2017 Volume 81 Issue 6 Pages 870-878
Background: Hydrogen sulfide (H2S) exerts beneficial actions against the development of cardiovascular disease. Diallyl trisulfide (DATS) is an organic polysulfide found in garlic oil that liberates H2S under physiological conditions. This study investigated whether DATS modulates endothelial cell function, as well as revascularization processes in a mouse model of hind-limb ischemia.
Methods and Results: Wild-type (WT), endothelial nitric oxide synthase-deficient (eNOS-KO) and Akt1-heterogenic deficient (Akt-Het) mice were subjected to unilateral hindlimb ischemia (HLI). DATS or a vehicle control was injected into the abdomen of mice for up to 10 days following HLI induction. Treatment with DATS enhanced blood flow recovery and capillary density in the ischemic limbs of WT mice. This was accompanied by a reduction in apoptotic activity and oxidative stress in the ischemic muscles. DATS also increased the phosphorylation of Akt and eNOS in ischemic muscles. In contrast to WT mice, DATS did not improve blood flow of eNOS-KO and Akt-Het mice. In cultured human umbilical vein endothelium cells, DATS decreased apoptotic activity and oxidative stress under hypoxic conditions, and stimulated the phosphorylation of Akt and eNOS. Inhibition of Akt or NOS signaling reversed DATS-stimulated eNOS phosphorylation and blocked the effects of DATS on apoptosis and oxidative stress.
Conclusions: These observations suggest that DATS promotes revascularization in response to HLI through its ability to stimulate the Akt-eNOS signaling pathway.
Peripheral artery disease (PAD) resulting from atherosclerosis is quickly becoming a global health concern due to an ever-aging population. As a result, a large number of patients with critical limb ischemia (CLI) require amputation of affected limbs. This causes not only a reduced quality of life, but also a decline in life span.1 Therefore, augmenting new blood vessel formation is important for salvaging ischemic tissue and benefiting public health.
Historically, hydrogen sulfide (H2S) was considered a toxic gas molecule. However, recent studies revealed that H2S is produced enzymatically in all mammalian species at low micromolar levels and acts as a gasotransmitter.2 The production of H2S is attributed to 3 endogenous enzymes: cystathionine γ-lyase (CGL or CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3MST).3–5 Recently, it was reported that H2S possesses antioxidant, anti-apoptotic, anti-inflammatory, and mitochondrial-protecting activities in various cell types, including cardiac myocytes6 and endothelial cells (ECs).7,8 H2S also exerts beneficial actions against the development of cardiovascular disease.9,10 We previously demonstrated that H2S protects against myocardial ischemia/reperfusion (I/R) injury via anti-apoptotic and anti-oxidant effects mediated, in part, by phosphatidylinositole-3 kinase/Akt, protein kinase C, and extracellular signal-regulated kinase 1/2, as well as via the activation and nuclear translocation of nuclear-factor-E2-related factor 2.11 Furthermore, increases in H2S via exogenous treatment or modulating endogenous production levels also limit the extent of myocardial I/R injury by decreasing myocardial inflammation and preserving mitochondrial functions.12 More recently, we reported that H2S protects against pressure overload-induced heart failure via activation of the vascular endothelial growth factor (VEGF)-Akt-endothelial nitric oxide synthase (eNOS) axis, which was accompanied by preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density.13 Together, these data suggest that H2S exerts protective actions on a variety of cardiovascular disorders, including vascular dysfunction and cardiac injury.
Diallyl trisulfide (DATS) is an organic polysulfide found in garlic oil. DATS is able to release H2S through interactions with biological thiols, including glutathione (GSH) within red blood cells.14 Historically, dietary consumption of garlic has been recognized as a beneficial agent for preventing several diseases, including cardiovascular disease. Epidemiological data have shown that garlic consumption is inversely correlated with the progression and severity of cardiovascular disease.15 Sulfide levels are decreased in skeletal muscles of patients with CLI.16 Recently, we demonstrated that DATS protects the ischemic myocardium via preservation of endogenous levels of H2S in mice.17 However, it is currently unknown whether DATS can improve vascular diseases. In the present study, we investigated whether DATS induces blood vessel growth under ischemic conditions in vivo and tested whether it modulates endothelial functions in vitro.
