Atheroprotective WSS effects on human aortic endothelial cells in a new generation bioreactor

Abstract

Abstract Funding Acknowledgements None. Background Luminal arterial endothelial cells are mainly aligned along the blood flow direction, whereas the in vitro endothelial cells (EC) showed heterogeneous distribution. In atherosclerosis the altered blood flow, acting on mechano-transduction, contributes to endothelial dysfunction and barrier disorganization. A low wall shear stress (WSS) has a pro-atherosclerotic effect, whilst elevated WSS is atheroprotective. Purposes Aim of this work is to study the primary human aortic EC cells (HAOEC) capability to acquire a distribution reminiscent of the luminal alignment under atheroprotective WSS using a prototypal new generation bioreactor. Methods We have developed a novel device compatible with culture inside standard sterile incubator, allowing to apply complex WSS patterns in controlled conditions. The bioreactor includes a sample housing and a parallel plate flow chamber and is controlled by a custom electronic interface actuating motor and pump. The cells were settled over PMMA supports and coating conditions were setup. Atheroprotective WSS patterns were tested. We compared HAOEC submitted to high flow (1.2 Pa for 24h) vs. ramps with different slopes (3h, 8h, 24h) followed by steady-state flow (1.2Pa for 24h, 48h). Viability and distribution of Acridine Orange-labelled HAOEC were assessed over bioreactor supports at both beginning and end of the experiments using fluorescence microscope. Hematoxylin/Eosin and Coomassie Blue staining were tested after the experiment to evaluate the shape and cell orientation (directionality FiJi plugin). Confocal microscopy on HAOEC labelled with Phalloidin–TRITC, anti-CD31/PECAM1-AlexaFluor488 and DAPI displayed the flow-related changes. Results HAOEC settled over naked sterile PMMA supports mainly detached under WSS, independently from the cell number. Coating with extracellular matrix components (i.e., human laminin, bovine fibronectin, chicken collagen type II, human collagen type I) showed the superiority of collagen type I to obtain homogeneously distributed, flow-resistant cell monolayers. In static condition, HAOEC on coated supports demonstrated ≥50% detachment within 24h, those submitted to a direct atheroprotective WSS of 1.2Pa almost completely detached. Consistently, to ramps with high slope (range 0.2-1,2Pa in 3h or 8h) followed by1.2Pa WSS corresponded 60%-90% cell detachment. Conversely, a 24h ramp (range 0.2-1,2Pa) followed by 24h or 48h of 1.2Pa WSS preserved the HAOEC monolayer adherence, and aligned cells were found. Microscopy comparative analysis of selected areas before/after WSS showed a partial spatial reorganization, with changes in the F-actin cytoskeleton and CD31 distribution. Conclusion We have optimized a protocol for studying HAOEC under atheroprotective WSS. In perspective further increasing the duration of the stimulus under constant WSS will achieve full alignment and provide a pseudo-physiologic model for multidirectional atherogenic WSS studies.