NAD<sup>+</sup> Enhancer Nicotinamide Riboside Alters Extracellular Purine Metabolism in Human Endothelial Cells.

Nicotinamide adenine dinucleotide (NAD⁺) is essential for maintaining homeostasis in all types of cells, including endothelium, and depletion of its pool can impair bioenergetics and stress response, contributing to cardiovascular disorders. Nicotinamide riboside (NR) effectively restores the intracellular NAD⁺ pool, supporting endothelial integrity, but the molecular mechanisms remain incompletely elucidated, particularly regarding extracellular adenine nucleotide catabolism, purinergic signaling, and their effects on immune cell adhesion. In this study, we aimed to investigate the effects of NR on intracellular nucleotides, extracellular adenine nucleotide catabolism, and adhesive properties in the cultured murine (H5V) and human (HMEC-1) microvascular endothelial cell line. We demonstrated that NR treatment significantly increased intracellular NAD⁺ concentrations without changes in the energy status of endothelial cells. We also showed that NR treatment accelerated extracellular hydrolysis of ATP and AMP and decreased the rate of adenosine deamination in endothelial cells. Moreover, we observed CD73 activity and adenosine-related reduced adhesion of T-lymphocytes, monocytes and platelets to the NR-treated endothelial monolayer. Our findings highlight a previously unrecognized role of NR in maintaining endothelial homeostasis, showing that NR is not only a potent intracellular NAD⁺ booster in endothelial cells but also affects extracellular nucleotide metabolism in a way that promotes cytoprotective adenosine formation.