal ion-induced inflammation. In addition, the approach is essential to the development of targeted intervention strategies, as for example in the luminal surface modification of vascular prostheses to permit endothelial cell seeding.

The role of the microcirculation in multiple organ dysfunction syndrome (MODS): a review and perspective

Major advances in intensive care medicine during the past two decades have altered the spectrum of disease encountered by intensive care physicians, anaesthesiologists, traumatologists and pathologists. One of the most important manifestations of severe trauma or infections is the multiple organ dysfunction syndrome (MODS), a life-threatening condition that often ends in multiple organ failure (MOF) and death. Evidence gathered from clinical and morphological observations in humans, taken together with experimental animal studies and a vast accumulation of in vitro data, clearly indicate that the microcirculation lies at the centre of this complex process, which results in peripheral vascular insufficiency, inadequate oxygen delivery to vital organs, and hence, severe organ dysfunction. The multifunctional nature of the endothelium makes it a prime candidate for study of the pathomechanisms of MODS. This paper reviews the evidence for the hypothesis that the microcirculation, and in particular its endothelial component, has a central role in the pathogenesis of MODS. The evidence is reviewed principally from the standpoints of classical morbid anatomy and cell pathobiology.

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.

Neuronal networks in vitro: formation and organization on biofunctionalized surfaces

Receptor-mediated recognition of substrate molecules is a prerequisite for nerve cells in order to find their target structures in vivo and leads to formation of neuronal connections and networks. In order to study these mechanisms under in vitro conditions, we cultured embryonic hippocampal neurons or neuronal cell lines, SH-SY5Y and PCC7-PCC7-Mz1, onto biofunctionalized surfaces. Micropatterning on polymer surfaces, glass- and silicone-oxide-based chip materials was performed in a micrometer range by microcontact printing using polydimethylsiloxane (PDMS) stamps. Hippocampal neurons were found to form networks on chip surfaces under serum-free conditions and remained functional for more than a week. Human neuroblastoma cells SH-SY5Y as well as PCC7-Mz1 stem cells were found to follow microcontact printed pattern on polystyrene surfaces. Both cell lines showed neuronal marker expression and were cultured for up to 7 days with serum containing culture medium. Widths of 3–5 µm of coating lines were found to enhance single cell spreading along the pattern. The techniques described in this study may be useful in promoting nerve cell regeneration and organization following transection due to trauma or surgery. The neuronal alignment and network formation in vitro may furthermore serve as a model system in the field of biosensors.

The role of metal corrosion in inflammatory processes: induction of adhesion molecules by heavy metal ions

Prosthetic devices undergo corrosion processes after implantation including the release of certain amounts of metal ions into the adjacent tissues. On reaching the bloodstream, a systemic influence of those ions may be envisaged. Cell adhesion molecules (CAMs) are recognized as an essential component of the mechanisms of endothelial damage. To study the influence of selected heavy metals on human umbilical vein endothelial cells (HUVEC) EIA methods were used to evaluate cellular expression of E-selectin, ICAM-1, VCAM-1 and GMP-140 under the influence of high (cytotoxic) very low (non-cytotoxic) concentrations of Zn, Ni, Co and Cr. The de novo synthesis of CAMs was studied with the help of mRNA analysis. Intermediate voltage immuno electron-microscopical imaging was performed to detect the localization on the cell surface of the adhesion molecules E-selectin and ICAM-1 under the influence of cytokines, which represent important factors in inflammatory processes. Very low concentrations of metal ions, which gave no significant influence on cell morphology, elicited a significant expression of CAMs on endothelial cells in vitro. Thus, for example, zinc, nickel and cobalt ions in concentrations of 1×10-9 M increased the expression of endothelial E-selectin, compared to the control after a 5 h incubation. Similar findings were established for zinc, nickel and cobalt ions also with regard to ICAM-1, VCAM-1 and GMP-140. Northern blot analysis gave an increased ELAM-1 and ICAM-1 mRNA expression after incubation with high concentrations of zinc and nickel ions. The results should draw attention to possible effects of very low concentrations, which are released during processes of metal corrosion on prosthetic devices.

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.

Dynamic blood cell contact with biomaterials: validation of a flow chamber system according to international standards.

The increasing number of patients requiring prosthetic substitution of segments of the vascular system strongly supports the need to optimize a relevant, standardized testing panel for new materials designed for synthetic vascular prostheses. The ISO gives the standard requirements for testing biomaterials provided for implantation. Our primary interest was the establishment of a reliable in vitro panel as a useful and relevant screening system for vascular implant devices to evaluate blood/device interactions under flow conditions. The aim of the present study was to evaluate influences of different flow conditions on blood cell–biomaterial interactions with special emphasis on the interactions of human granulocytes (PMN) and polymeric surfaces. PMN were isolated and vital cells were quantified by flow cytometrical analysis directly before, as well as immediately after the experiments. The viscosity of the final cellular suspension was analysed by using a computerized cone-plate rheometer. As reference materials we used FEP-teflon, PVC-DEHD, PU, PP and PE. Dacron and ePTFE synthetic vascular protheses were tested in a comparative way to those references. The adhesion processes were observed over a period of 40 minutes under arterial (shear stress 0.74 Pa) and venous (shear stress 0.16 Pa) flow conditions in a parallel plate flow chamber system under highly standardized conditions and laminar flow. The cells were observed with the help of inverse light microscopy. Cell behaviour was recorded and analysed in both analogue (video) and digital (imaging system) modes. Samples of the cell suspensions were obtained at regular time intervals and analysed by enzyme linked immuno sorbent assay (ELISA) to quantify LTB4 release. Irrespective of the material, approximately 3 to 4 times more PMN adhered to the biomaterial surfaces under venous flow conditions compared to the arterial. Shear intensity did not influence the running order of biomaterials with respect to cell numbers. This response in descending order at the end of the experiments was as follows: PU, PVC-DEHD, PP, PE and ePTFE. The biochemical analyses indicate that in the system used only a weak effect on LTB4 release induced by the different materials could be determined. A significant effect caused by flow conditions was not observed. Further experiments, both static as well as dynamic, must be performed for multiple, relevant parameters of haemocompatibility, for potential biomaterials as well as those currently in use in vascular prostheses.