Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells.

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 µM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Hydrogen Sulfide: A Novel Player in Airway Development, Pathophysiology of Respiratory Diseases, and Antiviral Defenses.

Hydrogen sulfide (H2S) is a biologically relevant signaling molecule in mammals. Along with the volatile substances nitric oxide (NO) and carbon monoxide (CO), H2S is defined as a gasotransmitter. It plays a physiological role in a variety of functions, including synaptic transmission, vascular tone, angiogenesis, inflammation, and cellular signaling. The generation of H2S is catalyzed by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). The expression of CBS and CSE is tissue specific, with CBS being expressed predominantly in the brain, and CSE in peripheral tissues, including lungs. CSE expression and activity are developmentally regulated, and recent studies suggest that CSE plays an important role in lung alveolarization during fetal development. In the respiratory tract, endogenous H2S has been shown to participate in the regulation of important functions such as airway tone, pulmonary circulation, cell proliferation or apoptosis, fibrosis, oxidative stress, and inflammation. In the past few years, changes in the generation of H2S have been linked to the pathogenesis of a variety of acute and chronic inflammatory lung diseases, including asthma and chronic obstructive pulmonary disease. Recently, our laboratory made the critical discovery that cellular H2S exerts broad-spectrum antiviral activity both in vitro and in vivo, in addition to independent antiinflammatory activity. These findings have important implications for the development of novel therapeutic strategies for viral respiratory infections, as well as other inflammatory lung diseases, especially in light of recent significant efforts to generate controlled-release H2S donors for clinical therapeutic applications.

Heme-dependent Metabolite Switching Regulates H2S Synthesis in Response to Endoplasmic Reticulum (ER) Stress.

Substrate ambiguity and relaxed reaction specificity underlie the diversity of reactions catalyzed by the transsulfuration pathway enzymes, cystathionine ß-synthase (CBS) and γ-cystathionase (CSE). These enzymes either commit sulfur metabolism to cysteine synthesis from homocysteine or utilize cysteine and/or homocysteine for synthesis of H2S, a signaling molecule. We demonstrate that a kinetically controlled heme-dependent metabolite switch in CBS regulates these competing reactions where by cystathionine, the product of CBS, inhibits H2S synthesis by the second enzyme, CSE. Under endoplasmic reticulum stress conditions, induction of CSE and up-regulation of the CBS inhibitor, CO, a product of heme oxygenase-1, flip the operating preference of CSE from cystathionine to cysteine, transiently stimulating H2S production. In contrast, genetic deficiency of CBS leads to chronic stimulation of H2S production. This metabolite switch from cystathionine to cysteine and/or homocysteine renders H2S synthesis by CSE responsive to the known modulators of CBS: S-adenosylmethionine, NO, and CO. Used acutely, it regulates H2S synthesis; used chronically, it might contribute to disease pathology.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (ß-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

Endogenous CBS-H2S Pathway Contributes to the Development of CCI-Induced Neuropathic Pain.

Studies showed a complex relationship between hydrogen sulfide (H2S) and neuropathic pain. In this study, the relationship between endogenous CBS-H2S pathway in L4-6 spinal cord and neuropathic pain was explored. A total of 163 adult Kunming mice were used in this study. CBS expression and H2S formation in L4-6 spinal cord were detected in the development of neuropathic pain firstly. Then, effect of AOAA, an CBS inhibitor, on treatment of neuropathic pain by chronic construction injury surgery (CCI) was detected. Pain thresholds and activation of NF-κB(p65), ERK1/2 and CREB were measured as biomarks of neuropathic pain. Results showed that CCI surgery significantly upregulated protein expression of CBS and H2S formation. Correlation analysis showed pain thresholds had negative relationships with protein expression of CBS and H2S formation. Treatment with AOAA, a CBS inhibitor, inhibited CCI-induced upregulation of CBS expression and H2S formation (P < 0.05). Further, AOAA significantly decreased activation of NF-κB(p65), ERK1/2 and CREB pathway, and reversed CCI-induced allodynia (P < 0.05). This indicated that CBS-H2S pathway promoted the development of neuropathic pain. CBS-H2S pathway could be a promising target for treatment of neuropathic pain.