For in vivo experiments, the DATS was dissolved in 100% dimethyl sulfoxide (DMSO) and further diluted in sterile saline to obtain the correct dosage to be delivered in a volume of 100 μL. The vehicle control consisted of a solution of 1% DMSO in sterile saline. For in vitro experiments, DATS was dissolved in 100% DMSO and further diluted in EC basal medium (EBM-2; Lonza Japan), and the resulting concentration of DMSO was also 1%. EBM-2 containing 1% DMSO was used for medium with the vehicle control group.
Protocol of Mouse Hindlimb Ischemia ModelAll protocols were approved by the Institutional Animal Care and Use Committee at Nagoya University and Emory University. Male C57 BL/6J (WT) mice (8–10 weeks’ old, Charles River Laboratories), eNOS-deficient (eNOS-KO; C57/BL6J background) mice, and Akt1-heterogenic deficient (Akt-Het; C57/BL6J background) mice were used in this study. Unilateral hindlimb ischemia (HLI) was induced by permanent ligation of the femoral artery. Then, mice were randomly divided into 2 groups. Mice in the vehicle-control group were injected intraperitoneally (i.p.) with 1% DMSO. The DATS group was injected with DATS (500 μg･kg−1･day−1 i.p.) for up to 10 days following HLI induction. Laser Doppler perfusion image (LDI) was performed immediately after the operation, and at days 3, 7, 14, and 21 postoperation to evaluate blood perfusion in ischemic hindlimbs, as previously described.18 In addition, L-NG-Nitroarginine methyl ester (L-NAME) (100 mg･kg−1･day−1 i.p.; Sigma-Aldrich) was administered daily to a group of mice to inhibit eNOS activation, and wortmannin (1 mg･kg−1･day−1 i.p.; Merck Millipore) was administered daily to another group of mice to inhibit Akt activation. Mice were euthanized at days 3, 7, 14, and 21 post-HLI induction, and muscles from ischemic limbs and blood samples were harvested 1 h after injection of vehicle or DATS. No mice died during the experimentation.
HistologyIschemic muscles of mice treated with the vehicle control or DATS were collected at postoperative day 21. Frozen sections of 6-μm thickness were prepared using a cryostat (Leica CM3050 S, Leica Biosystems) and stained with an anti-CD31 antibody (Becton Dickinson) to detect capillary ECs. The number of capillary ECs was counted by fluorescence microscopy, as previously described.18
Measurement of H2S and Sulfane SulfurFree H2S and sulfane sulfur levels were measured in isolated muscle tissue and blood of using a combined gas chromatography-chemiluminesence approach, as previously described.17
Protocol of DATS Administration to HUVECsHuman umbilical vein ECs (HUVECs) were purchased from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD, USA). HUVECs were cultured in EBM-2 supplemented with EGM-2 MV at 37℃ in 5% CO2. At confluence, the culture medium of HUVECs was removed, and EBM-2 supplemented with EGM-2 MV and DATS (100 μmol/L) was added to the cells. We also added 2 mmol/L GSH to enhance the release of H2S from DATS. HUVECs were washed with phosphate buffer saline (PBS) and lysed in RIPA lysis buffer (Thermo Scientific) at 0, 5, 15, 30, and 60 min post-DATS administration.
Hypoxia and Serum-Starvation Assays With HUVECsBefore experiments, HUVECs were transferred to 4 chamber culture slides (BD Falcon) with EBM-2 supplemented with EGM-2 MV. At confluence, the culture medium was replaced with EBM-2 with or without DATS (100 μmol/L). The cells were then incubated in a hypoxic chamber for 48 h. The hypoxic condition was induced using an Anaero Pack (Mitsubishi Gas Chemical Co., Inc.). In addition, we administrated L-NAME (10 μmol/L) or wortmannin (10 nmol/L) to the culture medium and incubated in the hypoxic chamber for 48 h.
Dihydroethidium (DHE) StainingWe performed DHE staining (Invitrogen Molecular Probes) to detect reactive oxygen species (ROS) production. Non-ischemic from sham and ischemic muscles were collected at postoperative day 3. Both 6-μm thick frozen sections and HUVECs, cultured under hypoxic and serum-starved conditions, were prepared. Each sample was pretreated with 100 μmol/L rotenone to inhibit mitochondrial electron transport and suppress ROS production, originating from the mitochondrial respiratory chain. Subsequently, samples were subjected to DHE staining (1 μmol/L), and analyzed by fluorescence microscopy.
