Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259.

OBJECTIVE: Cardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. APPROACH AND RESULTS: We interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-ß-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. CONCLUSION: Downregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

Tetramethylpyrazine protects mitochondrial function by up-regulation of TFAM and inhibition of neuronal apoptosis in a rat model of surgical brain injury.

Objectives: Mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage and mutation is widely accepted as one of the pathological processes of neurodegenerative diseases. As an mtDNA binding protein, mitochondrial transcription factor A (TFAM) maintains the integrity of mtDNA through transcription, replication, nucleoid formation, damage perception, and DNA repair. In recent works, the overexpression of TFAM increased the mtDNA copy count, promoted mitochondrial function, and improved the neurological dysfunction of neurodegenerative diseases. The role of TFAM in neurodegenerative diseases has been well explained. However, the role of TFAM after surgical brain injury (SBI) has not been studied. In this work, we aimed to study the role of TFAM in the brain after SBI and its mechanism of action. Materials and Methods: One hour after the occurrence of SBI, tetramethylpyrazine (TMP) was injected into the abdominal cavity of rats, and the brain was collected 48 hr later for testing. The evaluation included neurobehavioral function test, brain water content measurement, immunofluorescence, western blot, TUNEL staining, FJC staining, ROS test, and ATP test. Results: After SBI, the content of TFAM on the ipsilateral side increased and reached a peak at about 48 hr. After intraperitoneal injection of TMP in rats, 48 hr after SBI, the concentration of TFAM, Bcl-2, and adenosine triphosphate (ATP) increased; the content of caspase-3, reactive oxygen species (ROS), and cerebral edema decreased; and the nerve function significantly improved. Conclusion: TMP inhibited cell apoptosis after SBI in rats by up-regulating TFAM and protecting brain tissues.

Role of Lipocalin-2 in N1/N2 Neutrophil Polarization After Stroke.

BACKGROUND: Neutrophils and Lipocalin-2 (LCN2) play pivotal roles in cerebral ischemiareperfusion (I/R) injury. However, their contribution is not fully clarified. OBJECTIVE: This study aimed to explore the role of LCN2 and its association with neutrophil polarization in I/R injury. METHODS: A mouse model of middle cerebral artery occlusion (MCAO) was used to induce cerebral ischemia. LCN2mAb was administered 1 h and Anti-Ly6G was administered for 3d before MCAO. The role of LCN2 in the polarity transition of neutrophils was explored using an in vitro HL-60 cell model. RESULTS: LCN2mAb pretreatment had neuroprotective effects in mice. The expression of Ly6G was not significantly different, but the expression of N2 neutrophils was increased. In the in vitro study, LCN2mAb-treated N1-HL-60 cells induced N2-HL-60 polarization. CONCLUSION: LCN2 may affect the prognosis of ischemic stroke by mediating neutrophil polarization.

Gasotransmitter-Mediated Cysteinome Oxidative Posttranslational Modifications: Formation, Biological Effects, and Detection.

Significance: Gasotransmitters, including nitric oxide (NO), hydrogen sulfide (H2S) and sulfur dioxide (SO2), participate in various cellular processes via corresponding oxidative posttranslational modifications (oxiPTMs) of specific cysteines. Recent Advances: Accumulating evidence has clarified the mechanisms underlying the formation of oxiPTMs derived from gasotransmitters and their biological functions in multiple signal pathways. Because of the specific existence and functional importance, determining the sites of oxiPTMs in cysteine is crucial in biology. Recent advances in the development of selective probes, together with upgraded mass spectrometry (MS)-based proteomics, have enabled the quantitative analysis of cysteinome. To date, several cysteine residues have been identified as gasotransmitter targets. Critical Issues: To clearly understand the underlying mechanisms for gasotransmitter-mediated biological processes, it is important to identify modified targets. In this review, we summarize the chemical formation and biological effects of gasotransmitter-dependent oxiPTMs and highlight the state-of-the-art detection methods. Future Directions: Future studies in this field should aim to develop the next generation of probes for in situ labeling to improve spatial resolution and determine the dynamic change of oxiPTMs, which can lay the foundation for research on the molecular mechanisms and clinical translation of gasotransmitters. Antioxid. Redox Signal. 40, 145-167.

