Autophagy–urea cycle pathway is essential for the statin-mediated nitric oxide bioavailability in endothelial cells

Statins induce nitric oxide (NO) bioavailability by activating endothelial nitric oxide synthase via kinase- and calcium-dependent pathways in endothelial cells (ECs). However, their effect on the metabolism of L-arginine, the precursor for NO biosynthesis, and regulatory mechanism have not yet been investigated. In this study, we investigated the role of the autophagy–urea cycle-L-arginine pathway in simvastatin-mediated NO bioavailability in ECs. Griess’s assay was used to determine the NO bioavailability. Protein expression was assessed using Western blot analysis. Further, immunocytochemistry was performed to observe autophagosome formation, while conventional assay kits were used to quantify the levels of different intermediate substrates of the urea cycle. In ECs, treatment with simvastatin induced the activation of autophagy flux, as evidenced by the increased levels of microtubule-associated protein 1A/1B-light chain 3 II and autophagolysosome formation and decreased levels of p62. Inhibition of autophagy by ATG7 small interfering RNA (siRNA), chloroquine and bafilomycin A1 abolished simvastatin-induced NO bioavailability, EC proliferation, migration, and tube formation. Additionally, simvastatin increased the intermediate substrates levels of the urea cycle, including glutamate, acetyl-CoA, urea, and L-arginine, all of which were abrogated by chloroquine or bafilomycin A1. Genetic knockdown of argininosuccinate lyase using siRNA abrogated simvastatin-induced increase in NO bioavailability and EC-related functions. Moreover, inhibition of AMP-activated protein kinase (AMPK) and transient receptor potential vanilloid 1 (TRPV1) prevented simvastatin-induced activation of the autophagy–urea cycle pathway and NO production. Our findings suggest that simvastatin activates the autophagy–urea cycle pathway via TRPV1-AMPK signaling, which increases L-arginine bioavailability and ultimately promotes NO production in ECs.