8-Isoprostane AssayWe used the 8-isoprostane Enzyme Immunoassay Kit (Cayman Chemicals) according to the manufacturer’s instructions to evaluate 8-isoprostane production in non-ischemic from sham and ischemic muscles.
Measurement of Biological Anti-Oxidant Potential (BAP)We used the BAP test kit (Wismerll) according to the manufacturer’s instructions to measure BAP in serum collected from HLI-model mice at postoperative day 3, as previously described.19
TdT-Mediated dUTP Nick End Labeling (TUNEL) StainingTo examine the extent of apoptosis, we used the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics), according to the manufacturer’s instructions as previously described.19 The number of apoptotic cells was counted and expressed as the percentage of the total number of nuclei stained with 4,6-diamidino-2-phenylindole (DAPI).
Western Blot AnalysisTo evaluate Akt and eNOS activation, Western blot analysis was performed as previously described.17 Non-ischemic from sham and ischemic muscles collected at postoperative days 3, 7, 14, and 21 were homogenized, and lysates were used for Western blot analysis. HUVECs administered DATS and GSH were collected at 0, 5, 15, 30, and 60 min after administration and also homogenized for Western blot analysis.
Nitrate/Nitrite Fluorometric AssayWe used the Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemicals) according to the manufacturer’s instruction to evaluate the total concentrations of nitric oxide (NO) products in ischemic muscles.
Statistical AnalysisAll data are expressed as the mean±SEM. Statistical significance was evaluated with an unpaired Student’s t-test for comparisons between 2 means and with 1-way or 2-way analysis of variance (ANOVA) for comparisons among 3 or more means, using Prism 5 (GraphPad Software, Inc.). For the ANOVA, if a significant result was found, the Tukey (1-way ANOVA) or Bonferroni (2-way ANOVA) test was performed as a post-hoc analysis. For all data, a value of P<0.05 denotes statistical significance.
DATS has been shown to increase H2S and sulfane sulfur (storage form of H2S) in various tissues. We therefore assessed free H2S and sulfane sulfur levels in blood and muscles collected from mice subjected to HLI, which were treated with or without DATS. Mice treated with DATS for 3 days following HLI induction displayed significantly higher circulating sulfane sulfur levels (P<0.05; Figure 1A) and sulfane sulfur levels in the adductor muscle (Figure 1B) compared to vehicle-treated controls, but not in non-ischemic adductor muscles (Figure 1B). Furthermore, we assessed sulfane sulfur levels in blood and muscles on days 7 and 14 because DATS was injected for up to 10 days following HLI. Sulfane sulfur levels in blood and ischemic muscle were significantly higher in DATS-treated mice on day 7, but not on day 14 (Figure 1C,D). Free H2S levels significantly increased in the ischemic adductor muscle in DATS-treated mice on day 7 compared to vehicle-treated controls, but not in blood and muscles on days 3 (Figure 1E,F).
Measurement of sulfane sulfur and free hydrogen sulfide (H2S) in vivo. Concentration of sulfane sulfur in blood (A) and muscles (B) at 3 days post-hindlimb ischemia (HLI) induction. Concentration of sulfane sulfur in blood (C) and muscles (D) at 7 and 14 days post-HLI induction. (E) Concentration of circulating free H2S in blood at 3 days post-HLI induction. (F) Concentration of free H2S in ischemic and non-ischemic muscles at 3 days and 7 days post-HLI induction. The values shown are the mean±SEMs. The numbers inside the bars indicate the sample sizes.
We examined the effect of DATS on revascularization processes in response to HLI. For these experiments, mice were subjected to unilateral surgery-induced HLI and then treated with either DATS or the vehicle control (Figure 2A). Figure 2B shows representative LDI of the hindlimb blood flow after surgery in DATS-treated and vehicle-control mice. Blood flow recovery in the ischemic hindlimb was accelerated in mice treated with DATS compared to vehicle-treated mice. Quantitative analysis of LDI showed that treatment with DATS significantly increased blood flow in the ischemic limbs compared to controls on postoperative days 14 and 21 (P<0.05; Figure 2C).
Diallyl trisulfide (DATS) augments ischemia-induced angiogenesis. (A) Schema of the experimental protocols. (B) Representative pictures of Laser Doppler perfusion image (LDI) (B) and quantitative analysis of the ischemic/non-ischemic perfusion ratio (C) following hindlimb ischemia (HLI). (D) Representative capillaries stained with anti-CD31 antibodies (D) and quantitative analysis of capillary density (E) at 21 days following HLI induction. The values shown are the means±SEMs. *P<0.05 vs. vehicle-control mice. The numbers inside the bars indicate the sample sizes.
