Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Abstract

Hydrogen sulfide (H2 S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2 S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2 S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2 S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2 S, stimulated NO production whereas blockage of the activity of H2 S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2 S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2 S on ECs. Our results suggest that H2 S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2 S-induced angiogenesis.

Keywords: CSE; Cystathionine gamma-lyase; Endothelial cells; Hydrogen sulfide; Nitric oxide; eNOS.

Fig. 1

H₂S stimulated NO production in endothelial cells (ECs) and aortic tissues. (A) The effect of NaHS on NO production in ECs detected by Griess assay, n = 4, *P < 0.05 versus control. (B) The effects of NO synthase (NOS) inhibitor L-NAME (200 μM, 1 hr), cystathionine gamma-lyase (CSE) inhibitor PPG (10 mM, 4 hrs), NaHS (100 μM, 30 min) and L-cysteine (6 mM, 30 min.) on NO production detected by the Griess assay, n = 3–4, *P < 0.05 versus control, #P < 0.05 versus NaHS or L-cysteine-treated groups. (C) The effects of CSE knockdown or overexpression on NO production assessed by the Griess assay. (D) The efficiency of CSE knockdown or overexpression, determined by Western blot, n = 3–4, *P < 0.05 versus control. The effect of NaHS (100 μM) and L-arginine (1 mM) treatment on NO production in isolated aortic tissues (scale bar: 50 μm) (E) and cultured ECs (F) using diaminofluorescein fluorophore system (DAF-FM) fluorescent probe (scale bar: 200 μm), n = 3–4.

Fig. 2

H₂S stimulated the phosphorylation of endothelial NO synthase (eNOS) in endothelial cells (ECs). (A) The effect of NaHS treatment on eNOS phosphorylation. ECs were starved in Dulbecco's modified eagles medium (DMEM) medium free of serum for 24 hrs and treated with different concentrations of NaHS for 30 min. Western blot analysis was conducted using anti-phospho-eNOS and anti-total eNOS antibody, n = 3–4, *P < 0.05 versus control. (B) Time-dependent effect of NaHS treatment on the phosphorylation of eNOS. ECs were treated with NaHS (100 μM) for different periods (0–60 min.). At the end of each time-point, cells were collected and proteins lysates were analysed by Western blot, n = 3–4, *P < 0.05 versus control. (C) The effect of NaHS treatment on eNOS expression level in ECs. The ECs were treated with NaHS (100 μM) for 12–36 hrs, and then cells were collected and proteins were subjected to Western blot analysis. n = 3–4, *P < 0.05 versus control.

Fig. 3

H₂S-induced phosphorylation of p38 MAPK, Akt and ERK. Endothelial cells (ECs) were treated with NaHS (100 μM) for different times (0–60 min.). At the end of each time-point, cells were collected and proteins lysates were analysed by Western blot, using antibodies specific for the phosphorylated and total forms of (A) p38 MAPK, (B) Akt, and (C) ERK. Data were normalized to total protein level, n = 3–4, *P < 0.05 versus control.

Fig. 4

H₂S-stimulated endothelial NO synthase (eNOS) phosphorylation is dependent on p38 MAPK and Akt. Endothelial cells (ECs) were pre-treated with (A) SB203580 (10 μM), (B) LY294002 (10 μM), and (C) U0126 (10 μM) for 1 hr and then treated with NaHS (100 μM) for 30 min. Cell lysates were harvested and the level of phosphorylated forms of p38 MAPK, Akt, ERK and eNOS were measured by Western blot. n = 3, *P < 0.05 versus control, #P < 0.05 versus NaHS-treated group.

Fig. 5

Crosstalk between p38 MAPK and Akt in H₂S-induced NO production. The p38 MAPK inhibitor inhibited Akt and NO production induced by H₂S. (A) Endothelial cells (ECs) were pre-treated with either SB203580 (10 μM) or LY294002 (10 μM) for 1 hr, and then treated with NaHS (100 μM) for 30 min. At the indicated time-point, the NOx generation was assessed by Griess assay, n = 3–4, *P < 0.05 versus control, #P < 0.05 versus NaHS. (B) The phosphorylated Akt was measured by Western blot after pre-treatment with SB203580 (10 μM) for 1 hr and NaHS (100 μM) treatment for 30 min., *P < 0.05 versus control, n = 3–4.

Fig. 6

H₂S-stimulated endothelial cell (EC) proliferation. (A) The effects of NaHS treatment on EC proliferation assessed using BrdU proliferation assay. n = 3, *P < 0.05 versus control. (B) The effects of cystathionine gamma-lyase (CSE) knockdown or overexpression on EC proliferation. n = 3, *P < 0.05 versus control. (C) The efficiency of endothelial NO synthase (eNOS) overexpression in ECs detected by Western blot, n = 3, *P < 0.05 versus Mock. (D) The effect of eNOS overexpression on EC proliferation. n = 3, *P < 0.05 versus Mock.

Fig. 7

H₂S interacts with NO to stimulate endothelial cell (EC) proliferation and angiogenesis. (A) The efficiency of endothelial NO synthase (eNOS) knockdown transfection in EC detected by Western blot. n = 3-4, *P < 0.05 versus control. (B) The effects of eNOS-knockdown (eNOS siRNA, 50 nM), NaHS (100 μM), and L-arginine (1 mM) treatments on EC proliferation evaluated by BrdU assay. n = 3-4, *P < 0.05 versus control, #P < 0.05 versus NaHS-treated group. (C) H₂S-NO interaction on EC tube formation. The effects of NaHS (100 μM) and L-NAME (200 μM) on tube formation of ECs (Scale Bar: 500 μm). (D) The effects of L-NAME (200 μM), L-arginine (1 mM) and NaHS (100 μM) on angiogenesis (scale bar: 200 μm), n = 3–4 mice for each group, *P < 0.05 versus control, #P < 0.05 versus NaHS-treated group. (E) The involvements of p38 MAPK and Akt in EC proliferation and tube formation. ECs were pre-treated with p38 MAPK inhibitor SB202190 (10 μM) and Akt inhibitor LY294002 (10 μM) for 1 hr, and treated with NaHS (100 μM) for 30 min. Cells (2 × 10⁴ cells) were seeded on Martigel for 12 hrs to assist the formation of capillary-like structure (scale bar, 500 μm). (F) Cells were pre-treated with LY294002 or SB202190 and NaHS. The cells were cultured for 24 hrs for measurement of proliferation rate using BrdU proliferation assay. n = 3–4, *P < 0.05 versus control.

Fig. 8

Schematic representation of proposed pathways of H₂S-induced NO production and angiogenesis.