L-cystathionine protects against oxidative stress and DNA damage induced by oxidized low-density lipoprotein in THP-1-derived macrophages.

Introduction: Oxidative stress in monocyte-derived macrophages is a significant pathophysiological process in atherosclerosis. L-cystathionine (L-Cth) acts as a scavenger for oxygen free radicals. However, the impact of L-Cth on macrophage oxidative stress during atherogenesis has remained unclear. This study aimed to investigate whether L-Cth affects oxidative stress in THP-1-derived macrophages and its subsequent effects on DNA damage and cell apoptosis. Methods: We established a cellular model of oxLDL-stimulated macrophages. The content of superoxide anion, H2O2, NO, and H2S in the macrophage were in situ detected by the specific fluorescence probe, respectively. The activities of SOD, GSH-Px, and CAT were measured by colorimetrical assay. The protein expressions of SOD1, SOD2, and iNOS were detected using western blotting. The DNA damage and apoptosis in the macrophage was evaluated using an fluorescence kit. Results: The results demonstrated that oxLDL significantly increased the content of superoxide anion and H2O2, the expression of iNOS protein, and NO production in macrophages. Conversely, oxLDL decreased the activity of antioxidants GSH-Px, SOD, and CAT, and downregulated the protein expressions of SOD1 and SOD2 in macrophages. However, treatment with L-Cth reduced the levels of superoxide anion, H2O2, and NO, as well as the protein expression of iNOS induced by oxLDL. Moreover, L-Cth treatment significantly enhanced GSH-Px, SOD, and CAT activity, and upregulated the expressions of SOD1 and SOD2 proteins in macrophages treated with oxLDL. Furthermore, both L-Cth supplementation and activation of endogenous L-Cth production suppressed DNA damage and cell apoptosis in oxLDL-injured macrophages, whereas inhibition of endogenous L-Cth exacerbated the deleterious effects of oxLDL. Conclusion: These findings suggest that L-Cth exerts a pronounced inhibitory effect on the oxidative stress, subsequent DNA damage and cell apoptosis in oxLDL-stimulated THP-1 monocytes. This study deepens our understanding of the pathogenesis of macrophage-related cardiovascular pathology.

Hydrogen Sulfide Regulates Macrophage Function in Cardiovascular Diseases.

Significance: Hydrogen sulfide (H2S) is an endogenous gasotransmitter that plays a vital role in immune system regulation. Recently, the regulation of macrophage function by H2S has been extensively and actively recognized. Recent Advances: The mechanisms by which endogenous H2S controls macrophage function have attracted increasing attention. The generation of endogenous H2S from macrophages is mainly catalyzed by cystathionine-γ-lyase. H2S is involved in the macrophage activation and inflammasome formation, which contributes to macrophage apoptosis, adhesion, chemotaxis, and polarization. In addition, H2S has redox ability and interacts with reactive oxygen species to prevent oxidative stress. Moreover, H2S epigenetically regulates gene expression. Critical Issues: In this article, the generation of endogenous H2S in macrophages and its regulatory effect on macrophage function are reviewed. In addition, the signal transduction targeting macrophages by H2S is also addressed. Finally, the potential therapeutic effect of H2S on macrophages is discussed. Future Directions: Further experiments are required to explore the involvement of endogenous H2S in the regulation of macrophage function in various physiological and pathophysiological processes and elucidate the mechanisms involved. Regarding the clinical translation of H2S, further exploration of the application of H2S in inflammation-related diseases is needed. Antioxid. Redox Signal. 38, 45-56.

Podoplanin: Its roles and functions in neurological diseases and brain cancers.