To investigate the extent of revascularization at the microcirculatory level, we also measured capillary densities in histological sections harvested from ischemic adductor muscles. Figure 2D shows representative photomicrographs of muscle tissues stained for expression of the EC marker, CD31. Quantitative analysis of CD31-positive cells revealed that on postoperative day 21, capillary densities in ischemic adductor muscles were significantly higher in DATS-treated mice compared to the densities observed in vehicle-treated controls (Figure 2E).
DATS Ameliorated Oxidative Damage and Apoptosis After Tissue IschemiaTo investigate ROS production in ischemic muscles, we analyzed DHE staining by fluorescence microscopy at 3 days post-HLI surgery or sham. Ischemic injury increased ROS production in adductor muscles more so in WT mice than in sham mice. However, this induction was attenuated by DATS treatment (Figure 3A). Next, we measured the levels of 8-isoprostane production in non-ischemic and ischemic muscles at 3 days post-HLI induction or sham. DATS treatment significantly decreased 8-isoprostane concentrations compared to vehicle treatment in non-ischemic and ischemic muscles (P<0.05; Figure 3B). Furthermore, the serum levels of BAP, an index of anti-oxidative activity, increased significantly following DATS treatment (Figure 3C) compared to vehicle treatment.
Impact of DATS on oxidative stress and apoptosis in vivo. (A) Representative fluorescence staining of ischemic and non-ischemic tissues with dihydroethidium (DHE, red) at 3 days post-HLI induction or sham. Quantitative analysis of 8-isoprostane concentrations (B) and the biological anti-oxidant potential (BAP) (C) in ischemic tissues and non-ischemic muscles. (D) Representative fluorescence staining (E) and quantitative analysis (F) of ischemic or non-ischemic tissues with TUNEL (green) and DAPI (blue) at 3 days post-HLI induction or sham. The values shown are the means±SEMs. The numbers inside the bars indicate sample sizes. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; DAPI, 4,6-diamidino-2-phenylindole. Other abbreviations as in Figure 2.
To evaluate cell viabilities after DATS treatment, we performed TUNEL staining in non-ischemic and ischemic tissues harvested at 3 days post-HLI induction or sham operation. Representative photographs of TUNEL-positive nuclei in non-ischemic and ischemic muscles are shown in Figure 3E. Quantitative analysis revealed that a significantly lower proportion of TUNEL-positive apoptotic cells were present in the ischemic muscles of DATS-treated mice, compared to that observed in vehicle-treated mice (P<0.05; Figure 3D). Thus, DATS ameliorated oxidative damage and apoptosis in ischemic tissues.
eNOS Activation Is Essential for DATS-Induced Revascularization In VivoIt is well established that the serine/threonine kinase Akt and its downstream target eNOS are crucial regulators of blood vessel growth and vascular cell function.20,21 The expression and phosphorylation of Akt and eNOS in ischemic adductor muscles of WT mice and non-ischemic adductor muscles of sham mice were assessed by Western blot analysis on postoperative day 3. DATS administration did not alter the phosphorylation of Akt at Thr-308 and eNOS at Ser-1177 in non-ischemic muscle of sham mice. DATS administration increased the phosphorylation of Akt at Thr-308 and eNOS at Ser-1177 in ischemic muscles (Figure 4A–C). Next, we investigated whether DATS increased the concentration of NO products, such as nitrate and nitrite, in ischemic tissues using the Nitrate/Nitrite Fluorometric Assay Kit. DATS administration increased the concentration of NO products in ischemic tissues on postoperative day 3, compared to that observed in vehicle-control mice (Figure 4D). In addition, we assessed Akt and eNOS phosphorylation in ischemic adductor muscles on postoperative days 7, 14, and 21. DATS administration increased Akt phosphorylation on days 7 and 14, and increased eNOS phosphorylation on day 7 (Figure 5A–C). DATS administration also increased the concentration of NO products on postoperative day 7, but not on days 14 and 21 (Figure 5D). Treatment with DATS had no effects on the total expression levels of the Akt and eNOS proteins in ischemic muscles.
