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Background: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide–producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE–hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. Methods: Endothelial-cell–specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial–mesenchymal transition. Results: Endothelial-cell–specific CSE knockout mice exhibited increased endothelial–mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial–mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. Conclusions: Our data demonstrate that endothelial CSE modulates endothelial–mesenchymal transition and ameliorate the severity of pressure-overload–induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.

Hydrogen sulfide is a gasotransmitter, with similarities to nitric oxide and carbon monoxide. Although the enzymes indicated below have multiple enzymatic activities, the focus here is the generation of hydrogen sulphide (H2S) and the enzymatic characteristics are described accordingly. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are pyridoxal phosphate (PLP)-dependent enzymes. 3-mercaptopyruvate sulfurtransferase (3-MPST) functions to generate H2S; only CAT is PLP-dependent, while 3-MPST is not. Thus, this third pathway is sometimes referred to as PLP-independent. CBS and CSE are predominantly cytosolic enzymes, while 3-MPST is found both in the cytosol and the mitochondria. For an authoritative review on the pharmacological modulation of H2S levels, see Szabo and Papapetropoulos, 2017 [8].

Haem oxygenase (heme,hydrogen-donor:oxygen oxidoreductase (α-methene-oxidizing, hydroxylating)), E.C. 1.14.99.3, converts heme into biliverdin and carbon monoxide, utilizing NADPH as cofactor.

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [12] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

AimsAdvanced age is unequivocally linked to the development of cardiovascular disease, however, the mechanisms leading to loss of endothelial cell regenerative capacity during aging remain poorly understood. Here we aimed to investigate novel mechanisms involved in endothelial cell senescence, that impact on endothelial cell transcription and the vascular repair response upon injuryMethods and resultsRNA sequencing of a unique collection of native endothelial cells from young and aged individuals, showed that aging (20 vs. 80 years) is characterized by p53- mediated reprogramming to promote the expression of senescence-associate genes. Molecular analysis revelead that p53 accumulated and acetylated in the nucleus of aged human endothelial cells to suppress glycolysis. Metabolic flux analysis identified an associated reduction in glucose uptake and ATP availability that inhibited the assembly of the telomerase complex, which was essential for proliferation. Nuclear translocation of p53 in aged endothelial cells was attributed to the loss of the vasoprotective enzyme, cystathionine γ-lyase (CSE), which physically anchored p53 in the cytosol. In mice, loss of endothelial cell CSE activated p53 and arrested vascular repair upon injury, while the AAV9 mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in human native aged endothelial cells maintained low p53 activity and re-activated telomerase to revert endothelial cell senescence.ConclusionOur data identified the interaction between CSE and p53 as a promising target to preserve vascular regeneration during aging.Key QuestionTo identify the mechanisms that regulate endothelial cell senescence under native conditions and their impact on vascular repair in aging.Key FindingLack of a physical interaction between CSE and p53 metabolically reprogrammes endothelial cells to reduce telomerase activity and halt endothelial cell regeneration.Take home messageInterventions to increase CSE expression represent a novel therapy against p53-induced endothelial cell cycle arrest and senescenseTranslational perspectiveEndothelial rejuvenation strategies could serve as promising therapies against age-related cardiovascular diseases. By investigating human native endothelial cells from young and aged individuals, we identified that the age-related nuclear accumulation of p53 reprograms endothelial cell metabolism, regulates telomerase activity and inhibits endothelial cell regeneration. Nuclear localization of p53 resulted from a loss of its interaction with the cysteine catabolizing enzyme cystathionine γ-lyase in the cytoplasm. Enhancing the physical interaction of p53 with CSE by gene therapy could revert endothelial cell senescence and activate endothelial reparative responses.

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide–related sulfane sulfur compounds (H 2 S n ), that exert their biological actions via cysteine S -sulfhydration of target proteins. This study set out to map the “ S -sulfhydrome” (ie, the spectrum of proteins targeted by H 2 S n ) in human endothelial cells. Methods: Liquid chromatography with tandem mass spectrometry was used to identify S -sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress–induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell–specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. Results: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H 2 S n donor, SG1002. The endothelial cell “ S -sulfhydrome” consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S -sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S -sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H 2 S n generation, impaired flow-induced dilatation, and failure to detect β3 integrin S -sulfhydration, all of which were rescued after the administration of an H 2 S n supplement. Conclusions: Vascular disease is associated with marked changes in the S -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H 2 S n supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

