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

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. 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. 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. 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 PGI_{2] levels. These results allow us to construct the physiological model of sequential intracellular pathways activated in the endothelium by MNA.