Role of Akt and endothelial nitric oxide synthase (eNOS) in DATS-induced revascularization in vivo. Representative immunoblots (A) and quantitative analysis (B,C) of eNOS-p, total eNOS, Akt-p, total Akt, and GAPDH in the muscle at 3 days post-HLI induction or sham. (D) The concentration of nitric oxide (NO) products in ischemic limbs at 3 days post-HLI induction. Representative photos (E) and clinical score (F) of eNOS knock out (KO) mice at 14 days post-HLI induction. Quantitative analysis of the ischemic/non-ischemic perfusion ratio in C57 BL/6J mice treated with L-NAME (G) and wortmannin (I). (H) Quantitative analysis of the ischemic/non-ischemic perfusion ratio in Akt-Het mice. Representative Western blot results (J) and quantitative analysis (K,L) of eNOS-p, total eNOS, Akt-p, total Akt, and GAPDH in ischemic tissues treated with vehicle, DATS, and DATS+wortmannin. The values shown are the means±SEMs. *P<0.05, **P<0.01 vs. the DATS with wortmannin group. The numbers inside the bars indicate sample sizes. Other abbreviations as in Figure 2.
DATS upregulates the Akt-eNOS signaling pathway in vivo and in vitro. Representative immunoblots (A) and quantitative analysis (B,C) of eNOS-p, total eNOS, Akt-p, total Akt, and GAPDH at 7, 14, and 21 days post-HLI induction. (D) Concentrations of NO products in ischemic limbs at 7, 14, and 21 days post-HLI induction. (E) Representative immunoblots of eNOS-p, total eNOS, Akt-p, total Akt, and β-actin in human umbilical vein endothelial cells (HUVECs) cultured with DATS+glutathione (GSH) (E) and GSH alone (F). Quantitative analysis of the ratio of Akt-p to total Akt (G) and eNOS-p to total eNOS (I) in HUVECs cultured with DATS and GSH. Quantitative analysis of the ratio of Akt-p to total Akt (H), and eNOS-p to total eNOS (J) in HUVECs treated with GSH alone. The values shown are the means±SEMs. The numbers inside the bars indicate sample sizes. Other abbreviations as in Figures 2,4.
To examine whether eNOS signaling is required for the actions of DATS on revascularization, we administered DATS to eNOS-KO mice. In contrast to WT mice, DATS treatment did not affect blood flow recovery in the ischemic limbs of eNOS-KO mice throughout the experimental period (data not shown). Because eNOS-KO mice exhibit severe ischemia-induced vascular insufficiency, which is accompanied by amputation (Figure 4E), we assessed lower limb functions and tissue salvage post-surgery using a clinical scoring system. Similarly, the index of severity of tissue ischemia after hindlimb surgery did not differ between DATS-treated eNOS-KO and vehicle-treated eNOS-KO mice (Figure 4F). We also analyzed the effect of DATS on blood flow recovery to ischemic muscles in WT mice receiving the NOS inhibitor, L-NAME, or the vehicle control. While DATS-treated WT mice showed an increased recovery of blood flow compared to vehicle-treated WT mice, treatment with L-NAME abrogated the increase in limb perfusion recovery in DATS-treated WT mice (Figure 4G). Therefore, the in vivo effect of DATS on ischemia-induced revascularization depended on eNOS.
Role of Akt in DATS-Induced RevascularizationTo further evaluate the involvement of the Akt-eNOS signaling axis in DATS-induced revascularization, we examined the effect of DATS on blood flow recovery to ischemic muscles in WT mice receiving the PI3K-inhibitor, wortmannin, or the vehicle control because wortmannin can also inhibit PI4K slightly, but mainly inhibit PI3K. Treatment with wortmannin also blocked increased limb perfusion in DATS-treated WT mice (Figure 4H). In addition, treatment with wortmannin significantly diminished the DATS-induced increase in Akt and eNOS phosphorylation in ischemic muscle tissues (Figure 4J–L). Furthermore, we analyzed the effect of DATS on blood flow recovery to ischemic muscles in Akt-Het mice. In contrast to WT mice, DATS treatment did not affect the blood flow recovery in ischemic limbs of Akt-Het mice throughout the experimental period (Figure 4I). These data suggested that Akt was also involved in DATS-induced eNOS activation and revascularization.
