Long term exposure to L-arginine accelerates endothelial cell senescence through arginase-II and S6K1 signaling

Abstract

L-arginine supplementation is proposed to improve health status or as adjunct therapy for diseases including cardiovascular diseases. However, controversial results and even detrimental effects of L-arginine supplementation are reported. We investigate potential mechanisms of L-arginine-induced detrimental effects on vascular endothelial cells. Human endothelial cells were exposed to a physiological (0.1 mmol/L) or pharmacological (0.5 mmol/L) concentration of L-arginine for 30 minutes (acute) or 7 days (chronic). The effects of L-arginine supplementation on endothelial senescence phenotype, i.e., levels of senescence-associated beta-galactosidase, expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, eNOS-uncoupling, arginase-II expression/activity, and mTORC1-S6K1 activity were analyzed. While acute L-arginine treatment enhances endothelial NO production accompanied with superoxide production and activation of S6K1 but no up-regulation of arginase-II, chronic L-arginine supplementation causes endothelial senescence, up-regulation of the adhesion molecule expression, and eNOS-uncoupling (decreased NO and enhanced superoxide production), which are associated with S6K1 activation and up-regulation of arginase-II. Silencing either S6K1 or arginase-II inhibits up-regulation/activation of each other, prevents endothelial dysfunction, adhesion molecule expression, and senescence under the chronic L-arginine supplementation condition. These results demonstrate that S6K1 and arginase-II form a positive circuit mediating the detrimental effects of chronic L-arginine supplementation on endothelial cells.

Figure 1. Chronic L-arginine supplementation promotes endothelial senescence, inflammation and eNOS-uncoupling.

Confluent young HUVECs were L-arginine-starved overnight followed by treatment with 0.1 mmol/L or 0.5 mmol/L L-arginine for 7 days (7 d) with change of medium every 48 hours. Cells were serum starved overnight prior to experiment. (A)SA-β gal assay. (B)Western blot analysis of ICAM-1/VCAM-1. Tubulin served as loading control. (C) Detection of O₂^.- and NO by DHE and DAF-2DA staining, respectively. The eNOS inhibitor L-NAME (1 mmol/L) was added to the cells 2 hours prior to the DAF-2DA staining. (D) Western blot analysis of eNOS protein levels with tubulin as loading control. The Western blot analysis of expression of ICAM-1, VCAM-1 and eNOS shown in (B) and (D), respectively, was performed with the same membrane. The quantification of the signals is presented as graphics below the corresponding images or blots. n represents the number of the repeated independent experiments. *p < 0.05 vs 0.1 mmol/L; #p < 0.05 vs 0.5 mmol/L. Scale bar = 200 μm

Figure 2. Acute L-arginine supplementation enhances production of both NO and superoxide anion

Cells were serum- and L-arginine-starved overnight followed by treatment with 0.1 mmol/L or 0.5 mmol/L L-arginine for 30 minutes. (A) Detection of O₂^.− and NO by DHE and DAF-2DA staining, respectively. (B) Western blot analysis of eNOS protein level with tubulin as loading control. (C) Western blot analysis of ICAM-1/VCAM-1 levels. The Western blot analysis shown in (B) and (C) was performed with the same membrane. Tubulin served as loading control. The quantification of the signals is presented as graphics at the corresponding right panels. *p < 0.05 vs 0.1 mmol/L. Scale bar = 200 μm

Figure 3. Chronic L-arginine supplementation induces Arg-II expression/activity and mTORC1-S6K1 signaling

Cells were treated as described in Fig.1. (A) Western blot analysis of Arg-II expression and arginase activity assay. (B) Western blots showing activation of mTORC1 and S6K1 as monitored by phosphorylation of S6K1-T389 and S6-S235/236, respectively. The graphics at the corresponding right panels present the quantification of the signals. *p < 0.05 vs 0.1 mmol/L

Figure 4. Acute L-arginine supplementation activates mTORC1-S6K1 signaling, but not Arg-II expression/activity

Endothelial cells were treated as described in Fig. 2. (A) Western Blots showing activation of mTORC1 and S6K1 as monitored by phosphorylation of S6K1-T389 and S6-S235/236, respectively. (B) Western Blot analysis of Arg-II expression and arginase activity assay. The graphics at the corresponding right panels present the quantification of the signals. *p < 0.05 vs 0.1 mmol/L

Figure 5. S6K1 and Arg-II form a positive regulatory circuit under the condition of chronic L-arginine supplementation

(A) Endothelial cells were treated as described in Fig. 1, except that the cells were transduced with the rAd/U6-LacZ^shRNA as control, rAd/U6-Arg-II^shRNA or -S6K1^shRNA after L-arginine-starvation and prior to the treatment with only 0.5 mmol/L of L-arginine for 7 days. Shown are Western blot analyses of S6-S235/236, total S6, Arg-II and S6K1, and the quantification of the signals. *p < 0.05 vs rAd/U6-LacZ^shRNA+0.5 mmol/L L-arginine control. (B) Endothelial cells were treated as described in Fig. 1, except that the cells were transduced with the rAd/U6-LacZ^shRNA as control or rAd/U6-Arg-II^shRNA48 hours prior to serum- and L-arginine-starvation. Shown are Western blot analyses of S6K1-T389, S6K1, S6-S235/236, total S6, Arg-II, tubulin, and the quantification of the signals. *p < 0.05 vs rAd/U6-LacZ^shRNA+0.1 mmol/L L-arginine

Figure 6. Arg-II and S6K1 mediate the chronic effect of L-arginine on cell senescence

Endothelial cells were treated as in Fig. 5. (A) SA-β gal assay. (B) Western blot analysis of eNOS, ICAM-1, VCAM-1. Tubulin served as loading control. (C) DHE staining for detection of O₂^.−, DAF-2DA staining for detection of NO. The graphics at the corresponding right panels present the quantification of the signals. *p < 0.05 vs rAd/U6-LacZshRNA control+0.5 mmol/L L-arginine. Scale bar = 200 μm

Figure 7. Arg-II and S6K1 mediate the chronic effect of L-arginine on cell senescence

Endothelial cells were treated as in Fig. 1 except that rapamycin (20 ng/ml) was added at same time as L-arginine (0.5 mmol/L). (A) SA-β gal assay. (B) DHE staining for detection of O₂^.−, DAF-2DA staining for detection of NO. (C) Western blot analysis of VCAM-1, ICAM-1, eNOS, Arg-II, S6K1-T389, S6K1, S6-S235/236 and S6. Tubulin served as loading control. The bar graphs are the quantifications of signals from the corresponding experiments shown in the left panels. *p < 0.05 vs L-arginine alone as the control (Con). Scale bar = 200 μm