The H2S-generating enzymes cystathionine ß-synthase and cystathionine γ-lyase play a role in vascular development during normal lung alveolarization.

The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. Recent studies revealed that H2S administration limited perturbations to lung structure in experimental animal models of bronchopulmonary dysplasia (BPD), partially restoring alveolarization, limiting pulmonary hypertension, limiting inflammation, and promoting epithelial repair. No studies have addressed roles for endogenous H2S in lung development. H2S is endogenously generated by cystathionine ß-synthase (Cbs) and cystathionine γ-lyase (Cth). We demonstrate here that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization and that alveolarization is blunted in Cbs(-/-) and Cth(-/-) mouse pups, where a 50% reduction in the total number of alveoli was observed, without any impact on septal thickness. Laser-capture microdissection and immunofluorescence staining indicated that Cbs and Cth were expressed in the airway epithelium and lung vessels. Loss of Cbs and Cth led to a 100-500% increase in the muscularization of small- and medium-sized lung vessels, which was accompanied by increased vessel wall thickness, and an apparent decrease in lung vascular supply. Ablation of Cbs expression using small interfering RNA or pharmacological inhibition of Cth using propargylglycine in lung endothelial cells limited angiogenic capacity, causing a 30-40% decrease in tube length and a 50% decrease in number of tubes formed. In contrast, exogenous administration of H2S with GYY4137 promoted endothelial tube formation. These data confirm a key role for the H2S-generating enzymes Cbs and Cth in pulmonary vascular development and homeostasis and in lung alveolarization.

Gene transfer of cystathionine ß-synthase into RVLM increases hydrogen sulfide-mediated suppression of sympathetic outflow via KATP channel in normotensive rats.

Hydrogen sulfide has been shown to have a sympathoinhibitory effect in the rostral ventrolateral medulla (RVLM). The present study examined the function of cystathionine ß-synthase (CBS)/hydrogen sulfide system in the RVLM, which plays a crucial role in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding CBS (AdCBS) or enhanced green fluorescent protein (AdEGFP) were transfected into the RVLM in normotensive rats. Identical microinjection of AdCBS into the RVLM had no effect on systolic blood pressure and heart rate (HR) in conscious rats. Acute experiments were performed at day 7 after gene transfer in anesthetized rats. Microinjection of the CBS inhibitors hydroxylamine (HA) or amino-oxyacetate into the RVLM produced an increase in the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and HR. There was a potentiation of the increases in RSNA, MAP, and HR because of the CBS inhibitors in AdCBS-injected rats compared with AdEGFP-injected rats. Pretreatment with pinacidil, a ATP-sensitive potassium (KATP) channel activator, abolished the effects of HA in two groups. Microinjection of glibenclamide, a KATP channel blocker, produced increases in RSNA, MAP, and HR in AdCBS-injected rats. No changes in behavior were observed in AdEGFP-injected rats. Furthermore, Western blot analysis indicated an increase in the expression of sulfonylurea receptor 2 and inward rectifier K(+) 6.1 in AdCBS-injected rats. These results suggest that the increase in KATP channels in the RVLM may be responsible for the greater sympathetic outflow and pressor effect of HA in AdCBS-injected rats compared with AdEGFP-injected rats.

Hypoxia-inducible factors regulate human and rat cystathionine ß-synthase gene expression.