DATS Stimulates Akt and eNOS In VitroThe effect of DATS on the activating phosphorylation of Akt at Thr-308 in ECs was assessed by Western blot analysis. Consistent with previous observations,22 treatment with DATS alone did not affect the phosphorylation of Akt and eNOS (data not shown). Because DATS releases H2S when it is converted by GSH, we added DATS with GSH to the medium of cultured HUVECs. Treatment with DATS with GSH stimulated the phosphorylation of Akt and eNOS in a time-dependent manner, with maximal Akt and eNOS phosphorylation occurring after 30 min (Figure 5E,G,I). We confirmed that the administration of GSH alone did not alter Akt and eNOS phosphorylation (Figure 5F,H,J). DATS and GSH had no effect on Akt and eNOS protein levels.
Role of Akt-eNOS Signaling in the DATS-Induced Decrease in Apoptosis and Oxidative Stress In VitroTo test whether DATS affects endothelial apoptosis and oxidative stress, HUVECs were treated with DATS or the vehicle control under hypoxic conditions. Analysis of apoptotic activity by TUNEL staining demonstrated that DATS treatment significantly reduced the frequency of TUNEL-positive cells (Figure 6A,C). To investigate whether Akt-eNOS signaling participates in DATS-induced EC apoptosis, HUVECs were treated with wortmannin or L-NAME, and EC apoptotic activity was assessed. Pre-treatment with wortmannin or L-NAME reversed the inhibitory effects of DATS on the frequency of TUNEL-positive cells (Figure 6A,C).
Anti-oxidative and anti-apoptotic effects of DATS through Akt-eNOS signaling in cultured HUVECs) under stress conditions. (A) Representative fluorescence staining in cultured HUVECs by TUNEL (green) and DAPI (blue) staining. (B) Representative fluorescence staining in cultured HUVECs with DHE (red) and DAPI (blue). (C) Quantitative analysis of the ratio of TUNEL-positive cells to all cells (DAPI-stained). (D) Quantitative analysis of the ratio of DHE-positive cells to all cells (DAPI-stained). The values shown are the means±SEMs. The numbers inside the bars indicate sample sizes. Abbreviations as in Figures 2–5.
We next examined anti-oxidative effects of DATS in HUVECs. HUVECs were treated with DATS or the vehicle control under hypoxic conditions, after which DHE staining was performed. Treatment with DATS significantly decreased the prevalence of DHE-positive cells compared to that observed in vehicle-control cells (Figure 6B,D). Furthermore, pre-treatment with wortmannin or L-NAME reversed the inhibitory effects of DATS on the frequency of DHE-positive cells (Figure 6B,D). Thus, these results indicated that Akt-eNOS signaling was required for EC responses to DATS.
The major findings of the present study are: (1) DATS treatment augmented ischemia-induced revascularization in vivo; (2) DATS treatment ameliorated oxidative damage and apoptosis in ischemic muscles in mice; and (3) Akt-eNOS signaling is required for the DATS-induced decrease in apoptosis and oxidative stress, both in vivo and in vitro. These data indicated that DATS exerts beneficial actions on revascularization.
It is well established that Akt and its downstream target eNOS are crucial regulators of blood vessel growth and vascular functions.23,24 Here, we showed that DATS ameliorated oxidative damage and apoptosis in ECs, and stimulated Akt and eNOS phosphorylation in ECs. Inhibition of Akt signaling reversed the stimulatory effects of DATS on these EC functions and eNOS phosphorylation. Thus, the stimulation of EC function by DATS may depend on its ability to activate Akt-eNOS signaling. Furthermore, DATS promoted revascularization in ischemic muscle tissues in vivo, which was associated with increased Akt and eNOS phosphorylation. Of note, the impact of DATS on blood vessel recruitment was abolished in eNOS-KO mice. Collectively, these data clearly suggest that the DATS-induced Akt-eNOS regulatory axis can attenuate oxidative damage and apoptosis under conditions of ischemia, thereby accelerating revascularization. Additionally, H2S interacts with numerous signaling targets such as the ATP-sensitive K+ (KATP) channel located on the surface of cell membranes and mitochondria in many cell types, including neurons, smooth muscle cells, and cardiac myocytes.2,25 It has been reported that KATP channels are affected by H2S in a dose-dependent manner,26 and that endogenous H2S is a key regulator of KATP channel activity,10 leading to ECs and smooth muscle cell hyperpolarization and vasorelaxation.27 NaHS inhibited angiotensin II-induced vascular smooth muscle cell proliferation and collagen generation in spontaneously hypertensive rats.28 Therefore, the effects of H2S on smooth muscle cells may also contribute to augmenting ischemia-induced angiogenesis in part.