Podoplanin is a small mucin-like glycoprotein involved in several physiological and pathological processes in the brain including development, angiogenesis, tumors, ischemic stroke and other neurological disorders. Podoplanin expression is upregulated in different cell types including choroid plexus epithelial cells, glial cells, as well as periphery infiltrated immune cells during brain development and neurological disorders. As a transmembrane protein, podoplanin interacts with other molecules in the same or neighboring cells. In the past, a lot of studies reported a pleiotropic role of podoplanin in the modulation of thrombosis, inflammation, lymphangiogenesis, angiogenesis, immune surveillance, epithelial mesenchymal transition, as well as extracellular matrix remodeling in periphery, which have been well summarized and discussed. Recently, mounting evidence demonstrates the distribution and function of this molecule in brain development and neurological disorders. In this review, we summarize the research progresses in understanding the roles and mechanisms of podoplanin in the development and disorders of the nervous system. The challenges of podoplanin-targeted approaches for disease prognosis and preventions are also discussed.

Endogenous Hydrogen Sulfide Persulfidates Caspase-3 at Cysteine 163 to Inhibit Doxorubicin-Induced Cardiomyocyte Apoptosis.

Doxorubicin (DOX) is an efficient antitumor anthracycline drug, but its cardiotoxicity adversely affects the prognosis of the patients. In this study, we explored whether endogenous gasotransmitter hydrogen sulfide (H2S) could protect against DOX-induced cardiomyocyte apoptosis and its mechanisms. The results indicated that DOX significantly downregulated endogenous H2S production and endogenous synthetase cystathionine γ-lyase (CSE) expression and obviously stimulated the apoptosis in H9C2 cells. The supplement of H2S donor sodium hydrosulfide (NaHS) or overexpression of CSE inhibited DOX-induced H9C2 cell apoptosis. DOX enhanced the activities of caspase family members in cardiomyocytes, while NaHS attenuated DOX-enhanced caspase-3, caspase-2, and caspase-9 activities by 223.1%, 73.94%, and 52.29%, respectively. Therefore, taking caspase-3 as a main target, we demonstrated that NaHS or CSE overexpression alleviated the cleavage of caspase-3, suppressed caspase-3 activity, and inhibited the cleavage of poly ADP-ribose polymerase (PARP). Mechanistically, we found that H2S persulfidated caspase-3 in H9C2 cells and human recombinant caspase-3 protein, while the thiol-reducing agent dithiothreitol (DTT) abolished H2S-induced persulfidation of caspase-3 and thereby prevented the antiapoptotic effect of H2S on caspase-3 in H9C2 cells. The mutation of caspase-3 C148S and C170S failed to block caspase-3 persulfidation by H2S in H9C2 cells. However, caspase-3 C163S mutation successfully abolished the effect of H2S on caspase-3 persulfidation and the corresponding protection of H9C2 cells. Collectively, these findings indicate that endogenous H2S persulfidates caspase-3 at cysteine 163, inhibiting its activity and cardiomyocyte apoptosis. Sufficient endogenous H2S might be necessary for the protection against myocardial cell apoptosis induced by DOX. The results of the study might open new avenues with respect to the therapy of DOX-stimulated cardiomyopathy.

Nitric oxide inhibits endothelial cell apoptosis by inhibiting cysteine-dependent SOD1 monomerization.

Endothelial cell apoptosis is an important pathophysiology in many cardiovascular diseases. The gasotransmitter nitric oxide (NO) is known to regulate cell survival and apoptosis. However, the mechanism underlying the effect of NO remains unclear. In this research, by targeting cytosolic copper/zinc superoxide dismutase (SOD1) monomerization, we aimed to explore how NO inhibited endothelial cell apoptosis. We showed that treatment with the NO synthase (NOS) inhibitor nomega-nitro-l-arginine methyl ester hydrochloride (L-NAME) significantly decreased the endogenous NO content of endothelial cells, facilitated the formation of SOD1 monomers, inhibited dismutase activity, and promoted reactive oxygen species (ROS) accumulation in human umbilical vein endothelial cells (HUVECs); by contrast, supplementation with the NO donor sodium nitroprusside (SNP) upregulated NO content, prevented the formation of SOD1 monomers, enhanced dismutase activity, and reduced ROS accumulation in L-NAME-treated HUVECs. Mechanistically, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a specific reducer of cysteine thiol, increased SOD1 monomer formation, thus preventing the NO-induced increase in dismutase activity and the decrease in ROS. Furthermore, SNP inhibited HUVEC apoptosis caused by the decrease in endogenous NO, whereas TCEP abolished this protective effect of SNP. In summary, our data reveal that NO protects endothelial cells against apoptosis by inhibiting cysteine-dependent SOD1 monomerization to enhance SOD1 activity and inhibit oxidative stress.