Increased catalytic activity of CBS (cystathionine ß-synthase) was recently shown to mediate vasodilation of the cerebral microcirculation, which is initiated within minutes of the onset of acute hypoxia. To test whether chronic hypoxia was a stimulus for increased CBS expression, U87-MG human glioblastoma and PC12 rat phaeochromocytoma cells were exposed to 1% or 20% O2 for 24-72 h. CBS mRNA and protein expression were increased in hypoxic cells. Hypoxic induction of CBS expression was abrogated in cells transfected with vector encoding shRNA targeting HIF (hypoxia-inducible factor) 1α or 2α. Exposure of rats to hypobaric hypoxia (0.35 atm; 1 atm=101.325 kPa) for 3 days induced increased CBS mRNA, protein and catalytic activity in the cerebral cortex and cerebellum, which was blocked by administration of the HIF inhibitor digoxin. HIF-binding sites, located 0.8 and 1.2 kb 5' to the transcription start site of the human CBS and rat Cbs genes respectively, were identified by ChIP assays. A 49-bp human sequence, which encompassed an inverted repeat of the core HIF-binding site, functioned as a hypoxia-response element in luciferase reporter transcription assays. Thus HIFs mediate tissue-specific CBS expression, which may augment cerebral vasodilation as an adaptive response to chronic hypoxia.

Neonatal colonic inflammation sensitizes voltage-gated Na(+) channels via upregulation of cystathionine ß-synthetase expression in rat primary sensory neurons.

The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood, and treatment remains difficult. We have previously reported that colon-specific dorsal root ganglion (DRG) neurons were hyperactive in a rat model of IBS induced by neonatal colonic inflammation (NCI). This study was designed to examine plasticity of voltage-gated Na(+) channel activities and roles for the endogenous hydrogen sulfide-producing enzyme cystathionine ß-synthetase (CBS) in chronic visceral hyperalgesia. Abdominal withdrawal reflex (AWR) scores were recorded in response to graded colorectal distention in adult male rats as a measure of visceral hypersensitivity. Colon-specific DRG neurons were labeled with 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate and acutely dissociated for measuring Na(+) channel currents. Western blot analysis was employed to detect changes in expressions of voltage-gated Na(+) (Na(V)) channel subtype 1.7, Na(V)1.8, and CBS. NCI significantly increased AWR scores when compared with age-matched controls. NCI also led to an ~2.5-fold increase in Na(+) current density in colon-specific DRG neurons. Furthermore, NCI dramatically enhanced expression of Na(V)1.7, Na(V)1.8, and CBS in colon-related DRGs. CBS was colocalized with Na(V)1.7 or -1.8 in colon-specific DRG neurons. Administration of O-(carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, remarkably suppressed Na(+) current density and reduced expression of Na(V)1.7 and Na(V)1.8. More importantly, intraperitoneal or intrathecal application of AOAA attenuated AWR scores in NCI rats in a dose-dependent manner. These data suggest that NCI enhances Na(+) channel activity of colon DRG neurons, which is most likely mediated by upregulation of CBS expression, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.

High-fat diet stimulates hepatic cystathionine ß-synthase and cystathionine γ-lyase expression.

Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) catalyze homocysteine (Hcy) metabolism via the trans-sulfuration pathway. They are also responsible for hydrogen sulfide (H2S) production via desulfuration reactions. The liver contributes significantly to the regulation of Hcy and H2S homeostasis, which might participate in many physiological and pathological processes. The aim of this study was to investigate the effect of a high-fat diet (HFD) on hepatic CBS and CSE expression and its impact on Hcy and H2S metabolism. Mice (C57BL/6) fed a HFD (60% kcal fat) for 5 weeks developed fatty liver. The mRNA and protein levels of CBS and CSE in the liver were significantly elevated in mice fed a HFD. Subsequently the metabolism of Hcy by CBS and CSE was increased in the liver, and its level decreased in the circulation. Increased CBS and CSE expression also caused a significant elevation in H2S production in the liver. The level of lipid peroxides was elevated, indicating oxidative stress, while the level of total glutathione remained unchanged in the liver of HFD-fed mice. Upregulation of the trans-sulfuration pathway might play an adaptive role against oxidative stress by maintaining total glutathione levels in the liver.

Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases, including Parkinson's disease. The present study attempted to investigate the effect of hydrogen sulfide (H(2)S) on 6-hydroxydopamine (6-OHDA)-induced ER stress in SH-SY5Y cells. We found in the present study that exogenous application of sodium hydrosulfide (NaHS; an H(2)S donor, 100 µM) significantly attenuated 6-OHDA (50 µM)-induced cell death. NaHS also reversed the upregulation of cleaved poly(ADP-ribose) polymerase and caspase 9 in 6-OHDA-treated cells. Consistent with its cytoprotective effects, NaHS markedly reduced 6-OHDA induced-ER stress responses, including the upregulated levels of eukaryotic initiation factor-2α phosphorylation, glucose-regulated protein 78, and C/EBP homologous protein expression. The protective effect of H(2)S on ER stress was attenuated by blockade of Akt activity with an Akt inhibitor or inhibition of heat shock protein (Hsp)90 with geldanamycin but not by suppression of ERK1/2 with PD-98059. Blockade of Akt also significantly decreased the protein abundance of Hsp90 in SH-SY5Y cells. Moreover, overexpression of cystathionine ß-synthase (a main H(2)S-synthesizing enzyme in the brain) elevated the Hsp90 protein level and suppressed 6-OHDA-induced ER stress. In conclusion, the protective effect of H(2)S against 6-OHDA-induced ER stress injury in SH-SY5Y cells involves the Akt-Hsp90 pathway.

Ischemia/reperfusion reduces transcription factor Sp1-mediated cystathionine beta-synthase expression in the kidney.

Cystathionine beta-synthase (CBS) is a key enzyme that catalyzes the rate-limiting step for homocysteine (Hcy) metabolism via the trans-sulfuration pathway and is also responsible for the production of H(2)S through the desulfhydration reaction. Our recent studies demonstrate that renal ischemia/reperfusion decreased the CBS activity leading to Hcy accumulation and H(2)S reduction in the kidney, which in turn contributed to kidney injury. Both Hcy and H(2)S play important roles in physiological and pathological processes. In this study we investigated the molecular mechanism by which CBS activity was regulated in the kidney. The left kidney of Sprague-Dawley rat was subjected to 45 min of ischemia followed by 6 h of reperfusion. Ischemia/reperfusion caused a significant decrease in CBS mRNA and protein levels in the kidney. As a consequence, there was a marked reduction in the CBS enzyme activity. Transfection of kidney proximal tubular cells with transcription factor (Sp1) small interfering RNA caused a marked reduction in CBS mRNA, indicating a pivotal role for Sp1 in regulating CBS expression in kidney cells. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay detected a lower Sp1 activity in kidneys subjected to ischemia/reperfusion as compared with that in a sham-operated group. ERK-mediated phosphorylation of Sp1 was responsible for a decreased transcriptional activity of Sp1 in the kidney upon ischemia/reperfusion. These results suggest that reduced kidney CBS gene expression during ischemia/reperfusion is mediated via a decrease in Sp1 transcriptional activity. Regulation of CBS-mediated Hcy and H(2)S homeostasis may offer a renal protective effect against ischemia/reperfusion injury.

Endogenous hydrogen sulfide is involved in asymmetric dimethylarginine-induced protection against neurotoxicity of 1-methyl-4-phenyl-pyridinium ion.

