Figure 1.
H2S promotes endothelial cell migration and microvessel tube formation.
(A) Representative micrographs of monolayer wounding assay in HUVECs treated with or without NaHS (50 µM) at 0 h and 6 h after treatment. (B) Statistical analysis of the scratch wounding assay. (C) Cell migration was also assessed by transwell boyden chamber assay. Shown are the representative micrographs and the values (D) of the migrated cells treated with 50 µM NaHS. (E) Representative micrographs of microvessel tube formation in HUVECs treated or not treated with NaHS (50 µM). Statistical analysis of tube length (F) and branching points (G). Data represent the means ± SE of five independent experiments. Each experiment was performed in duplicate. *P<0.05.
Figure 2.
H2S induces reorganization of the actin cytoskeleton in HUVECs.
(A) The effects of H2S on the actin cytoskeleton and on paxillin-containing focal adhesions. Serum-starved HUVECs were stimulated or not by 50 µM NaHS for indicated times. Thereafter, immunofluorescence analysis was performed using DAPI (blue) to stain nucleus, rhodamine-conjugated phalloidin (red) to stain F-actin and a mouse anti-paxillin monoclonal antibody followed by Alexa Fluor-labeled secondary antibody (green) to stain paxillin-contained focal adhesions. An overlay of fluorescent signals, generating yellow color in areas of colocalization, is shown on the right column. The thin arrows indicate lamellipodia, arrowheads indicate cell protrusions and thick arrows show paxillin-containing focal adhesions. Scale bars, 10 µm. Representative blots (B) and statistical values (C) showing that NaHS (50 µM) treatment has no effect on the phosphorylation of paxillin. The results are representative of three independent experiments. Values are means ± SE. NS, not significant.
Figure 3.
H2S selectively activates the Rho GTPase Rac1 in HUVECs but does not directly activate Rac1 in vitro.
(A, B) Pull down assays were conducted to detect the activity of Rho GTPases. Serum-starved HUVECs were treated with 50 µM NaHS for indicated times. Cells were then lysed and incubated with GST-PAK1-PBD-bound or GST-Rhotekin-RBD-bound glutathione beads. GTP-loaded Rac1 and Cdc42 bound to GST-PAK1-PBD (A) and GTP-loaded RhoA bound to GST-Rhotekin RBD (B) were detected by western blotting with antibodies against Rac1, Cdc42 or RhoA (upper panel). GTPγS loaded as positive control. Total cell lysates were also probed with the same antibodies to demonstrate that equal amounts of total protein were used in individual assays (lower panel). The results are representative of three independent experiments. (C) The time course for Rac1 activation by H2S. ELISA-based GTPase activation assays (G-LISA) of Rac1 were performed at various times after treatment with 50 µM NaHS. (D) In vitro interaction of H2S with Rac1. Human recombinant Rac1 was allowed to react with different concentrations of NaHS, and G-LISA assays were performed to detect the level of GTP-bound Rac1. The data are presented as means ± SE of three independent experiments each performed in triplicate. *P<0.05 vs control. NS, not significant.
Figure 4.
Dominant negative Rac1 inhibits the H2S-induced reorganization of the actin cytoskeleton in HUVECs.
(A) HUVECs were transfected with plasmids encoding EGFP only (EGFP) or dominant-negative Rac1 (EGFP-Rac1-T17N). Western blot analysis showed manifest levels of the EGFP-Rac1 fusion protein in HUVECs transfected with EGFP-Rac1-T17N vector but not in the cells transfected with EGFP control vector. (B) Transfected cells were starved and then treated with or without 50 µM NaHS for 12 min. Cells were then fixed and stained with rhodamine-labeled phalloidin for F-actin and anti-EGFP antibody for EGFP-tagged Rac1. Representative images are shown. Thin arrows point to the formed lamellipodia. Thick arrows indicate the fragments of disrupted F-actin around the nucleus. Scale bars, 10 µm. (C, D) Rac1 siRNA decreased the expression of Rac1 both on the mRNA levels (C) and protein levels (D). (C) Electroporation was used to transfect HUVECs with siRNA. 48 h after transfection, mRNA transcripts of Rac1 and GAPDH as measured by real-time PCR were significantly reduced by siRNA separately. (D) Representative blots were shown on the protein levels after 72 h transfection. Transfection of HUVECs with Rac1 siRNA and GAPDH siRNA specifically knocked down the expression of their respective target genes without affecting each other’s targets. Values represent the means ± SE, n = 9. *P<0.05.