Endogenous Taurine Downregulation Is Required for Renal Injury in Salt-Sensitive Hypertensive Rats via CBS/H2S Inhibition.

Although taurine is known to exert an antihypertensive effect, it is unclear whether it is involved in the mechanism for hypertension-related target organ injury. To reveal the role of endogenous taurine in renal injury formation during salt-sensitive hypertension and clarify its mechanisms, both salt-sensitive Dahl rats and salt-resistant SS-13BN rats were fed a high-salt diet (8% NaCl) and given 2% taurine for 6 weeks. Rat systolic blood pressure (SBP) was measured by the tail-cuff method and artery catheterization. Kidney ultrastructure was observed under an electron microscope. Taurine content and mRNA and protein levels of taurine synthases, cysteine dioxygenase type 1 (CDO1) and cysteine sulfinic acid decarboxylase (CSAD), were decreased in Dahl rats fed a high-salt diet. However, taurine supplementation and the resulting increase in renal taurine content reduced the increased SBP and improved renal function and structural damage in high-salt diet-fed Dahl rats. In contrast, taurine did not affect SS-13BN SBP and renal function and structure. Taurine intervention increased the renal H2S content and enhanced cystathionine-ß-synthase (CBS) expression and activity in Dahl rats fed a high-salt diet. Taurine reduced the renin, angiotensin II, and aldosterone contents and the levels of oxidative stress indices in Dahl rat renal tissues but increased antioxidant capacity, antioxidant enzyme activity, and protein expression. However, taurine failed to achieve this effect in the renal tissue of SS-13BN rats fed a high-salt diet. Pretreatment with the CBS inhibitor HA or renal CBS knockdown inhibited H2S generation and subsequently blocked the effect of taurine on renin, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) levels in high-salt-stimulated Dahl renal slices. In conclusion, the downregulation of endogenous taurine production resulted in a decrease in the renal CBS/H2S pathway. This decrease subsequently promoted renin-angiotensin-aldosterone system (RAAS) activation and oxidative stress in the kidney, ultimately contributing to renal injury in salt-sensitive Dahl rats.

[Effect of blood-letting puncture of "Well-points" on hippocampal mitophagy-related protein expression in rats with acute brain injury due to hypobaric hypoxia].

OBJECTIVE: To study the effect of blood-letting puncture at "Well-points" of the twelve meridians on hippocampal mitophagy of hypobaric hypoxia-induced brain injury (HHIBI) rats, so as to explore its biological mechanisms underlying improvement of high altitude hypoxia-induced brain injury. METHODS: Male SD rats were randomly divided into normal control group （n=9）， and model and blood-letting groups which were further divided into 6, 12, 24, 48 and 72 h subgroups (n=9 in each subgroup). The HHIBI model was established by putting the rats into a hypobaric hypoxia chamber (equivalent to 5 000 m above sea level).The blood-letting groups were given blood-letting therapy at "Shaoshang"(LU11), "Shangyang"(LI1), "Zhongchong"(PC9), "Guanchong"(SJ1), "Shaochong"(HT9), "Shaoze"(SI9), once a day for 7 days. H.E. staining was used to observe the histopatholo-gical changes of hippocampus tissue. Serum hypoxia inducible factor(HIF)-1α and vascular endothelial growth factor(VEGF) contents were assayed using ELISA, and the expression levels of hippocampal Beclin-1 and LC3-Ⅱ proteins detected using Western blot. RESULTS: Compared with the normal control group, the levels of serum HIF-1α and VEGF at each time point, and the expressions of hippocampal Beclin-1 at 12 and 24 h, LC3-Ⅱat each time point were significantly increased in the model group (P<0.05, P<0.01); while in comparison with the model group, the levels of serum HIF-1α and VEGF contents, and the expressions of Beclin-1 at 12 h, LC3-Ⅱ at 24, 48 and 72 h were further significantly up-regulated in the blood-letting group (P<0.01, P<0.05). H.E. staining revealed that the pyramidal cells in the hippocampal CA1 region had a disordered arrangement, and some of them presented swelling with loose and pale cytoplasm or vacuolation at 6, 12 and 24 h, and showed indistinct nucleolus, irregular shape, pyknosis and deep staining and an obvious edema at 48 and 72 h, which was relatively milder in the blood-letting group. CONCLUSION: Blood-letting of "Well-points" can up-regulate serum HIF-1α and VEGF contents and hippocampal Beclin-1 and LC3-Ⅱ (mitophagy related proteins) expressions in HHIBI rats, which may contribute to its effect in reducing hypoxic brain injury.