Over the last two decades, hydrogen sulfide (H2S) has emerged as an endogenous regulator of a broad range of physiological functions. H2S belongs to the class of molecules known as gasotransmitters, which typically include nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). The present article reviews the regulation of these enzymes as well as the pathways of their enzymatic and nonenzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g., NO) and reactive oxygen species are also outlined. Next, the various biological targets and signaling pathways are outlined, with special reference to H2S or oxidative posttranscriptional modification (persulfidation or sulfhydration) of proteins and the effect of H2S on various channels and intracellular second messenger pathways, the regulation of gene transcription and translation, and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed, including the regulation of membrane potential, endo- and exocytosis, regulation of various cell organelles (endoplasmic reticulum, Golgi, mitochondria), regulation of cell movement, cell cycle, cell differentiation, and physiological aspects of regulated cell death. Next, the physiological roles of H2S in various cell types and organ systems are overviewed, including the role of H2S in red blood cells, immune cells, the central and peripheral nervous systems (with focus on neuronal transmission, learning, and memory formation), and regulation of vascular function (including angiogenesis as well as its specialized roles in the cerebrovascular, renal, and pulmonary vascular beds) and the role of H2S in the regulation of special senses, vision, hearing, taste and smell, and pain-sensing. Finally, the roles of H2S in the regulation of various organ functions (lung, heart, liver, kidney, urogenital organs, reproductive system, bone and cartilage, skeletal muscle, and endocrine organs) are presented, with a focus on physiology (including physiological aging) but also extending to some common pathophysiological conditions. From these data, a wide array of significant roles of H2S in the physiological regulation of all organ functions emerges and the characteristic bell-shaped biphasic effects of H2S are highlighted. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified.

The single curative measure for heart failure patients is total heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have pinpointed a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes,in vivo,in vitroandex vivo.Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the centre of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues.The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Background/Introduction Oleuropein (oleu), the main polyphenolic constituent of olive, has cardioprotective effects against ischemia (I) – reperfusion (R) injury (IRI) when administered chronically. Purpose We aimed to assess the cardioprotection afforded by acute administration of oleu, to evaluate the underlying mechanism and whether it could enhance or imbed the cardioprotective manifestation of ischemic postconditioning (PostC). Methods Male rabbits were subjected to I/R (30/180 min) and randomized to 7 groups: (i) Control (ii) PostC: 8 cycles of 30-sec I/R at the onset of R; (iii) Oleu (100 mg/kg, iv bolus) at the 20th min of I; (iv) Oleu+NOS inhibitor (L-NAME, 10mg/kg); (v) Oleu+PI3K/Akt inhibitor (wortmannin, 60 μg/kg); (vi) Oleu+JAK2 inhibitor (AG490, 6μg/kg/min) and (vii) Oleu+PostC. Additionally, male C57BL6/J mice, subjected to I/R (30/180 min) and randomized to 4 groups: (i) Control; (ii) PostC: 3 cycles of 10-sec I/R at the onset of R; (iii) Oleu (350 mg/kg, iv bolus) at the 20th min of I; (iv) Oleu+PostC. In both animal models, infarct size (IS) expressed as percentage of infarct to area at risk ratio (I/R, %) was determined. Oleu's effect on cardiomyocytes was measured by MTT assay in adult rat cardiomyocytes exposed to simulated I (SI) and reoxygenation. We also assessed the effect of oleu on mitochondrial permeability transition pore (mPTP) through calcium retention capacity (CRC) assay and on cGMP accumulation in rat aortic smooth muscle cells. In a mouse model of IRI, we explored the effect of oleu on the recruitment of inflammatory monocytes and neutrophils in the IR heart using flow cytometry, whereas the effect of oleu on Nrf-2 signaling pathway-related genes was analyzed by western blot. Results In both animal models acute oleu administration reduced significantly the IS compared to the control group. None of the inhibitors of the classic cardioprotective pathways influence its IS limiting effects in rabbits. Combination of oleu with PostC caused further limitation of IS compared to PostC in both animal models (I/R Rabbits: 14.6±0.9 vs. 26.7±2.7%, p Conclusion(s) Acute oleu administration combined with PostC provides robust and synergistic cardioprotection in experimental models of IRI by inducing antioxidant defense genes through Nrf-2 axis and independently of the classic cardioprotective signaling pathways. Funding Acknowledgement Type of funding sources: None.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Aims Glycogen synthase kinase 3 beta (GSK3β) link with the mitochondrial Permeability Transition Pore (mPTP) in cardioprotection is debated. We investigated the role of GSK3β in ischaemia (I)/reperfusion (R) injury using pharmacological tools. Methods and results Infarct size using the GSK3β inhibitor BIO (6-bromoindirubin-3'-oxime) and several novel analogues (MLS2776-MLS2779) was determined in anaesthetized rabbits and mice. In myocardial tissue GSK3β inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3β localization was determined in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from Langendorff-perfused murine hearts (30'I/10'R or normoxic conditions). Calcium retention capacity (CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species (ROS) formation, ATP production, or hydrolysis were measured in SSM at baseline. Cyclosporine A (CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller infarct size compared with control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3β inhibition was confirmed at the 10th min and 2 h of reperfusion, while up-regulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3β was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent. Conclusion Pharmacological inhibition of GSK3β attenuates infarct size beyond mPTP inhibition.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 μΜ (p = 7 x 10−6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens., Author summary Multidrug- resistant Pseudomonas (P.) aeruginosa is one of the most common causes of ventilator-associated pneumonia (VAP), which consist one of the most common causes of sepsis in hospitalized patients. The increasing antimicrobial resistance of P. aeruginosa necessitates the investigation of new treatment strategies. In this study we highlight host derived hydrogen sulfide (H2S), produced by cystathionine-γ-lyase (CSE), as a new protective mechanism against Pseudomonas aeruginosa infection. We have shown that serum H2S levels above 5.3μM can discriminate survivors of P.aeruginosa related sepsis with high sensitivity and is correlated with reduced mortality. We verified this finding in an animal model of CSE knockout mice. The protective role of endogenous H2S resides in the enhancement of neutrophil recruitment and the phagocytic activity of neutrophils, as well as by inhibiting the cell-to-cell communication system of P. aeruginosa (quorum sensing system), rendering it more vulnerable to phagocytosis.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Background and purpose l-cysteine or hydrogen sulfide (H2 S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30-100 μM. Here, we have investigated the signalling involved in the H2 S-induced contraction. Experimental approach Vascular response to NaHS or l-cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ-lyase (CSE-/- ), soluble guanylyl cyclase (sGCα1 -/- ) and endothelial nitric oxide synthase (eNOS-/- ) knock-out mice. The cAMP, cGMP and inosine 3',5'-cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated. Key results CSE-derived H2 S-induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2 S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell-permeable analogue of cIMP elicits concentration-dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE-/- mice, confirms that H2 S-induced contraction involves cIMP. Conclusion and implications The endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE-derived H2 S that is mediated by cIMP.

