WP8: Disease Models

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The great majority of studies exploring adverse effects of ENMs have been conducted in study settings mimicking effects in healthy individuals, but only part of the world’s population can be categorised into this group.

A significant part of the population has impaired cardiovascular (such as atherosclerosis or myocardial ischemia) or pulmonary (such as asthma or COPD) functions, health conditions that may make people more susceptible to health risks from particulate exposure.

Therefore, this work package is designed to identify the key characteristics of nanomaterials that control their fate and biological effects in cells and tissue from susceptible individuals.

Outcomes of the workpackage

The great majority of studies exploring potential pathogenic effects of ENM have been conducted using study settings mimicking effects in healthy individuals. However, individuals suffering from chronic illnesses, such as cardiovascular or pulmonary diseases, are thought to be more susceptible to develop health problems from particulate exposure. Therefore, the objectives of WP8 were to explore the effects of ENM exposure in diseased cells, tissues, or organisms either in vitro by using cultured human endothelial cells as well as a 3D in vitro airway model with cells from healthy and diseased human donors and or in vivo in murine cardiovascular disease and asthma models.

Findings from the first part of this WP demonstrate that the endothelial glycocalyx, a structure that constitutes a glycoprotein-polysaccharide meshwork coating the luminal surface of blood vessels, effectively controls ENM-endothelium interactions in the microvasculature. Constituents of the endothelial glycocalyx were found to physically cover endothelial adhesion and signalling molecules thereby preventing the endothelial attachment and uptake of ENM as well as their translocation through the vessel wall. Conversely, a degraded endothelial glycocalyx, e.g., under the pathological condition of ischemia-reperfusion, enables interactions of ENM with the microvascular endothelium, underlining the relevance of the endothelial glycocalyx for the protection of the tissue from blood-borne ENM in vivo. The results of two other studies strongly suggest that the surface functionalization of ENM determines their association with as well as their distribution pattern within atherosclerotic lesion in vivo, most likely by regulating the molecular composition of the biomolecule corona. The individual association behaviour of differently functionalized ENM appears to be based on a different affinity to molecular and cellular structures present in the atherosclerotic lesion environment.

The goal of the second part of this WP was to investigate the health effects of CuO ENM in both healthy and asthmatic individuals, and to elucidate the relevant molecular pathways involved in these effects. Using a murine asthma model, it was found that CuO ENM induced an acute inflammatory response when entering the lungs. Core CuO and its functionalized versions caused a significant influx of leukocytes, cells that protect our body against foreign invaders, into the airways of asthmatic subjects. Furthermore, relevant signalling molecules, that promote and mediate inflammatory reactions, were found in elevated levels in lung tissue of asthmatic mice as compared to their respective controls, especially in response to core CuO. Although the results obtained were qualitatively similar across the tested materials, it was found that surface modifications changed the immunomodulatory potential of the core CuO material. Based on the analysis of cells and solutes from the lower respiratory tract as well as on histological evaluation, the severity of pulmonary inflammation decreased in the order CuO-NH3 > core CuO > CuO-COOH > CuO-PEG. Work using the MucilAir™ in vitro 3D human airway model focussed on the secretion of proinflammatory cytokines as a marker for the development of an inflammatory response in the airways upon exposure to core CuO and CuO-COOH. The co-culturing of fully-differentiated human airway cells at an air-liquid interface in a 3D configuration that harbour a variety of physiologically relevant cellular mechanisms makes it a relevant in vitro model that resembles the in vivo situation as close as possible. The results show that a 1h-exposure through air to high concentrations of CuO and CuO-COOH NPs seemed to provoke an inflammatory response, hallmarked by a strong increase in the secretion of relevant proinflammatory mediators. Interestingly, exposure to CuO-COOH exposure was less toxic to the cells and also led to a less proinflammatory response. Despite the difference in the proinflammatory response between exposure to core CuO and CuO-COOH ENM, the analysis of gene expression profiles revealed a large overlap in affected pathways. Generally, stress-activated pathways and pathways involved in a proinflammatory response were upregulated. Another important, yet unexpected outcome of this study was the finding that cells that originated from healthy donors appeared to be more prone to develop an inflammatory response through CuO ENM exposure than cells from asthmatic individuals, while the latter showed a higher sensitivity for cytotoxicity. Taken together, the experimental setup used allows relevant exposure through air and is anticipated to enable relevant predictions of the bioavailability and adverse effects of inhaled compounds because the results do not need a translation step from dispersed, and thus altered, nanomaterials to real-life exposure. In conclusion, this WP demonstrated that combining the MucilAir™ model with an air exposure system is a practical and relevant approach to assess ENM inhalation toxicity in vitro, thus enabling the translation from in vivo animal data towards potential hazards of ENM exposure through air in humans.

In summary, WP8 has identified the endothelial glycocalyx as an important barrier against interactions of blood-borne ENM with the vessel wall and their translocation to extravascular tissue, verified the importance of the surface chemistry of ENM for their microdistribution in tissue and their association with cellular targets in atherosclerotic lesions, improved the understanding of how selected materials affect individuals with a pre-existing health condition, such as asthma, and identified similarities and dissimilarities between in vitro and in vivo models.