NO-stimulated cGMP Accumulation Research Articles

PL12 Molecular mechanisms and therapeutic implications of the pro-angiogenic effect of hydrogen sulfide

Introduction Angiogenesis is a key process in a variety of biological processes ranging from wound healing to tumor biology. Hydrogen sulfide (H 2 S) and nitric oxide (NO) are recognized as essential, interacting, endogenous gaseous signaling molecules. Here we review the existing data demonstrating the effect of exogenous and endogenous H 2 S on angiogenesis and overview the molecular pathways involved. We are also presenting a range of therapeutic context where these mechanisms can be exploited in the future. Methods Murine bEnd.3 endothelial cells were used for in vitro studies; burn-induced wound healing in rats and mice was used for in vivo studies. Results Exposure of endothelial cells to a H 2 S donor increased intracellular cGMP in a NO-dependent manner, activated protein kinase G (PKG) and induced the phosphorylation of its downstream substrate, VASP. Inhibition of eNOS or PKG abolished the H 2 S-stimulated angiogenic response. Thus, there is a convergence of the vascular actions of H 2 S and NO to the cGMP/PKG pathway. Silencing of the H 2 S-producing enzyme cystathionine-gamma-lyase (CSE) abolished NO-stimulated cGMP accumulation and angiogenesis, indicating the requirement of H 2 S in the vascular actions of NO. VEGF, a pro-angiogenic and vasorelaxant hormone stimulated the production of both NO and H 2 S: VEGF-induced angiogenic and vasorelaxant responses were attenuated either by the inhibition of NO or by the silencing of CSE. A key site of the cooperative interaction between NO and H 2 S involves the cGMP/PKG pathway: H 2 S exerted a potent inhibitory effect on phosphodiesterase 5 activity in vitro leading to cGMP elevation and PKG activation. Another site of the interaction between H 2 S and NO is Akt: H 2 S increased the phosphorylation (activation) of Akt, as well as the phosphorylation of eNOS at Ser1177. In vivo H 2 S administration promoted post-burn wound healing, while reduced H 2 S production (mice deficient in CSE) delayed it. H 2 S -mediated and angiogenesis in vivo was suppressed by pharmacological inhibition (L-NAME) or genetic ablation of eNOS (eNOS −/− mice). Conclusions H 2 S is a pro-angiogenic endogenous hormone. Administration of H 2 S promotes wound healing. NO and H 2 S are mutually required for the control of angiogenesis and wound healing. H 2 S supplementation may be beneficial to facilitate post-burn wound healing. Potential modes of delivery may include topical or systemic administration. However, the angiogenic effect of H 2 S is not expected to be optimal in conditions where endogenous NO production is impaired (i.e. conditions associated with endothelial dysfunction syndrome). In such conditions, simultaneous supplementation of both gasotransmitters may be possible. In addition to wound healing, the pro-angiogenic effects of H 2 S may be utilized to improve revascularization following ischemic conditions, whereas inhibition of H 2 S production (and associated angiogenic responses) may be of therapeutic interest in conditions associated with pathological increases in angiogenesis (e.g. in the context of tumor angiogenesis or in diabetic retinopathy).

NO-H2S interactions involve cGMP

Results We have shown that H2S increases cGMP in both endothelial and smooth muscle cells. However, H2S does not activate sGC or alter NO-induced sGC activity. Interestingly, H2S inhibits phosphodiesterase (PDE) activity; although it reduces PDE activity of several PDE H2S is most effective and potent against PDE-5. IN line with the ability of H2S to increase cellular cGMP, we observed that exposure of cells to H2S leads to activation of cGMPdependent protein kinase and VASP phosphorylation. As both NO and H2S promote angiogenesis and vasodilation we explored their interactions in the vessel wall in the context of these two biological processes. Inhibition of eNOS or PKG reduced the H2S-stumlated angiogenic properties of endothelial cells, as well as H2S–stimulated vasorelaxation, suggesting a prominent role for cGMP/ PKG pathways in H2S signaling. On the other hand, silencing of the H2S-producing enzyme cystathionine-g-lyase (CSE) reduced NO-stimulated cGMP accumulation, angiogenesis and smooth muscle relaxation, proving that NO requires H2S to manifest its effects. Finally, H2Sinduced wound healing and angiogenesis in vivo was suppressed by pharmacological inhibition or genetic ablation of eNOS. Conclusion Inhibition of the production of one gasotransmitter (NO or H2S) reduces the ability of the other to elevate cGMP and to trigger angiogenesis and vasodilation. These observations establish the existence of a positive, synergistic cross-talk between H2S and NO in vascular tissues.