It has been shown that NO produced by eNOS is protective against various vascular diseases including PAD.29 Although H2S and NO have been shown to function through independent signaling pathways in vivo and in vitro, crosstalk appears to occur between the H2S and NO signaling pathways. For instance, a recent report showed that H2S activates eNOS phosphorylation through an Akt-dependent mechanism, leading to NO production in HUVECs.30 In addition, H2S donors such as SG-1002 or DATS significantly increase NO production through activation of the Akt-eNOS-NO pathway in pressure overload-induced heart failure in mice.13,31 CSE-KO mice exhibit exacerbated myocardial and hepatic I/R injury with downregulating eNOS-NO pathway, and H2S therapy restore eNOS function and NO bioavailability, and attenuated I/R injury in CSE-KO mice.32 Moreover, NO upregulates the H2S-generating enzyme, CSE, in vascular smooth muscle cells.33 Consistent with these findings, the current study showed that DATS increased eNOS phosphorylation, leading to NO production in both ischemic muscles and cultured ECs. Our data also indicated that the influence of DATS on EC behavior and vessel growth is mediated, at least in part, through its ability to activate eNOS. These observations suggested that eNOS acts as a crucial mediator of the protective actions of DATS on the vasculature.
In vivo findings obtained to elucidate the role of H2S in regulating angiogenesis have provided conflicting results. In agreement with our data, several investigators reported that H2S exhibits proangiogeic effect in vivo and in vitro.33–36 NaHS promoted neovascularization by Akt phosphorylation in vivo Matrigel plug assay in mice, improved EC functions such as cell growth, migration, scratched wound healing, and tube-like structure formation in cultured ECs,34 and augmented revascularization by upregulating VEGF, VEGF-R2, and Akt phosphorylation in a rat model of HLI.37 Endogenous and exogenous H2S stimulates angiogenesis-related properties of ECs’ blood vessel formation in vivo through a KATP channel/MAPK pathway.36 In contrast, it has been reported that DATS can inhibit tumor growth in vitro and in vivo by inhibiting neovascularization.38–40 The reason for this discrepancy is possibly due to differences in assay systems utilized to study angiogenesis. It is also possible that H2S differentially regulates pathological and physiological angiogenesis, as has been proposed to explain the effects of statins on vascularization.41 As another possibility, difference of administered DATS concentration should be considered. According to a safety data sheet of DATS, oral LD50 is 100 mg/kg in mouse although the toxicological effects of DATS have not been fully studied. In our experiences, DATS (500 µg/kg) clearly augmented ischemia-induced angiogenesis in a mice model of HLI. Previously, it has been reported that DATS treatment to HUVECs increased cell death and inhibited EC functions such as tube formation and migration, VEGF secretion, VEGF receptor-2 protein levels, and Akt phoshorylation in a concentration-dependent manner.22 In our preliminary study, administration of DATS (50 µmol/L) alone into HUVECs actually exhibited similar results, as described above (data not shown), because the IC50 of DATS is approximately 4 µmol/L in HUVECs.22 However, administration of DATS (50 µmol/L) with GSH clearly inhibited oxidative damage and apoptosis in ECs, and upregulated the Akt-eNOS signaling pathway in ECs. Importantly, DATS needs GSH to release H2S. These data indicate that a high concentration of DATS itself may have cytotoxicity but released H2S from DATS is able to exhibit cytoprotective effects via upregulation of the Akt-eNOS-NO signaling pathway. Of importance, it has been established that activation of the Akt-eNOS signaling pathway confers a pro-angiogenic phenotype in ischemic hindlimbs. Taken together, these observations suggest that the induction of Akt-eNOS signaling by an appropriate concentration of DATS as an H2S donor can facilitate revascularization responses in the setting of tissue ischemia.
In conclusion, our data document that DATS functions as a novel regulator of vascular responses to ischemia through eNOS activation in ECs by increasing the bioavailability of H2S. Therapeutic approaches aimed at augmenting exogenous H2S using H2S-releasing agents could be potentially useful for treating cardiovascular diseases.
We gratefully acknowledge the technical assistance of Yoko Inoue.
This work was supported by the Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology, 2013.
None.
