Abstract
Lifestyle and genetic factors can lead to the development of atherosclerosis and, ultimately, cardiovascular adverse events. Rodent models are commonly used to investigate mechanism(s) of atherogenesis. However, the 3Rs principles, aiming to limit animal testing, encourage the scientific community to develop new physiologically relevant in vitro alternatives. Leveraging the 96-chip OrganoPlate®, a microfluidic platform, we have established a three-dimensional (3D) model of endothelial microvessels-on-a-chip under flow using primary human coronary arterial endothelial cells. As functional readout, we have set up an assay to measure the adhesion of monocytes to the lumen of perfused microvessels. For monitoring molecular changes in microvessels, we have established the staining and quantification of specific protein markers of inflammation and oxidative stress using high content imaging, as well as analyzed transcriptome changes using microarrays. To demonstrate its usefulness in systems toxicology, we leveraged our 3D vasculature-on-a-chip model to assess the impact of the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product, and the 3R4F reference cigarette on the adhesion of monocytic cells to endothelial microvessels. Our results show that THS 2.2 aerosol-conditioned medium had a reduced effect on monocyte-endothelium adhesion compared with 3R4F smoke-conditioned medium. In conclusion, we have established a relevant 3D vasculature-on-a-chip model for investigating leukocyte-endothelial microvessel adhesion. A case study illustrates how the model can be used for product testing in the context of systems toxicology-based risk assessment. The current model and its potential further development options also open perspectives of applications in vascular disease research and drug discovery.
Highlights
The endothelium is a single layer of cells at the interface between circulating blood and organ tissues, which plays critical roles in vascular processes, such as barrier permeability (Radeva and Waschke, 2018), vasotone regulation, leukocyte adhesiveness and extravasation, blood clotting and angiogenesis (Favero et al, 2014)
Rapid vessel formation and vessel stability for at least 8 days without ingrowth of human coronary artery endothelial cell (HCAEC) into the extracellular matrix (ECM) were achieved with a seeding density of 106 cells/mL together with collagen I 4 mg/mL ECM cultured in MV2 medium
To show stable vessel formation and endothelial marker expression, endothelial microvessels were fixed at different time points; 3D reconstructions show the reproducible formation of a complete vessel after 2 days
Summary
The endothelium is a single layer of cells at the interface between circulating blood and organ tissues, which plays critical roles in vascular processes, such as barrier permeability (Radeva and Waschke, 2018), vasotone regulation, leukocyte adhesiveness and extravasation, blood clotting and angiogenesis (Favero et al, 2014). Endothelial dysfunction, which increases permeability, adhesiveness and transmigration of leukocytes as well as the accumulation of fatty streaks in the subendothelial compartment (Insull, 2009), is a hallmark of atherosclerosis, characterized by the development of plaques that can become unstable and rupture, resulting in adverse cardiovascular events (Favero et al, 2014). The development of atherosclerosis and its prevention by therapeutic interventions is studied extensively in humans and in rodent models. The identification of potent drug compounds for therapeutic intervention and the testing of compounds/products for toxicological safety or risk assessments require high-throughput settings for a pre-screening phase that is generally not conducted in vivo. The perspective of the 3R principles, “replacement, reduction, and refinement,” which aim to limit animal testing (Burden et al, 2015), encourages the scientific community to develop
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