In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (HLiquid chromatography with tandem mass spectrometry was used to identifyThree paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing HVascular disease is associated with marked changes in the

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5'-triphosphate) to cGMP (cyclic guanylyl monophosphate). It contains the heme binding H-NOX (heme-nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its

Hydrogen sulfide (H2S) is now recognized as an endogenous signaling gasotransmitter in mammals. It is produced by mammalian cells and tissues by various enzymes - predominantly cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - but part of the H2S is produced by the intestinal microbiota (colonic H2S-producing bacteria). Here we summarize the available information on the production and functional role of H2S in the various cell types typically associated with innate immunity (neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, basophils, eosinophils) and adaptive immunity (T and B lymphocytes) under normal conditions and as it relates to the development of various inflammatory and immune diseases. Special attention is paid to the physiological and the pathophysiological aspects of the oral cavity and the colon, where the immune cells and the parenchymal cells are exposed to a special "H2S environment" due to bacterial H2S production. H2S has many cellular and molecular targets. Immune cells are "surrounded" by a "cloud" of H2S, as a result of endogenous H2S production and exogenous production from the surrounding parenchymal cells, which, in turn, importantly regulates their viability and function. Downregulation of endogenous H2S producing enzymes in various diseases, or genetic defects in H2S biosynthetic enzyme systems either lead to the development of spontaneous autoimmune disease or accelerate the onset and worsen the severity of various immune-mediated diseases (e.g. autoimmune rheumatoid arthritis or asthma). Low, regulated amounts of H2S, when therapeutically delivered by small molecule donors, improve the function of various immune cells, and protect them against dysfunction induced by various noxious stimuli (e.g. reactive oxygen species or oxidized LDL). These effects of H2S contribute to the maintenance of immune functions, can stimulate antimicrobial defenses and can exert anti-inflammatory therapeutic effects in various diseases.

Background: Hydrogen sulfide (H 2 S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. Methods: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E–deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H 2 S bioavailability, and amino acid profiling were also performed with human material. Results: The endothelial cell–specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H 2 S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1β. Consistent with the loss of H 2 S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R–target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l -cystathionine negatively correlated with vascular reactivity and H 2 S levels, indicating its potential use as a biomarker for vascular disease. Conclusions: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H 2 S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H 2 S are lost owing to the phosphorylation and inhibition of the enzyme.

Background Osteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H2S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development. Methods 3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST−/− meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST−/− cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated. Results 3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST−/− mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes. Conclusion 3-MST-generated H2S protects against joint calcification and experimental OA. Enhancing H2S production in chondrocytes may represent a potential disease modifier to treat OA.

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel–PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel–PIP2 interactions. H2S regulation was attenuated when channel–PIP2 interactions were strengthened and was increased when channel–PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel–PIP2 interactions and channel gating.