P23 The angiogenic and vasorelaxant effect of H2S require the endogenous production of NO, while the angiogenic and vasorelaxant effect of NO require the endogenous production of H2S

Background Hydrogen sulfide (H2S) and nitric oxide (NO) are recognized as essential endogenous gaseous signaling molecules involved in a variety of homeostatic and disease processes. Recent studies suggest that multiple interactions exist between H2S and NO, and indicate that these interactions are crucial in regulating biological responses of vascular cells. However, the precise nature and mechanisms of the interactions between these two gasotransmitters remain incompletely understood. The goal of the current study was to investigate whether angiogenesis and vasodilation require the simultaneous production of H2S and NO. Methods Cultured murine bEnd.3 endothelial cells were used for the studies. CSE was silenced using siRNA. eNOS and PKG were inhibited by pharmacological inhibitors. H2S production was measured by the methylene blue method. Burn-induced wound healing was measured in rats by planimetry. Results Exposure of endothelial cells to a H2S donors (NaHS) increased intracellular cGMP in a NO-dependent manner and activated protein kinase G (PKG) as evidenced by phosphorylation of its downstream substrate, VASP. Inhibition of eNOS or PKG abolished the H2S-stimulated angiogenic response. Inhibition of eNOS also attenuated H2S-stimulated vasorelaxation. These findings demonstrate the requirement of NO in vascular H2S signaling, and the convergence of the vascular actions of H2S and NO to the cGMP/PKG pathway. Conversely, silencing of the H2S-producing enzyme cystathionine-gamma-lyase (CSE) abolished NO-stimulated cGMP accumulation and angiogenesis and attenuated the NO-induced vasorelaxation, indicating the requirement of H2S in the vascular actions of NO. Moreover, acetylcholine-induced, endothelium-dependent relaxations were attenuated after silencing of CSE. VEGF, a pro-angiogenic and vasorelaxant hormone is associated with the production of both NO and H2S: VEGF-induced angiogenic and vasorelaxant responses are attenuated either by the inhibition of NO or by the silencing of CSE. A key site of the cooperative interaction between NO and H2S involves the cGMP/PKG pathway: H2S exerted a potent inhibitory effect on phosphodiesterase 5 (PDE5) activity in vitro leading to cGMP elevation and PKG activation. Another site of the interaction between H2S and NO is Akt: H2S increased the phosphorylation (activation) of Akt, as well as the phosphorylation of eNOS at Ser1177. The in vivo relevance of the current findings is highlighted by the fact that H2S-induced wound healing in rats and angiogenesis in the matrigel plug model in mice, were suppressed by pharmacological inhibition (L-NAME) or genetic ablation of eNOS (eNOS−/− mice). Conclusions In conclusion, NO and H2S are mutually required for the control of angiogenesis and vasorelaxation.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.

Inhibition of Nitric Oxide–Stimulated Vasorelaxation by Carbon Monoxide-Releasing Molecules

Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation.

Regulation of soluble guanylyl cyclase by phosphorylation

Results In vitro kinase assays revealed that the α1, but not the β1, subunit of sGC is a PKG substrate and that the phosphorylation site is located within the first 360 residues of the α1. A constitutively active form of PKG stimulated incorporation of 32P into the α1 subunit in vivo. In addition, PKG could be detected in sGC immunoprecipitates, suggesting that the two proteins interact in cells. Serine to alanine mutation of putative PKG sites revealed that Ser64 is the main phosphorylation site for PKG. After generating a phospho-specific antibody for Ser64, we could demonstrate that phosphorylation of endogenous sGC on Ser64 increases in cells and tissues exposed to NO. Phosphorylation of sGC in cells following SNP stimulation was blocked by the peptide inhibitor of PKG, DT-3. Wild-type (wt) sGC when co-expressed in COS cells with a constitutively active form of PKG exhibited lower basal and NOstimulated cGMP accumulation. In contrast, the S64A α1/ β1 sGC was resistant to the PKG-induced reduction in activity. Moreover, co-cultures of endothelial cells with smooth muscle expressing a phosphorylation-resistant form of sGC, exhibited higher levels of cGMP (both basally and following stimulation with bradykinin) compared to co-cultures expressing wild-type sGC. Phosphorylation of the α1 subunit did not alter its ability to form heterodimers with β1. Using purified sGC and mutants, we observed that the S64D α1 phosphomimetic/β1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D α1/β1 compared to wt sGC. Expression of a phosphorylation deficient form of sGC showed reduced desensitization to acute NO exposure and allowed for greater VASP phosphorylation.