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is profoundly protective against 1-methy-4-phenylpyridinium ion (MPP+)-induced neurotoxicity. Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of MPP+; while hydrogen sulfide (H2S) is a pivotal endogenous antioxidant. This study is to assess the potential role of endogenous H2S in the neuroprotection of ADMA against MPP+-induced toxicity in PC12 cells. We showed that ADMA prevented MPP+-induced inhibition of endogenous H2S generation through inhibiting the down-regulation of cystathionine-ß-synthetase (CBS, the major enzyme responsible for endogenous H2S generation in PC12 cells) expression and activity elicited by MPP+. ADMA obviously attenuated MPP+-triggered accumulation of intracellular ROS, dissipation of mitochondrial membrane potential (MMP), release of cytochrome c (Cyt-c), and downregulation of Bcl-2 protein expression in PC12 cells. Inhibition of CBS activity by amino-oxyacetate and CBS silencing with a short hairpin RNA vector targeting rat CBS gene reversed the protective action of ADMA against MPP+-caused cytotoxicity, ROS overproduction, and MMP loss in PC12 cells. These results indicate that the protection of ADMA against MPP+-mediated neurotoxicity involves the melioration of MPP+-induced inhibition of endogenous H2S generation. Our findings suggest that modulation of H2S production provide new therapeutic targets for the treatment of neurodegenerative disease, such as Parkinson's disease.

Cystathionine ß-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine ß-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of CBS in HCC development and immune evasion have not been systematically investigated, and their underlying mechanisms remain elusive. Here, we investigated the roles of CBS in tumor cells and tumor microenvironment of HCC. METHODS: 236 HCC samples were collected to detect the expression of CBS, cleaved Caspase-3 and paired related homeobox 2 (PRRX2) and the number of immune cells. HCC cell lines were employed to examine the effects of CBS on cellular viability, apoptosis and signaling in vitro. Cbs heterozygous knockout mice, C57BL/6 mice, nude mice and non-obese diabetic severe combined immunodeficiency mice were used to investigate the in vivo functions of CBS. RESULTS: Downregulation of CBS was observed in HCC, and low expression of CBS predicted poor prognosis in HCC patients. CBS overexpression dramatically promoted cellular apoptosis in vitro and inhibited tumor growth in vivo. Activation of the Cbs/H2S axis also reduced the abundance of tumor-infiltrating Tregs, while Cbs deficiency promoted Tregs-mediated immune evasion and boosted tumor growth in Cbs heterozygous knockout mice. Mechanistically, CBS facilitated the expression cleaved Caspase-3 in tumor cells, and on the other hand, suppressed Foxp3 expression in Tregs via inactivating IL-6/STAT3 pathway. As a transcription factor of IL-6, PRRX2 was reduced by CBS. Additionally, miR-24-3p was proven to be an upstream suppressor of CBS in HCC. CONCLUSIONS: Our results indicate the antitumor function of CBS in HCC by inactivation of the PRRX2/IL-6/STAT3 pathway, which may serve as a potential target for HCC clinical immunotherapy.

S-box and T-box riboswitches and antisense RNA control a sulfur metabolic operon of Clostridium acetobutylicum.

The ubiGmccBA operon of Clostridium acetobutylicum is involved in methionine to cysteine conversion. We showed that its expression is controlled by a complex regulatory system combining several RNA-based mechanisms. Two functional convergent promoters associated with transcriptional antitermination systems, a cysteine-specific T-box and an S-box riboswitch, are located upstream of and downstream from the ubiG operon, respectively. Several antisense RNAs were synthesized from the downstream S-box-dependent promoter, resulting in modulation of the level of ubiG transcript and of MccB activity. In contrast, the upstream T-box system did not appear to play a major role in regulation, leaving antisense transcription as the major regulatory mechanism for the ubiG operon. The abundance of sense and antisense transcripts was inversely correlated with the sulfur source availability. Deletion of the downstream promoter region completely abolished the sulfur-dependent control of the ubiG operon, and the expression of antisense transcripts in trans did not restore the regulation of the operon. Our data revealed important insights into the molecular mechanism of cis-antisense-mediated regulation, a control system only rarely observed in prokaryotes. We proposed a regulatory model in which the antisense RNA controlled the expression of the ubiG operon in cis via transcriptional interference at the ubiG locus.

