Hydrogen sulphide and angiogenesis: mechanisms and applications

Abstract

In vascular tissues, hydrogen sulphide (H(2)S) is mainly produced from L-cysteine by the cystathionine gamma-lyase (CSE) enzyme. Recent studies show that administration of H(2)S to endothelial cells in culture stimulates cell proliferation, migration and tube formation. In addition, administration of H(2)S to chicken chorioallantoic membranes stimulates blood vessel growth and branching. Furthermore, in vivo administration of H(2)S to mice stimulates angiogenesis, as demonstrated in the Matrigel plug assay. Pathways involved in the angiogenic response of H(2)S include the PI-3K/Akt pathway, the mitogen activated protein kinase pathway, as well as ATP-sensitive potassium channels. Indirect evidence also suggests that the recently demonstrated role of H(2)S as an inhibitor of phosphodiesterases may play an additional role in its pro-angiogenic effect. The endogenous role of H(2)S in the angiogenic response has been demonstrated in the chicken chorioallantoic membranes, in endothelial cells in vitro and ex vivo. Importantly, the pro-angiogenic effect of vascular endothelial growth factor (but not of fibroblast growth factor) involves the endogenous production of H(2)S. The pro-angiogenic effects of H(2)S are also apparent in vivo: in a model of hindlimb ischaemia-induced angiogenesis, H(2)S induces a marked pro-angiogenic response; similarly, in a model of coronary ischaemia, H(2)S exerts angiogenic effects. Angiogenesis is crucial in the early stage of wound healing. Accordingly, topical administration of H(2)S promotes wound healing, whereas genetic ablation of CSE attenuates it. Pharmacological modulation of H(2)S-mediated angiogenic pathways may open the door for novel therapeutic approaches.

Figure 1

Concentration-dependent increase in the cell index by hydrogen sulphide (H₂S) in human umbilical vein endothelial cells, as measured by the XCelligence cell-microelectronic sensing technique method. Cells were cultured in gelatin pretreated 96-well E-plates. Cells were attached and grown overnight and subjected to vehicle or various concentrations of H₂S (10–300 µM) and cell index was continuously recorded over 5 h. Please note the concentration-dependent increase in cell index in response to H₂S (10–300 µM). At a higher concentration tested (1000 µM) (not shown), the stimulatory effect of H₂S was less pronounced that with 300 µM, consistent with the type of bell-shaped concentration-response curve often seen with H₂S. Responses shown are the mean ± SEM of n = 6 wells for each condition.

Figure 2

Increase by hydrogen sulphide (H₂S) of tube formation in the Matrigel assay in cultured human umbilical vein endothelial cells in vitro. Cells were plated at a density of 15 000 cells/well in 96-well plates, precoated with 50 µL of growth factor-reduced Matrigel in the presence of H₂S (60 µM) or vehicle, in complete medium. Following a 24 h-incubation, tube formation was quantified by image analysis software (Scion Image Release Beta 4.0.2) and expressed as a percentage of control. H₂S treatment significantly (P < 0.05) increased tube formation.

Figure 3

Exogenously applied and endogenously produced hydrogen sulphide (H₂S) promotes angiogenesis in the chicken chorioallantoic membrane. (A) Membranes were treated with H₂S (240 pmol·egg⁻¹) or the H₂S synthesis inhibitor dl-propylargylglycine (PAG) (300 mmol·egg⁻¹) or the combination of the two agents for 48 h and vascular network length and branching were determined. Note the enhancement of angiogenesis in response to H₂S, the inhibition of angiogenesis by PAG, and the lack of effect of PAG on the pro-angiogenic effect of H₂S. n = 36–45; *P < 0.05 versus vehicle. Panel (B) shows representative photomicrographs. BCA, beta-cyano-L-alanine.

Figure 4

Time-course showing the effect of hydrogen sulphide (H₂S) (60 µM) on a number of signalling pathways involved in the pro-angiogenic effect of H₂S in vascular endothelial cells. Please note the differential time-courses of the response. ERK, extracellular signal regulated kinase.

Figure 5

Signalling pathways involved in the pro-angiogenic effect of hydrogen sulphide (H₂S) in vascular endothelial cells. ERK, extracellular signal regulated kinase; K_ATP, ATP-sensitive potassium channel.

Figure 6

Incubation with vascular endothelial growth factor (VEGF) and hydrogen sulphide (H₂S) does not produce an additive effect on endothelial migration. Endothelial cells were allowed to migrate for 4 h in response to VEGF (20 ng·mL⁻¹), H₂S (60 µM) or a combination of the two. n = 5; *P < 0.05 versus vehicle.