Protein Kinase G Phosphorylates Soluble Guanylyl Cyclase on Serine 64 and Inhibits Its Activity

Binding of nitric oxide (NO) to soluble guanylyl cyclase (sGC) leads to increased cGMP synthesis that activates cGMP-dependent protein kinase (PKG). Herein, we tested whether sGC activity is regulated by PKG. Overexpression of a constitutively active form of PKG (DeltaPKG) stimulated (32)P incorporation into the alpha1 subunit. Serine to alanine mutation of putative sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we observed that endogenous sGC phosphorylation on Ser 64 increases in cells and tissues exposed to NO, in a PKG-inhibitable manner. Wild-type (wt) sGC coexpressed with DeltaPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A alpha1/beta1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D alpha1 phosphomimetic /beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wild-type sGC. Expression of a phosphorylation-deficient sGC showed enhanced responsiveness to endothelium-derived NO, reduced desensitization to acute NO exposure, and allowed for greater VASP phosphorylation. We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.

Activation of soluble guanylyl cyclase (sGC) by the NO/heme-independent activator HMR1766 in cells

Most vascular diseases are characterized by an increase in the production of reactive oxygen species (ROS). The resulting oxidative stress reduces the biological activity of endothelial-derived nitric oxide (NO). In the present study we have evaluated the responsiveness of sGC in cells exposed to HMR1766, a NO-independent sGC activator that preferentially stimulates the oxidized/heme-free enzyme. Pre-treatment of rat aortic smooth muscle cells (RASMC) with 500 μM of H2O2 reduced the ability of both sodium nirtroprusside (SNP; a nitric oxide donor) and BAY 41–2272 (a NO-independent, heme-dependent sGC activator) to increase intracellular cGMP levels. In contrast, pre-treatment of cells with H2O2 significantly increased the HMR1766-stimulated cGMP accumulation. Similarly, pre-treatment of RASMC with SIN-1 (500 μM), a peroxynitrite donor, enhanced sGC activation by HMR1766. Adenovirus-mediated overepxression of catalase or superoxide dismutase in RASMC did not alter the responsiveness to HMR1766. To study if HMR1766 stimulates cGMP accumulation in cells expressing heme-deficient sGC we generated histidine 105 to phenylalanine (H105F) or cycteine (H105C) mutants of the sGC β1 subunit. In wild-type sGC-expressing cells NO-stimulated cGMP accumulation was blocked by ODQ, while HMR1766-stimulated cGMP was enhanced by ODQ. The H105F mutant was not sensitive to NO, as it is heme-deficient, but exhibited a 10-fold increase in cGMP accumulation after stimulation with HMR1766; this effect of HMR1766 was not affected by ODQ pretreatment. The α1/H105C enzyme was basally much more active than wt sGC and could be further stimulated with HMR1766, but not SNP. The above data indicate that HMR1766 is capable of activating oxidized and heme-free sGC in vivo.

Insulin-stimulated hydrogen peroxide increases guanylate cyclase activity in vascular smooth muscle.

Insulin resistance is associated with vascular disease. Physiological concentrations of insulin inhibit cultured vascular smooth muscle cell (VSMC) contraction and migration by increasing nitric oxide (NO)-stimulated cGMP accumulation. The failure to do so in insulin-resistant states may aggravate vascular disease. We sought to determine the mechanism of insulin's increase in cGMP accumulation. Isobutylmethylxanthine, an inhibitor of phosphodiesterase activity, inhibited the decline in cGMP levels measured by immunoassay in cGMP-loaded cultured rat aortic VSMCs, but 1 nmol insulin did not. Thus, insulin's increase in cGMP accumulation is due to stimulated production, not inhibited hydrolysis and/or efflux. Insulin, which increases the NADH/NAD+ ratio in these cells, stimulated superoxide anion (O2-) accumulation measured by lucigenin luminescence to 256+/-25% (P<0.05) by a process that was blocked by the NADH oxidase inhibitor diphenyliodonium (DPI) and enhanced by the superoxide dismutase inhibitor diethyldithiocarbonate (DETCA). Insulin also stimulated hydrogen peroxide (H2O2) accumulation measured by horseradish peroxidase/luminol luminescence to 221+/-22% (P<0.05) by a DETCA-sensitive mechanism. H2O2 (100 micromol/L) in the absence of insulin increased NO-stimulated cGMP accumulation to 151+/-11% (P<0.05). Insulin alone increased NO-stimulated cGMP accumulation to 183+/-17% (P<0.05), and this was blocked by either DPI or DETCA. We conclude that insulin increases NADH oxidase-derived O2- production in cultured rat VSMCs. This did not cause the expected scavenging of NO resulting in the reduction of NO-stimulated guanylate cyclase activity, but enough O2- was metabolized to H2O2 to increase overall NO-stimulated cGMP production.