Endogenous sulfur dioxide is a novel inhibitor of hypoxia-induced mast cell degranulation.

Introduction: Mast cell (MC) degranulation is an important step in the pathogenesis of inflammatory reactions and allergies; however, the mechanism of stabilizing MC membranes to reduce their degranulation is unclear. Methods: SO2 content in MC culture supernatant was measured by HPLC-FD. The protein and mRNA expressions of the key enzymes aspartate aminotransferase 1 (AAT1) and AAT2 and intracellular AAT activity were detected. The cAMP level in MCs was detected by immunofluorescence and ELISA. The release rate of MC degranulation marker ß-hexosaminidase was measured. The expression of AAT1 and cAMP, the MC accumulation and degranulation in lung tissues were detected. Objectives: To exam whether an endogenous sulfur dioxide (SO2) pathway exists in MCs and if it serves as a novel endogenous MC stabilizer. Results: We firstly show the existence of the endogenous SO2/AAT pathway in MCs. Moreover, when AAT1 was knocked down in MCs, MC degranulation was significantly increased, and could be rescued by a SO2 donor. Mechanistically, AAT1 knockdown decreased the cyclic adenosine monophosphate (cAMP) content in MCs, while SO2 prevented this reduction in a dose-independent manner. Pretreatment with the cAMP-synthesizing agonist forskolin or the cAMP degradation inhibitor IBMX significantly blocked the increase in AAT1 knockdown-induced MC degranulation. Furthermore, in hypoxia-stimulated MCs, AAT1 protein expression and SO2 production were markedly down regulated, and MC degranulation was activated, which were blunted by AAT1 overexpression. The cAMP synthesis inhibitor SQ22536 disrupted the suppressive effect of AAT1 overexpression on hypoxia-induced MC degranulation. In a hypoxic environment, mRNA and protein expression of AAT1 was significantly reduced in lung tissues of rats. Supplementation of SO2 elevated the cAMP level and reduced perivascular MC accumulation and degranulation in lung tissues of rats exposed to a hypoxic environment in vivo. Conclusion: SO2 serves as an endogenous MC stabilizer via upregulating the cAMP pathway under hypoxic circumstance.

Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling.

Sulfur dioxide (SO2) has emerged as a physiological relevant signaling molecule that plays a prominent role in regulating vascular functions. However, molecular mechanisms whereby SO2 influences its upper-stream targets have been elusive. Here we show that SO2 may mediate conversion of hydrogen peroxide (H2O2) to a more potent oxidant, peroxymonosulfite, providing a pathway for activation of H2O2 to convert the thiol group of protein cysteine residues to a sulfenic acid group, aka cysteine sulfenylation. By using site-centric chemoproteomics, we quantified >1000 sulfenylation events in vascular smooth muscle cells in response to exogenous SO2. Notably, ~42% of these sulfenylated cysteines are dynamically regulated by SO2, among which is cysteine-64 of Smad3 (Mothers against decapentaplegic homolog 3), a key transcriptional modulator of transforming growth factor ß signaling. Sulfenylation of Smad3 at cysteine-64 inhibits its DNA binding activity, while mutation of this site attenuates the protective effects of SO2 on angiotensin II-induced vascular remodeling and hypertension. Taken together, our findings highlight the important role of SO2 in vascular pathophysiology through a redox-dependent mechanism.

