Impact of TLR3 activation on endothelial functions: insights into iNOS-dependent mechanisms

This study aimed to identify the role of inducible nitric oxide synthase (iNOS) in regulating endothelial cell (EC) function within the toll-like receptor 3 (TLR3) signaling pathway.

To activate TLR3 and inhibit iNOS, the TLR3 agonist poly(I: C) and the selective iNOS inhibitor 1400 W were employed. Levels of iNOS, eNOS, and reactive oxygen species (ROS) were measured, and mitochondrial membrane potential (ΔΨm) was assessed to evaluate mitochondrial function. Cell proliferation, cell survival/growth, and cell death were evaluated using the BrdU assay, serial DAPI-based cell counting, and the apoptosis/death readout, respectively; caspase-3 activity was also measured.

Activation of TLR3 increased iNOS levels while concurrently decreasing eNOS expression in an iNOS-independent manner. TLR3 activation also elevated intracellular ROS production and induced mitochondrial dysfunction and impaired cell survival through an iNOS-dependent mechanism. Early proliferative activity was transiently increased (24 h) in an iNOS-dependent manner, while ongoing cell death led to a net reduction in cell number.

iNOS is a major contributor to ROS production within the TLR3 pathway but exerts no notable influence on eNOS expression. These findings identify iNOS as a selective therapeutic target for TLR3-triggered vascular disorders, potentially obviating the need for eNOS inhibition.