Luminal Surface Plasma Treatment of Closed Cylindrical Microchannels: A Tool toward the Creation of On-Chip Vascular Endothelium.

On-chip vascular microfluidic models provide a great tool to study aspects of cardiovascular diseases in vitro. To produce such models, polydimethylsiloxane (PDMS) has been the most widely used material. For biological applications, its hydrophobic surface has to be modified. The major approach has been plasma-based surface oxidation, which has been very challenging in the case of channels enclosed within a microfluidic chip. The preparation of the chip combined a 3D-printed mold with soft lithography and commonly available materials. We have introduced the high-frequency low-pressure air-plasma surface modification of seamless channels enclosed within a PDMS microfluidic chip. The plasma treatment modified the luminal surface more uniformly than in previous works. Such a setup enabled a higher degree of design freedom and a possibility of rapid prototyping. Further, plasma treatment in combination with collagen IV coating created a biomimetic surface for efficient adhesion of vascular endothelial cells as well as promoted long-term cell culture stability under flow. The cells within the channels were highly viable and showed physiological behavior, confirming the benefit of the presented surface modification.

Myeloperoxidase mediated alteration of endothelial function is dependent on its cationic charge.

Endothelial cell (EC) glycocalyx (GLX) comprise a multicomponent layer of proteoglycans and glycoproteins. Alteration of its integrity contributes to chronic vascular inflammation and leads to the development of cardiovascular diseases. Myeloperoxidase (MPO), a highly abundant enzyme released by polymorphonuclear neutrophils, binds to the GLX and deleteriously affects vascular EC functions. The focus of this study was to elucidate the mechanisms of MPO-mediated alteration of GLX molecules, and to unravel subsequent changes in endothelial integrity and function. MPO binding to GLX of human ECs and subsequent internalization was mediated by cell surface heparan sulfate chains. Moreover, interaction of MPO, which is carrying a cationic charge, with anionic glycosaminoglycans (GAGs) resulted in reduction of their relative charge. By means of micro-viscometry and atomic force microscopy, we disclosed that MPO can crosslink GAG chains. MPO-dependent modulation of GLX structure was further supported by alteration of wheat germ agglutinin staining. Increased expression of ICAM-1 documented endothelial cell activation by both catalytically active and also inactive MPO. Furthermore, MPO increased vascular permeability connected with reorganization of intracellular junctions, however, this was dependent on MPO's catalytic activity. Novel proteins interacting with MPO during transcytosis were identified by proteomic analysis. Altogether, these findings provide evidence that MPO through interaction with GAGs modulates overall charge of the GLX, causing modification of its structure and thus affecting EC function. Importantly, our results also suggest a number of proteins interacting with MPO that possess a variety of cellular localizations and functions.