Figure 2

3-MP mimics the proangiogenic property of H₂S. (A) Aortic rings were harvested from rats and cultured for 7 d in collagen gel in Opti-MEM medium containing 1% FBS in the presence or absence of 3-MP (10 µmol/L to 1 mmol/L) or NaHS (30 µmol/L), followed by the quantification of microvessels. Both 3-MP and NaHS increased microvessel formation (*p < 0.05 versus vehicle; n = 4/group), although at the increase of 3-MP from 100 µmol/L to 1 mmol/L did not elicit an additional increase in microvessel formation; in fact, a tendency for a decrease was noted. This type of concentration response is consistent with the often-reported bell-shaped pharmacological effect of H₂S where higher concentrations exert inhibitory effects on various cell functions. (B) bEnd3 cells were seeded in 12-well plates and cultured overnight. Following starvation, a wound scratch was made. Cells were subsequently incubated with 3-M (10 µmol/L to 1 mmol/L) or NaHS (30 µmol/L) for an additional 48 h, followed by the evaluation of scratch wound healing (µm). Both 3-MP and NaHS facilitated scratch wound healing responses (*p < 0.05 versus vehicle; n = 4/group). (C–D) The effects of 3-MP (10 µmol/L to 1 mmol/L) or NaHS (30 µmol/L) on bEnd3 cell migration (C) and cell proliferation (D). Both 3-MP and NaHS facilitated these responses (*p < 0.05 versus vehicle; n = 4/group), although at the highest concentration of 3-MP the cell proliferation response was no longer stimulated, consistent with the often-reported bell-shaped pharmacological effect of H₂S, where higher concentrations exert inhibitory effects on various cell functions. (E) bEnd3 cells were exposed to 100 µmol/L 3-MP for the indicated times. Cell lysates were analyzed by SDS/PAGE. PVDF membranes were blotted by using rabbit polyclonal antibodies against phosphorylated (Ser473) or total Akt. Increased levels of phosphorylated Akt were detected upon 3-MP treatment, starting at 30 min. The blot shows a representative experiment of n = 3 independent determinations conducted on different experimental days (F) The lentiviral shRNA vector targeting 3-MST was transfected into bEnd3 cells. The shRNA vector effectively inhibited the expression of 3-MST gene at the protein level, as shown by Western blot analysis (inset in F). Following 3-MST silencing, cells were seeded at the density of 3,000 cells per well in xCELLigence plates and proliferation was monitored for 72 h. Downregulation of 3-MST markedly reduced the proliferation rate of the bEnd3 cells. The graph shows a representative experiment of n = 3 independent determinations conducted on different experimental days; each data point represents mean ± SEM of n = 6 xCELLigence wells. (G–H) Cell lysates from wild-type cells, nontargeting (NT) shRNA cells and 3-MST shRNA cells were analyzed by SDS/PAGE and the analysis of the blots revealed low detectable levels of (G) phosphorylated Akt (ser473) and (H) phosphorylated VASP (ser239), a marker of protein kinase G activation. There was an inhibition of both of these responses in cells with 3-MST downregulation. The blots show a representative experiment of n = 3 independent determinations conducted on different experimental days. In all bar graphs, mean ± SEM values are shown.