Figure 5.
Dominant negative Rac1 and Rac1 siRNA both decrease H2S-promoted cell migration and tube formation in HUVECs.
Representative micrographs (A) and statistical analysis (B) of scratch wounding assays showing that expression of dominant-negative Rac1 (T17N) and Rac1 siRNA reduced the cell migration rate promoted by 50 µM NaHS. Representative graphs (C) and statistical data (D) of migrated cells in transwell boyden chamber assay indicating that dominant-negative Rac1 (T17N) and Rac1 siRNA affected the cell migration promoted by 50 µM NaHS. Representative pictures (E) and statistical data of tube length (F) and branching points (G) manifesting that dominant-negative Rac1 (T17N) and Rac1 siRNA blunted the microvessel tube formation in three-dimensional culture. Data represent the means ± SE of five independent experiments. Each experiment was performed in duplicate. *P<0.05.
Figure 6.
H2S signals through VEGFR-PI3K pathway to mediate the activation of Rac1 and to promote cell migration of HUVECs.
Representative blots (A) and statistical analysis (B) showing the effects of NaHS (50 µM) treatment on the phosphorylation of Akt, cofilin and MAPKs including ERK, p38 and JNK. (C) The action of pharmacologic inhibiting on the increased phosphorylation of Akt, ERK and cofilin triggered by NaHS and the respective statistical data (D). (E) G-LISA assays measuring the effect of recombinant VEGF (10 ng/ml) and different chemical inhibitors, including SU5416, LY294002, U0126 and NSC23766, on the activation of Rac1. (F) Statistical data of transwell boyden chamber assay showing that the promotion effect of NaHS on endothelial cell migration was inhibited by SU5416, LY294002 and U0126. VEGFR inhibitor-SU5416 (5 µM), PI3K inhibitor- LY294002 (10 µM), MEK inhibitor- U0126 (10 µM), Rac1 inhibitor- NSC23766 (50 µM). Data represent the means ± SE. *P<0.05. NS, not significant.
Figure 7.
The p110α isoform of PI3K is required for H2S-induced Rac1 activation and endothelial cell migration.
(A) HUVECs transfected with p110α, p110β, p110γ, p110δ or scrambled siRNAs were lysed and analyzed by immunoblotting for PI3K subunits and for Akt phosphorylation on site of Ser473. SiRNA oligonucleotides specifically knocked down expression of their target genes, and GAPDH was used as a loading control. (B) Densitometric analysis of three independent immunoblots to assess Akt phosphorylation levels and the levels of the p110 isoforms following the knockdown experiments. (C) G-LISA assays for Rac1 activation following the action of different p110 isoforms. 50 µM NaHS-induced Rac1 activation was prevented by p110α siRNA. Statistical data (D) and representative micrographs (E) of transwell boyden chamber assays showing that the promotion effect of NaHS on endothelial cell migration was inhibited by p110α siRNA. (G) G-LISA assays for Rac1 activation were also performed following the expression of dominant negative Akt (DN-Akt). Data showing that NaHS (50 µM) induced Rac1 activation cannot be prevented by DN-Akt. (H) The expression of the HA-tag protein demonstrates that cells were successfully transfected. CV, control vector. Data represent the means ± SE. **P<0.01. *P<0.05. NS, not significant.
Figure 8.
Dominant negative Rac1 and Rac1 siRNA abolish the phosphorylation of cofilin initiated by H2S, but have no effect on that of ERK.
Representative blots (A) and statistical analysis (B) showing the effects of dominant negative Rac1 on the phosphorylation of cofilin (Ser3) and ERK (Thr202/Tyr204). (C) Representative blots and statistical analysis (D) showing Rac1 siRNA on the phosphorylation of cofilin (Ser3), ERK (Thr202/Tyr204). Values represent means ± SE; n = 3 in each group. *P<0.05. NS, not significant.
Figure 9.
Model illustrating the underlying signal transduction pathways of H2S that affect the migration of endothelial cells.