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. N itric oxide (NO) and hydrogen sulfide (H 2 S) are two endogenous gasotransmitters whose regulatory roles in the cardiovascular system include vasorelaxation and stimulation of angiogenesis (1, 2). In endothelial cells, NO is synthesized by the endothelial isoform of NO synthase (eNOS). The principal pathway of NO signaling involves binding to the heme moiety of the soluble guanylyl cyclase (sGC) and production of the second messenger cyclic guanosine 5′-monophosphate (cGMP), followed by the activation of protein kinase G (PKG) (3, 4). However, vascular H 2 S is generated from L-cysteine by two pyridoxal 5′-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST); activation of the ATP-dependent potassium channel (K ATP channel), modulation of cell metabolism, and posttranslational protein modifications via sulfhydration have been identified as some of its key signaling pathways (5-7).It is generally assumed that the signaling pathways of NO and H 2 S are independent. In the ...

Yang et al. show that a disulfide isoform of HMGB1, with a role in TLR4 signaling, physically interacts with and binds MD-2. MD-2 deficiency in macrophage cell lines or in primary mouse macrophages stimulated with HMGB1 implicates MD-2 in TLR4 signaling. They also identify an HGMB1 peptide inhibitor, P5779, which when administered in vivo can protect mice from acetaminophen-induced hepatoxicity, ischemia/reperfusion injury, and sepsis.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H 2 S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H 2 S. Replacement of H 2 S or overexpression of the H 2 S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H 2 S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H 2 S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE −/− mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocininduced diabetes in rats resulted in a decrease in the circulating level of H 2 S; replacement of H 2 S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H 2 S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H 2 S catabolism form a positive feed-forward cycle. H 2 S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

The gaseous mediator hydrogen sulfide (H 2 S) is synthesized mainly by cystathionine gammalyase in the heart and plays a role in the regulation of cardiovascular homeostasis. Here we first overview the state of the art in the literature on the cardioprotective effects of H 2 S in various models of cardiac injury. Subsequently, we present original data showing the beneficial effects of parenteral administration of a donor of H 2 S on myocardial and endothelial function during reperfusion in a canine experimental model of cardiopulmonary bypass. Overview of the literature demonstrates that various formulations of H 2 S exert cardioprotective effects in cultured cells, isolated hearts and various rodent and large animal models of regional or global myocardial ischemia and heart failure. In addition, the production of H 2 S plays a role in myocardial pre-and post-conditioning responses. The pathways implicated in the cardioprotective action of H 2 S are multiple and involve K ATP channels, regulation of mitochondrial respiration, and regulation of cytoprotective genes such as Nrf-2. In the experimental part of the current article, we demonstrate the cardioprotective effects of H 2 S in a canine model of cardiopulmonary bypass surgery. Anesthetized dogs were subjected hypothermic cardiopulmonary bypass with 60 minutes of hypothermic cardiac arrest in the presence of either saline (control, n=8), or H 2 S infusion (1 mg/ kg/h for 2 h). Left ventricular hemodynamic variables (via combined pressure-volumeconductance catheter) as well as coronary blood flow, endothelium-dependent vasodilatation to acetylcholine and endothelium-independent vasodilatation to sodium nitroprusside were measured at baseline and after 60 minutes of reperfusion. Ex vivo vascular function and high-energy phosphate contents were also measured. H 2 S led to a significantly better recovery of preload recruitable stroke work (p<0.05) after 60 minutes of reperfusion. Coronary blood flow was also significantly higher in the H 2 S group (p<0.05). While the vasodilatory response to sodium nitroprusside was similar in both groups, acetylcholine resulted in a significantly higher increase in coronary blood flow in the H 2 S-treated group (p<0.05) both in vivo and ex vivo. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript high-energy phosphate contents were better preserved in the H 2 S group. Additionally, the cytoprotective effects of H 2 S were confirmed also using in vitro cell culture experiments in H9c2 cardiac m...

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Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the β cells of the islets of Langerhans. β cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in β cell destruction, including Fas, perforin, and TNF-α. Evidence for Fas-mediated lysis of β cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on β cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.

Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the `Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ~60 μM), tannic acid (IC50: ~40 μM) and benserazide (IC50: ~30 μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ~3 μM) and NSC67078 (IC50: ~1 μM), while aurintricarboxylic acid (IC50: ~3 μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ~1 μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ~6 μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ~6 μM), tannic acid (IC50: ~20 μM), benserazide (IC50: ~20 μM), and NSC67078 (IC50: ~0.3 μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ~300 μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300 μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300 μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100 μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50 mg/kg/day s.q.) prevented tumor growth. In silico docking simulations...

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