Figure 7

H₂S is required for vascular endothelial growth factor (VEGF)-stimulated, but not fibroblast growth factor (FGF)-stimulated endothelial cells (EC) migration; role of K_ATP channels in the response. The EC were serum starved overnight. Cells were then treated with the K_ATP channel inhibitors glibenclamide (10 µM) or 5-HD (100 µM) for 30 min. Cells were then trypsinized, placed in transwells and allowed to migrate for 4 h in the presence of vehicle, VEGF (20 ng·mL⁻¹) (A) or FGF-2 (10 ng·mL⁻¹) (B). n = 5; *P < 0.05 versus vehicle and #P < 0.05 versus VEGF. In the experiments shown in (C), EC were treated with the cystathionine gamma-lyase inhibitor dl-propylargylglycine (PAG) (3 mM) for 30 min. Cells were then placed in transwells and allowed to migrate for 4 h in the presence of vehicle or VEGF (20 ng·mL⁻¹). n = 5; *P < 0.05 versus vehicle and $P < 0.05 versus VEGF.

Figure 8

The angiogenic actions of vascular endothelial growth factor (VEGF) are regulated by endogenously produced hydrogen sulphide (H₂S) in the in vitro aortic ring angiogenesis assay. Aortic ring explants from cystathionine gamma-lyase (CSE) wild-type (WT) or knockout (KO) mice were incubated in the presence or absence of VEGF (20 ng·mL⁻¹). Representative photomicrographs are presented in (A), and quantification of the response is shown in the right panel (B). Please note the substantially lower number of new microvessels in the CSE-deficient groups. From Papapetropoulos et al. 2009, reproduced with permission.

Figure 9

A proposed pathway of the interaction between nitric oxide (NO) and hydrogen sulphide (H₂S) in endothelial cells during angiogenesis. Vascular endothelial growth factor (VEGF) receptor activation increases NO production, leading to soluble guanylyl cyclase activation and increased cGMP synthesis. At the same time VEGF receptor activation triggers the generation of increased amounts of H₂S; H₂S inhibits cGMP-degrading phosphodiesterases leading to a further elevation of cGMP that in turn activates cGMP-dependent protein kinase and promotes angiogenesis. Because H₂S increases the levels of intracellular calcium, VEGF-stimulated H₂S production might enhance NO production. The relationship between these pathways and the canonical pathway of VEGF receptor activation-induced stimulation of the intracellular signalling axis of Raf, MAK and extracellular signal regulated kinase (ERK)1/2 remains to be delineated in future studies. PDE, phosphodiesterase; sGC, soluble guanylyl cyclase.

Figure 10

Hydrogen sulphide (H₂S) promotes re-epithelialization in burn wounds. Rats received a 30% total body surface area dorsal full-thickness scald burn under deep anaesthesia. Animals were treated daily with subcutaneous injections of vehicle, or H₂S (50 µg·cm⁻²·day⁻¹) at four equally spaced sites in the transition zone between burn eschar and healthy tissue. Planimetric measurement of the wound surface and re-epithelialization, as well as the ratio of wound contraction was performed. n = 6; *P < 0.05 versus control.

Figure 11

Hydrogen sulphide (H₂S) promotes collateral vessel formation and regional blood flow after femoral artery occlusion in the rat hind limb ischaemia model. (A) Representative post-mortem angiograms obtained 4 weeks after surgery. There was more collateral vessel formation in the ischaemic left hindlimb of the rats treated with NaHS at a dose of 100 µmol·kg⁻¹·day⁻¹. Arrow denotes the site of ligation at the femoral artery. The arrowhead indicates the typical ‘corkscrew’ appearance of collateral vessels. (B) Quantitative analysis of collateral vessel development was performed by measuring the total length of the contrast-opacified vessels. The angiographic score was significantly greater in the rats receiving NaHS (50 and 100 µmol·kg⁻¹·day⁻¹) than in the control animals (*P < 0.05). Data represent the mean ± SEM of three to five experiments in each group. (C) Blood flow measured with microsphere assay. The regional blood flow in ischaemic limb was standardized to tissue weight and is represented as the ratio of fluorescence intensity in the ischaemic hind limb to that of the contralateral nonischaemic hind limb in each animal. NaHS treatment (20 and 50 µmol·kg⁻¹·day⁻¹) significantly improved regional blood flow in the ischaemic limb (*P < 0.05). Data represent the mean ± SEM of seven to nine experiments (from Wang et al., 2010; reproduced with permission).