Clinical Significance of CBS and CCL21 in Gallbladder Adenocarcinomas and Squamous Cell/Adenosquamous Carcinomas.

Gallbladder cancer (GBC) is a rare disease with high mortality. However, no biomarkers for the carcinogenesis, progression, prognosis, and early diagnosis are clinically available. This study investigated the expressions of cystathionine-ß-synthase (CBS) and C-C chemokine receptor 7 (CCR7) protein and their clinical and pathologic significances in gallbladder squamous cell/adenosquamous carcinomas (SC/ASC) and adenocarcinomas (AC). CBS and chemokine ligand 21 (CCL21) expression was measured using immunohistochemistry in 69 SC/ASCs and 146 ACs. A significantly high percentage of patients with an age above 45 years, lymph node metastasis, and invasion was observed in the SCs/ASCs compared with ACs (P<0.05). Both AC and SC/ASC patients with positive CBS and CCL21 expression exhibited a high tumor-lymph node-metastasis stage, lymph node metastasis, and invasion compared with patients with negative CBS and CCL21 expression (P<0.05 or P<0.01). SC/ASC patients with positive CBS expression was prone to have a larger tumor size than those with negative expression (P<0.05). Positive CBS and CCL21 expression correlated with poor differentiation and larger tumor size in AC patients. Positive CBS and CCL21 are closely associated with a decreased overall survival in SC/ASC and AC patients (P<0.05 or P<0.01) and were independent factors for a poor-prognosis. Both CBS and CCL21 showed a good overall diagnostic performance for SC/ASC (AUC=0.742 and AUC=0.764, respectively) and AC (AUC=0.734 and AUC=0.718, respectively). In conclusion, positive CBS and CCL21 expression are closely associated with the clinical severity and poor prognosis in GBC, and can be a marker for the diagnosis of AC and SC/ASC type of GBC.

Analysis of Vascular Hydrogen Sulfide Biosynthesis.

With potent vasodilatory and pro-angiogenic properties, hydrogen sulfide (H2S) is now accepted as the third gasotransmitter after nitric oxide (NO) and carbon monoxide. Endogenous H2S is mainly synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). Akin to previous studies showing hormonal regulation of NO biosynthesis, we first reported that uterine and systemic artery H2S biosynthesis is regulated by exogenous estrogens in an ovariectomized sheep model of estrogen replacement therapy, specifically stimulating CBS, but not CSE, expression, in uterine (UA) and mesenteric (MA), but not carotid (CA), arteries in ovariectomized nonpregnant sheep. We have found significantly elevated H2S biosynthesis due to CBS upregulation under estrogen-dominant physiological states, the proliferative phase of menstrual cycle and pregnancy in primary human UAs. Our studies have pioneered the role of H2S biology in uterine hemodynamics regulation although there is still much that needs to be learned before a thorough elucidation of a role that H2S plays in normal physiology of uterine hemodynamics and its dysregulation under pregnancy complications can be determined. In this chapter we describe a series of methods that we have optimized for analyzing vascular H2S biosynthesis, including (1) real-time quantitative PCR (qPCR) for assessing tissue and cellular levels of CBS and CSE mRNAs, (2) immunoblotting for assessing CBS and CSE proteins, (3) semiquantitative immunofluorescence microscopy to specifically localize CBS and CSE proteins on vascular wall and to quantify their cellular expression levels, and (4) methylene blue assay for assessing H2S production in the presence of selective CBS and CSE inhibitors.

Cystathione ß-Synthase Is Increased in Thyroid Malignancies.

