Nanomechanical sensing of the endothelial cell response to anti-inflammatory action of 1-methylnicotinamide chloride

AM Kolodziejczyk,¹ GD Brzezinka,¹ K Khurana,^1,2 M Targosz-Korecka,¹ M Szymonski<sup>1

1</sup>Research Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Krakow, Poland; ²Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, Australia

Background: There is increasing evidence that cell elastic properties should change considerably in response to chemical agents affecting the physiological state of the endothelium. In this work, a novel assay for testing prospective endothelium-targeted agents in vitro is presented.
Materials and methods: The proposed methodology is based on nanoindentation spectroscopy using an atomic force microscope tip, which allows for quantitative evaluation of cell stiffness. As an example, we chose a pyridine derivative, 1-methylnicotinamide chloride (MNA), known to have antithrombotic and anti-inflammatory properties, as reported in recent in vivo experiments.
Results: First, we determined a concentration range of MNA in which physiological parameters of the endothelial cells in vitro are not affected. Then, cell dysfunction was induced by incubation with tumor necrosis factor-alpha (TNF-α) and the cellular response to MNA treatment after TNF-α incubation was studied. In parallel to the nanoindentation spectroscopy, the endothelium phenotype was characterized using a fluorescence spectroscopy with F-actin labeling, and biochemical methods, such as secretion measurements of both nitric oxide (NO), and prostacyclin (PGI₂) regulatory agents.
Conclusion: We found that MNA could reverse the dysfunction of the endothelium caused by inflammation, if applied in the proper time and to the concentration scheme established in our investigations. A surprisingly close correlation was found between effective Young's modulus of the cells and actin polymerization/depolymerization processes in the endothelium cortical cytoskeleton, as well as NO and PGI₂ levels. These results allow us to construct the physiological model of sequential intracellular pathways activated in the endothelium by MNA.

Keywords: endothelium, cell stiffness, 1-methylnicotinamide chloride, atomic force microscopy, nanomechanical sensing