Iris Hermanns

Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock

In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn2+ and Cd2+ are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni2+ and Co2+ only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn(2+) and Cd(2+) are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni(2+) and Co(2+) only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.

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.

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.

MODULATION OF ENDOTOXIN-INDUCED NEUTROPHIL TRANSENDOTHELIAL MIGRATION BY ALVEOLAR EPITHELIUM IN A DEFINED BILAYER MODEL

Within the alveolus, epithelial cells, due to their close association with endothelial cells, can potentially influence endothelial cell responsiveness during inflammation and their interaction with leukocytes. To investigate this, three lung epithelial cell lines (A549, Calu-3, or NCI-H441) were grown with endothelium on opposing surfaces of Transwell filters and the formation and stability of bilayers was rigorously evaluated. All epithelial lines disrupted endothelial monolayer formation on filters with 3- or 5-µm pores by breaching the filter, and this occurred regardless of seeding density, matrix composition, or duration of culture. Endothelial disruption was not detectable by electrical resistance or permeability measurements but required cell-specific staining with immunofluorescence and microscopy. Distinct bilayers formed only on filters with 0.4-µm pores and only with A549 cells and human umbilical vein endothelial cells. Endotoxin (lipopolysaccharide [LPS]) stimulation of bilayers (4 hours) enhanced neutrophil transendothelial migration, but this was significantly decreased compared with the response of endothelium grown alone, irrespective of whether LPS exposure was via the epithelial or endothelial side of the bilayer. Down-modulation required epithelial-endothelial approximation and was not seen when these cells were separated by 0.5 to 1 mm. This study defines optimal conditions required for generation of intact bilayers of lung epithelial cells with endothelium for the study of leukocyte-transendothelial migration. Furthermore, it was demonstrated that lung epithelial cells can modulate endothelial cell responsiveness to an environmental inflammatory stimulus such as LPS and thus may have an important role in minimizing excessive and deleterious neutrophilic inflammation in the lung alveolus.

Age-related diagnostic value of D-dimer testing and the role of inflammation in patients with suspected deep vein thrombosis

Previous reports have investigated the impact of age on D-Dimer testing in elderly individuals with suspected deep vein thrombosis (DVT), but data on the age-related diagnostic value of D-dimer in a sample covering a broad age range are limited. The present study determined age-specifically the diagnostic accuracy of D-dimer and compared it to C-reactive protein (CRP), a marker of inflammation, in 500 patients with suspected DVT from the VTEval project (NCT02156401). Sensitivity of D-dimer was lower in patients < 60 years in comparison to patients ≥ 60 years (∆−16.8%), whereas specificity was 27.9% higher. Lowest levels of sensitivity were detected for female sex, unprovoked DVT, low thrombotic burden, and distal DVT. A fixed D-dimer threshold of 0.25 mg/L FEU resulted in elevated sensitivity for patients < 60 with a reduction of false negatives by 40.0% for proximal DVT and by 50.0% for distal DVT. In patients < 60 years, D-dimer and CRP demonstrated comparable diagnostic performance for both proximal and distal DVT (p > 0.05). In conclusion, these data outline a clinically-relevant limitation of D-dimer testing among younger patients with suspected DVT indicating a necessity for age-adapted cut-off values. Further research is required to decrypt the role of inflammation in the pathophysiology and diagnosis of venous thrombosis.

3.312 – Cell Culture Systems for Studying Biomaterial Interactions with Biological Barriers

The human body has numerous physical barriers that prevent most harmful or foreign compounds from entering the body. These barriers are formed by unique cell types, which through their location-specific biological cell characteristics prevent compounds from passing between or through them or selectively allow only specific compounds to move across the barrier that they form. Multiple cell types are involved that together form the functioning barrier in a particular organ or tissue. In many cases, in vitro human multicellular culture systems have been developed. These in vitro cell culture models have been extremely valuable in determining the toxic effects of novel compounds on cellular functions and in studying the mechanisms of barrier formation, the uptake and localization of the compounds within the cell, as well as the effects on cell–cell interactions. Moreover, they represent a major hurdle for micro- and nanoparticle biomaterials designed for drug and gene delivery to the body. The purpose of this chapter is to present an overview of the important barriers in the body and to describe in detail specific in vitro coculture models of the upper and lower respiratory units of the lung, models of the human skin and models to study the blood–brain barrier. How these models mimic the natural tissue, what criteria are used to validate the models, and how these model systems can be applied to the study of novel biomaterials, especially in micro- and nanoparticulate form, are described.

PO-58 - Cardiovascular risk profile in survivors of adult cancer - results from the general population study

INTRODUCTION The advancements in cancer treatment and detection of early cancer have resulted in steady increase of adult cancer survivors over the years. However, due to the long term toxic effects of chemotherapy and radiotherapy, the incidence of cardiovascular diseases (CVD) is increasing in survivors. Identifying risk factors and interventions to reduce the excess burden of CVD in this vulnerable population is urgently needed. AIM To investigate the cardiovascular risk factors (CVRFs), inflammation and coagulation profile in cancer survivors from a large population-based study. MATERIALS AND METHODS Presence of CVRFs and laboratory markers have been compared in individuals with (n=1,359) and without (n=13,626) history of cancer. Standard laboratory profile, including blood glucose and lipid profile, has been evaluated in 15,010 individuals from the Gutenberg Health Study (GHS). Coagulation factors, D-dimer and von Willebrand factor (vWF) activity were available in N=4,993. RESULTS The individuals with history of cancer were older compared to no history of cancer with mean age of 61,5years and 54.4years, respectively (p<0.001). Traditional CVRFs as diabetes (14% vs 8.8%), dyslipidemia (49.6% vs 43.7%) and hypertension (60.3 vs 48.7%) were more frequent whereas smoking was less frequent (14.5% vs 19.9%) in cancer survivors (p<0.001). The standard laboratory profile showed cancer survivors with lower erythrocyte, platelet and white blood cell counts and higher C-reactive protein (CRP), glucose, HbA1c and triglycerides levels (p<0.001). Multivariable logistic regression analysis adjusted for age, sex and CVRFs demonstrated an independent association with diabetes (odds ratio, OR: 1.24, 1.02-1.50; p=0.027) and higher CRP (OR: 1.01, 1.01-1.02; p=0.00071). Fibrinogen, FV, FVII, FVIII and FXI, D-dimer and vWF activity were higher in cancer survivors (p<0.001). Multivariable logistic regression confirmed an independent association with higher fibrinogen (OR: 1.002, 1.000-1.003) and vWF activity (OR: 1.005, 1.001-1.008). CONCLUSIONS This is the first study investigating CVRFs, inflammation and coagulation profile in individuals with history of cancer from a well characterized population-representative adult sample. It gives evidence for higher prevalence of CVRFs, particularly diabetes in this vulnerable population. Markers of inflammation as CRP and fibrinogen and vWF activity were higher in cancer survivors independent of the cardiovascular risk profile. These results underline the increased risk of CVD and need for development of cardio-oncology programs offering cardiovascular prevention.