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


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.


CONCLUSIONS
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.
Research Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Krakow, Poland.
A M
AM
Kolodziejczyk
G D
GD
Brzezinka
K
K
Khurana
M
M
Targosz-Korecka
M
M
Szymonski
D000199:Actins / Q000378:metabolism; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004548:Elasticity / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D011464:Epoprostenol / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000199:Actins; D000893:Anti-Inflammatory Agents; D014409:Tumor Necrosis Factor-alpha; D009536:Niacinamide; D009569:Nitric Oxide; D011464:Epoprostenol; D002118:Calcium; C024058:N(1)-methylnicotinamide
1-methylnicotinamide chloride; atomic force microscopy; cell stiffness; endothelium; nanomechanical sensing