Sulphenylation of CypD at Cysteine 104: A Novel Mechanism by Which SO2 Inhibits Cardiomyocyte Apoptosis.

Objectives: The study was designed to explore the role of endogenous gaseous signaling molecule sulfur dioxide (SO2) in the control of cardiomyocyte apoptosis and its molecular mechanisms. Methods: Neonatal mouse cardiac myocytes (NMCMs) and H9c2 cells were used in the cell experiments. The endogenous SO2 pathway including SO2 level and the expression of SO2-generating enzyme aspartate aminotransferase 1/2 (AAT1/2) were detected in NMCMs. The apoptosis of cardiomyocytes was examined by a TUNEL assay. The cleavage and the activity of apoptotic proteins caspase9 and caspase3 were measured. The content of ATP, the opening of mitochondrial permeability transition pore (mPTP), and the cytochrome c (cytc) leakage were detected by immunofluorescence. The sulphenylation of cyclophilin-D (CypD) was detected by biotin switch analysis. The four CypD mutant plasmids in which cysteine sites were mutated to serine were constructed to identify the SO2-affected site in vitro. Results: ISO down-regulated the endogenous SO2/AAT pathway of cardiomyocytes in association with a significant increase in cardiomyocyte apoptosis, demonstrated by the increases in apoptosis, cleaved-caspase3/caspase3 ratio, and caspase3 activity. Furthermore, ISO significantly reduced ATP production in H9c2 cells, but the supplement of SO2 significantly restored the content of ATP. ISO stimulated mPTP opening, resulting in an increase in the release of cytc, which further increased the ratio of cleaved caspase9/caspase9 and enhanced the protein activity of caspase9. While, the supplementation of SO2 reversed the above effects. Mechanistically, SO2 did not affect CypD protein expression, but sulphenylated CypD and inhibited mPTP opening, resulting in an inhibition of cardiomyocyte apoptosis. The C104S mutation in CypD abolished SO2-induced sulphenylation of CypD, and thereby blocked the inhibitory effect of SO2 on the mPTP opening and cardiomyocyte apoptosis. Conclusion: Endogenous SO2 sulphenylated CypD at Cys104 to inhibit mPTP opening, and thus protected against cardiomyocyte apoptosis.

Hydrogen sulfide regulates insulin secretion and insulin resistance in diabetes mellitus, a new promising target for diabetes mellitus treatment? A review.

BACKGROUND: Insulin resistance and impaired insulin secretion lead to disorders of glucose metabolism, which contributes to the development of diabetes. Hydrogen sulfide (H2S), a novel gasotransmitter, is found to play important roles in regulation of glucose metabolism homeostasis. AIM OF REVIEW: This study aimed to summarize and discuss current data about the function of H2S in insulin secretion and insulin resistance regulation as well as the underlying mechanisms. KEY SCIENTIFIC CONCEPTS OF REVIEW: H2S could be endogenously produced in islet ß cells, liver, adipose, skeletal muscles, and the hypothalamus, and regulates local and systemic glucose metabolism. It is reported that H2S suppresses insulin secretion, promotes or reduces the apoptosis of islet ß cells. It plays important roles in the regulation of insulin sensitivity in insulin responsive tissues. H2S inhibits glucose uptake and glycogen storage, and promotes or inhibits gluconeogenesis, mitochondrial biogenesis and mitochondrial bioenergetics in the liver. In adipose tissue, several investigators indicated that H2S promoted glucose uptake in adipocytes, while other studies reported that H2S inhibits this process. H2S has also been shown to promote adipogenesis, inhibit lipolysis, and regulate adiponectin and MCP-1 secretion from adipocytes. In skeletal muscle, H2S increases glucose uptake and improves insulin sensitivity. It is also observed that H2S modulates circadian-clock genes in muscle. Hypothalamic CBS/H2S pathway reduces obesity and improves insulin sensitivity via the brain-adipose interaction. Most studies indicated plasma H2S levels decreased in diabetic patients. However, the mechanisms by which H2S regulates systemic glucose metabolism remain unclear. Whether H2S acts as a new promising target for diabetes mellitus treatment merits further studies.