BACKGROUND: Cystathione ß-synthase (CBS) catalyzes the conversion of homocysteine and cysteine to hydrogen sulfide (H2S) and cystathione, via the trans-sulfuration pathway. CBS protein expression levels are increased in several different human malignancies, with increased protein expression correlating with parameters such as tumor stage, anaplasia, metastases, and chemotherapy resistance. MATERIALS AND METHODS: This study employed tissue microarrays to examine CBS expression in benign thyroid tissue, thyroid oncocytomas, thyroid follicular adenomas, and in follicular, papillary, anaplastic, and medullary thyroid carcinomas. RESULTS: CBS expression was increased in all thyroid carcinomas types compared to benign thyroid tissue, but not in thyroid follicular adenomas or oncocytomas. A similar pattern was observed for nicotinamide phosphoribosyltransferase (NAMPT) tissue microarray analysis comparing thyroid adenomas and follicular carcinomas. CONCLUSION: For the first time, we showed that an H2S-syntheszing enzyme plays a role in thyroid malignancies. Additionally, our data suggest that CBS and NAMPT immunohistochemistry may be useful in differentiating follicular adenomas from follicular carcinomas.

Influence of folate on arterial permeability and stiffness in the absence or presence of hyperhomocysteinemia.

OBJECTIVE: Elevated plasma total homocysteine (tHcy) is associated with risk for cardiovascular disease. A common cause of mild hyperhomocysteinemia (HHcy) is folate deficiency. We sought to determine whether folate deficiency per se increases arterial permeability (quantitative fluorescence microscopy) and stiffness (vessel elastigraph), and whether the effects of folate deficiency are more severe in the presence of mild HHcy. METHODS AND RESULTS: Heterozygous cystathionine beta-synthase (CBS)-deficient mice (CBS(+/-)) and their wild-type littermates (CBS(+/+)) were fed chow containing either standard (Con) or relatively low amounts of folate (LF) for 18+/-3 weeks. Liver folate (microg folate/g liver) and tHcy (microM), respectively, were 12+/-1 and 8+/-1 in CBS(+/+) Con mice (n=12), and 8+/-1 and 8+/-1 in CBS(+/+) LF animals (n=5). Carotid arterial permeability was &38% greater (P<0.05) in CBS(+/+) LF versus Con mice, but vascular stiffening was unaltered. Liver folate and tHcy, respectively, were 13+/-1 and 11+/-1 in CBS(+/-) Con mice (n=16), and 8+/-1 and 16+/-3 in CBS(+/-) LF animals (n=6). Carotid arterial dextran accumulation was &31% greater, and maximal strain in aortae was &20% lower (both P<0.05) in CBS(+/-) LF versus Con mice. CONCLUSIONS: Taken together, low folate (P<0.05) combined with mild HHcy (P<0.05) in CBS(+/-) mice produced more arterial dysfunction compared with low folate alone (ie, CBS(+/+) mice). These findings may be particularly relevant to elderly individuals because tHcy and deficiencies of folate metabolism increase with age.

Expression profiling of homocysteine junction enzymes in the NCI60 panel of human cancer cell lines.

Methionine metabolism provides two key cellular reagents: S-adenosylmethionine and glutathione, derived from the common intermediate, homocysteine. A majority of cancer cells exhibit a methionine-dependent phenotype whereby they are unable to grow in medium in which methionine is replaced by its precursor, homocysteine. Additionally, CpG island hypermethylation of tumor suppressor gene promoters is observed in a background of global hypomethylation in cancerous cells. In this study, we have profiled the expression levels of the homocysteine junction enzymes, methionine synthase (MS), MS reductase (MSR), and cystathionine beta-synthase (CBS) in the NCI60 panel of cancer cell lines. The doubling time of non-small lung cell cancer lines, which exhibit the lowest levels of MS within the panel, was significantly correlated with expression of MS. The ratio of MS to MSR varied over a 5-fold range in the different cell types, which may modulate methionine synthesis. Interestingly, markedly reduced CBS expression was seen in the methionine-dependent prostate cancer cell line, PC-3, but not in the methionine-independent cell line, DU-145. However, neither provision of the transsulfuration pathway product, cysteine, nor overexpression of CBS rescued the growth impairment, indicating that reduced CBS was not responsible for the methionine-dependent phenotype in this cell line.