Mechanism of erectogenic effect of the selective phosphodiesterase type 5 inhibitor, DA-8159.

DA-8159, a new Phosphodiesterase (PDE) 5 inhibitor, has exhibited potent erectogenic potential in a penile erection test in rats and anesthetized dogs. In this study, we investigated the mechanism of its erectogenic activity by measuring the activity of DA-8159 against a various PDE isozymes and assessing cGMP and cAMP formation in a rabbit corpus cavernosum in vitro. DA-8159 inhibited the PDE 5 activity in rabbit and human platelets, which the IC50 was 5.84 +/- 1.70 nM and 8.25 +/- 2.90 nM, respectively. The IC50 of DA-8159 on PDE 1, PDE 2, PDE 3 and PDE 6 were 870+/- 57.4 nM, 101 +/- 15 microM, 52.0 +/- 3.53 microM and 53.3 +/- 2.47 nM, respectively. This suggests that DA-8159 is a potent, highly selective, competitive inhibitor of PDE 5-catalyzed cGMP hydrolysis. The rates of cGMP hydrolysis catalyzed by human platelets-derived PDE 5 as a function of the cGMP concentration (5-100 nM) and two-fixed DA-8159 concentration (11.3 and 18.8 nM) were investigated in order to characterize the mode of PDE 5 inhibition by DA-8159. DA-8159 increased the apparent Km value for cGMP hydrolysis but had no effect on the apparent Vmax, indicating a competitive mode of inhibition. DA-8159 increased the cGMP concentrations in the rabbit corpus cavernosum dose dependently. In the presence of sodium nitroprusside (SNP), DA-8159 significantly stimulated the accumulation of cGMP when compared to the control level. This indicated that the enhancement of a penile erection by DA-8159 involved the relaxation of the cavernosal smooth muscle by NO-stimulated cGMP accumulation. In conclusion, DA-8159 is a selective inhibitor of PDE 5-catalyzed cGMP hydrolysis and the enhancement of a penile erection by DA-8159 is mediated by the relaxation of the cavernosal smooth muscle by the NO-stimulated cGMP accumulation.

Inactivation of endothelial derived relaxing factor by oxidized lipoproteins.

Endothelial cell derived relaxing factor (EDRF) mediated relaxation of blood vessels is impaired in vessels exposed to lipoproteins in vitro and in arteries of hyperlipidemic humans and animals. To investigate the mechanism by which lipoproteins impair the effects of EDRF, which is likely nitric oxide (NO) or a related molecule, we have bioassayed EDRF/NO activity by measuring its ability to increase cGMP accumulation in rat fetal lung cultured fibroblasts (RFL-6 cells). Low density lipoprotein modified by oxidation (ox-LDL) induced a concentration-dependent inhibition of EDRF activity that had been released from bovine aortic endothelial cells (BAEC) stimulated with bradykinin or the calcium ionophore A23187. In addition, lipoproteins directly impaired authentic NO-induced stimulation of cGMP accumulation in the detector cells; stimulation by sodium nitroprusside was unaffected. Ox-LDL or oxidized HDL3 were highly potent in blocking NO-stimulated cGMP accumulation with EC50's of approximately 1 microgram/ml. Lipid extracted from ox-LDL blocked NO-stimulated cGMP accumulation to about the same extent as intact ox-LDL, while the protein component of ox-LDL did not inhibit the cGMP response. These results suggest that the lipid component of oxidized lipoproteins inactivate EDRF after its release from endothelial cells.