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

Abstract

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.

Fig. 1

Simvastatin induces autophagy in HMECs. (A–F) HMECs were treated with simvastatin (1 μM) or various statins including rosuvastatin (1 μM), lovastatin (1 μM), and atorvastatin (1 μM) for the indicated times and treatments. (A–C) The representative images and quantitative results of Western blot analysis of LC3, p62 and α-tubulin in HMECs treated with simvastatin (1 μM) in time-course manner. (D–F) The representative images and quantitative results of Western blot analysis of LC3 at 9 h and p62 at 18 h in HMECs incubated with simvastatin, rosuvastatin (1 μM), lovastatin (1 μM), and atorvastatin (1 μM). (G–J) The representative images and quantitative results of (G and H) acridine orange staining and (I and J) LC3 puncta in HMECs treated with simvastatin for the indicated times. Arrowheads indicate (G) acidic vacuoles or (I) LC3 puncta. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group.

Fig. 2

Simvastatin promotes NO bioavailability by activating autophagy in ECs. HMECs were pretreated with ATG7 siRNA (100 nM) for 24 h or pretreated with autophagy inhibitor, chloroquine (CQ, 40 μM) and bafilomycin A (BafA1, 10 nM) for 2 h, and then treated with simvastatin (1 μM) for another 9 h or 18 h. (A–C) The representative images and quantitative results of Western blot analysis of LC3 at 9 h, and p62 and ATG7 at 18 h. (D) The intracellular levels of nitrite were evaluated by Griess’ assay. (F–H) The representative images and quantitative results of Western blot analysis of LC3, p62 and α-tubulin. (I) The intracellular levels of nitrite were evaluated by Griess’ assay. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 3

Autophagy mediates simvastatin-induced proliferation, migration, and tube formation in ECs. HMECs were pretreated with CQ (40 μM) or BafA1 (10 nM) for 2 h and then incubated with simvastatin (1 μM) for additional 18 h. (A) The EC proliferation was assessed using the MTT assay. (B–D) The EC migration and tube formation were evaluated by the wound healing assay, trans-well assay, and tube formation assay. (E–G) The representative images of (E and F) HMEC migration and (G) tube formation. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 4

Inhibition of autophagy abolishes the simvastatin-activated urea cycle activation in ECs. (A–E) HMECs were treated with simvastatin (1 μM) for the indicated times. The changes in the levels (Δ) of (A) glutamate, (B) acetyl-CoA, (C) L-arginine, (D) urea, and (E) ornithine were assessed as compared to vehicle-treated group. (F and G) HMECs were pretreated with ASL siRNA (80 nM) for 24 h and then incubated with simvastatin (1 μM) for additional 18 h, and the changes in the levels of (F) L-arginine and (G) urea were evaluated as compared to vehicle-treated group. (H) The levels of nitrite were determined by Griess’ assay. (I–L) HMECs were pretreated with CQ (40 μM) or BafA1 (10 nM) for 2 h and then incubated with simvastatin (1 μM) for another 18 h. The changes in the levels of (I) glutamate, (J) acetyl-CoA, (K) L-arginine, and (L) urea were evaluated as compared to vehicle-treated group. The value of Δ is defined by the concentration difference in each group from the group of 0 h or vehicle-treated group. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 5

Knockdown of ASL expression prevents the simvastatin-induced increases in EC proliferation, migration and tube formation. HMECs were pretreated with ASL siRNA (80 nM) for 24 h and then treated with simvastatin (1 μM) for another 18 h. (A) EC proliferation was examined by using the MTT assay. (B–D) The quantitative results of HMEC migration and tube formation were evaluated by the wound healing assay, trans-well assay, and tube formation assay. (E–G) The representative images of HMEC migration and tube formation. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 6

AMPK mediates simvastatin-induced activation of autophagy and urea cycle, and NO production in ECs. HMECs were pretreated with AMPK siRNA (100 nM) for 24 h or compound C (C.C., 10 μmol/L) for 2 h, then incubated with simvastatin (1 μM) for additional 9 h or 18 h. (A–D) The representative images and quantitative results of Western blot analysis of LC3 at 9 h, and p62 and AMPK at 18 h. (E–G) Western blot analysis of LC3 at 9 h and p62 at 18 h. (H and I) The representative images and quantitative results of LC3 puncta after treatment with simvastatin for 9 h. Arrowheads refer to (H) LC3 puncta. (J and K) The changes in the levels (Δ) of urea and L-arginine. The value of Δ is defined by the concentration difference in each group from vehicle-treated group. (L) The levels of nitrite in culture medium were analyzed by Griess’s assay. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 7

Inhibition of TRPV1 abolishes simvastatin-induced activation of autophagy and urea cycle, and NO production. HMECs were pretreated with TRPV1 pharmacological antagonist, capsazepine (CPZ, 10 μM) or SB-366791 (10 μM) for 2 h and then incubated with simvastatin (1 μM) for another 9 h or 18h. (A–C) Western blot analysis of LC3 at 9 h and p62 at 18 h. (D and E) The representative images and quantitative results of LC3 puncta after treatment with simvastatin for 9 h. Arrowheads refer to (D) LC3 puncta. (F and G) The changes in the levels (Δ) of urea and L-arginine were determined as compared with vehicle-treated group. The value of Δ is defined by the concentration difference in each group from vehicle-treated group. (H) The levels of nitrite in culture medium were assessed by Griess’ assay. One-way ANOVA followed by Fisher’s Least Significant Difference was used to compare data from more than two groups. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. the vehicle-treated group; ^#P < 0.05 vs. the simvastatin-treated group.

Fig. 8

Schematic illustration of the proposed molecular mechanisms by which simvastatin elicits TRPV1-AMPK signaling and in turn activates the autophagy–urea cycle pathway to induce an increase in L-arginine bioavailability, leading to NO production in ECs.