Persulfidation of transcription factor FOXO1 at cysteine 457: A novel mechanism by which H2S inhibits vascular smooth muscle cell proliferation.

INTRODUCTION: The proliferation of vascular smooth muscle cells (VSMCs) is an important physiological and pathological basis for many cardiovascular diseases. Endogenous hydrogen sulfide (H2S), the third gasotransmitter, is found to preserve vascular structure by inhibiting VSMC proliferation. However, the mechanism by which H2S suppresses VSMC proliferation has not been fully clear. OBJECTIVES: This study aimed to explore whether H2S persulfidates the transcription factor FOXO1 to inhibit VSMC proliferation. METHODS: After the proliferation of VSMC A7r5 cells was induced by endothelin-1 (ET-1), FOXO1 phosphorylation and proliferating cell nuclear antigen (PCNA) expression were detected by Western blotting, the degree of FOXO1 nuclear exclusion and PCNA fluorescent signals in the nucleus were detected by immunofluorescence, and the persulfidation of FOXO1 was measured through a biotin switch assay. RESULTS: The results showed that ET-1 stimulation increased cell proliferation, FOXO1 phosphorylation and FOXO1 nuclear exclusion to the cytoplasm in the cells. However, pretreatment with NaHS, an H2S donor, successfully abolished the ET-1-induced increases in the VSMC proliferation, FOXO1 phosphorylation, and FOXO1 nuclear exclusion to the cytoplasm. Mechanistically, H2S persulfidated the FOXO1 protein in A7r5 and 293T cells, and the thiol reductant DTT reversed this effect. Furthermore, the C457S mutation of FOXO1 abolished the H2S-induced persulfidation of FOXO1 in the cells and the subsequent inhibitory effects on FOXO1 phosphorylation at Ser256, FOXO1 nuclear exclusion to the cytoplasm and cell proliferation. CONCLUSION: Thus, our findings demonstrated that H2S might inhibit VSMC proliferation by persulfidating FOXO1 at Cys457 and subsequently preventing FOXO1 phosphorylation at Ser256.

Post-translational Modifications of IκBα: The State of the Art.

The nuclear factor-kappa B (NF-κB) signaling pathway regulates a variety of biological functions in the body, and its abnormal activation contributes to the pathogenesis of many diseases, such as cardiovascular and respiratory diseases and cancers. Therefore, to ensure physiological homeostasis of body systems, this pathway is strictly regulated by IκBα transcription, IκBα synthesis, and the IκBα-dependent nuclear transport of NF-κB. Particularly, the post-translational modifications of IκBα including phosphorylation, ubiquitination, SUMOylation, glutathionylation and hydroxylation are crucial in the abovementioned regulatory process. Because of the importance of the NF-κB pathway in maintaining body homeostasis, understanding the post-translational modifications of IκBα can not only provide deeper insights into the regulation of NF-κB pathway but also contribute to the development of new drug targets and biomarkers for the diseases.

Macrophage-derived sulfur dioxide is a novel inflammation regulator.

Macrophage-mediated inflammation is a key pathophysiological component of cardiovascular diseases, but the underlying mechanisms by which the macrophage regulates inflammation have been unclear. In our study, we, for the first time, showed an endogenous sulfur dioxide (SO2) production in RAW267.4 macrophages by using HPLC and SO2-specific fluorescent probe assays. Moreover, the endogenous SO2 generating enzyme aspartate aminotransferase (AAT) was found to be expressed by the macrophages. Furthermore, we showed that AAT2 knockdown triggered spontaneous macrophage-mediated inflammation, as represented by the increased TNF-α and IL-6 levels and the enhanced macrophage chemotaxis; these effects could be reversed by the treatment with a SO2 donor. Mechanistically, AAT2 knockdown activated the NF-κB signaling pathway in macrophages, while SO2 successfully rescued NF-κB activation. In contrast, forced AAT2 expression reversed AngII-induced NF-κB activation and subsequent macrophage inflammation. Moreover, treatment with a SO2 donor also alleviated macrophage infiltration in AngII-treated mouse hearts. Collectively, our data suggest that macrophage-derived SO2 is an important regulator of macrophage activation and it acts as an endogenous "on-off switch" in the control of macrophage activation. This knowledge might enable a new therapeutic strategy for cardiovascular diseases.

Endothelin-1 Downregulates Sulfur Dioxide/Aspartate Aminotransferase Pathway via Reactive Oxygen Species to Promote the Proliferation and Migration of Vascular Smooth Muscle Cells.

The regulatory mechanisms for proliferation and migration of vascular smooth muscle cells have not yet been clear. The present study was designed to investigate whether and how endothelin-1 (ET-1) impacted the generation of endogenous sulfur dioxide (SO2) in rat vascular smooth muscle cell (VSMC) proliferation and migration. Primary VSMCs and purified aspartate aminotransferase (AAT) protein were used in this study. We found that in the presence of ET-1, the expression of PCNA and Ki-67 was upregulated and the migration of VSMCs was promoted, while the AAT activity and SO2 levels in VSMCs were reduced without any changes in AAT1 and AAT2 expression. SO2 supplementation successfully prevented the ET-1-facilitated expression of PCNA and Ki-67 and the migration of VSMCs. Interestingly, ET-1 significantly increased reactive oxygen species (ROS) production in association with SO2/AAT pathway downregulation in VSMCs compared with controls, while the ROS scavenger N-acetyl-L-cysteine (NAC) and the antioxidant glutathione (GSH) significantly abolished the ET-1-stimulated downregulation of the SO2/AAT pathway. Moreover, the AAT activity was reduced in purified protein after the treatment for 2 h. However, NAC and GSH blocked the hydrogen peroxide-induced AAT activity reduction. In conclusion, our results suggest that ET-1 results in the downregulation of the endogenous SO2/AAT pathway via ROS generation to enhance the proliferation and migration of VSMCs.

L-Cystathionine Protects against Homocysteine-Induced Mitochondria-Dependent Apoptosis of Vascular Endothelial Cells.

The study was aimed at investigating the effects of L-cystathionine on vascular endothelial cell apoptosis and its mechanisms. Cultured human umbilical vein endothelial cells (HUVECs) were used in the study. Apoptosis of vascular endothelial cells was induced by homocysteine. Apoptosis, mitochondrial superoxide anion, mitochondrial membrane potential, mitochondrial permeability transition pore (MPTP) opening, and caspase-9 and caspase-3 activities were examined. Expression of Bax, Bcl-2, and cleaved caspase-3 was tested and BTSA1, a Bax agonist, and HUVEC Bax overexpression was used in the study. Results showed that homocysteine obviously induced the apoptosis of HUVECs, and this effect was significantly attenuated by the pretreatment with L-cystathionine. Furthermore, L-cystathionine decreased the production of mitochondrial superoxide anion and the expression of Bax and restrained its translocation to mitochondria, increased mitochondrial membrane potential, inhibited mitochondrial permeability transition pore (MPTP) opening, suppressed the leakage of cytochrome c from mitochondria into the cytoplasm, and downregulated activities of caspase-9 and caspase-3. However, BTSA1, a Bax agonist, or Bax overexpression successfully abolished the inhibitory effect of L-cystathionine on Hcy-induced MPTP opening, caspase-9 and caspase-3 activation, and HUVEC apoptosis. Taken together, our results indicated that L-cystathionine could protect against homocysteine-induced mitochondria-dependent apoptosis of HUVECs.