HDAC6 Enhances Endoglin Expression through Deacetylation of Transcription Factor SP1, Potentiating BMP9-Induced Angiogenesis.

Histone deacetylase 6 (HDAC6) plays a crucial role in the acetylation of non-histone proteins and is notably implicated in angiogenesis, though its underlying mechanisms were previously not fully understood. This study conducted transcriptomic and proteomic analyses on vascular endothelial cells with HDAC6 knockdown, identifying endoglin (ENG) as a key downstream protein regulated by HDAC6. This protein is vital for maintaining vascular integrity and plays a complex role in angiogenesis, particularly in its interaction with bone morphogenetic protein 9 (BMP9). In experiments using human umbilical vein endothelial cells (HUVECs), the pro-angiogenic effects of BMP9 were observed, which diminished following the knockdown of HDAC6 and ENG. Western blot analysis revealed that BMP9 treatment increased SMAD1/5/9 phosphorylation, a process hindered by HDAC6 knockdown, correlating with reduced ENG expression. Mechanistically, our study indicates that HDAC6 modulates ENG transcription by influencing promoter activity, leading to increased acetylation of transcription factor SP1 and consequently altering its transcriptional activity. Additionally, the study delves into the structural role of HDAC6, particularly its CD2 domain, in regulating SP1 acetylation and subsequently ENG expression. In conclusion, the present study underscores the critical function of HDAC6 in modulating SP1 acetylation and ENG expression, thereby significantly affecting BMP9-mediated angiogenesis. This finding highlights the potential of HDAC6 as a therapeutic target in angiogenesis-related processes.

Mechanisms Underlying the Hydrogen Sulfide Actions: Target Molecules and Downstream Signaling Pathways.

Significance: As a new important gas signaling molecule like nitric oxide (NO) and carbon dioxide (CO), hydrogen sulfide (H2S), which can be produced by endogenous H2S-producing enzymes through l-cysteine metabolism in mammalian cells, has attracted wide attention for long. H2S has been proved to play an important regulatory role in numerous physiological and pathophysiological processes. However, the deep mechanisms of those different functions of H2S still remain uncertain. A better understanding of the mechanisms can help us develop novel therapeutic strategies. Recent Advances: H2S can play a regulating role through various mechanisms, such as regulating epigenetic modification, protein expression levels, protein activity, protein localization, redox microenvironment, and interaction with other gas signaling molecules such as NO and CO. In addition to discussing the molecular mechanisms of H2S from the above perspectives, this article will review the regulation of H2S on common signaling pathways in the cells, including the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer, and activator of transcription (STAT) signaling pathway. Critical Issues: Although there are many studies on the mechanism of H2S, little is known about its direct target molecules. This article will also review the existing reports about them. Furthermore, the interaction between direct target molecules of H2S and the downstream signaling pathways involved also needs to be clarified. Future Directions: An in-depth discussion of the mechanism of H2S and the direct target molecules will help us achieving a deeper understanding of the physiological and pathophysiological processes regulated by H2S, and lay a foundation for developing new clinical therapeutic drugs in the future. Innovation: This review focuses on the regulation of H2S on signaling pathways and the direct target molecules of H2S. We also provide details on the underlying mechanisms of H2S functions from the following aspects: epigenetic modification, regulation of protein expression levels, protein activity, protein localization, redox microenvironment, and interaction with other gas signaling molecules such as NO and CO. Further study of the mechanisms underlying H2S will help us better understand the physiological and pathophysiological processes it regulates, and help develop new clinical therapeutic drugs in the future. Antioxid. Redox Signal. 40, 86-109.

Cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction: Toward an ultrasensitive split-type fluorescent immunoassay.

An ultrasensitive split-type fluorescent immunobiosensor has been reported based on a cascade signal amplification strategy by coupling chemical redox-cycling and Fenton-like reaction. In this strategy, Cu2+ could oxidize chemically o-phenylenediamine (OPD) to generate photosensitive 2, 3-diaminophenazine (DAP) and Cu+/Cu0. On one hand, the generated Cu0 in turn catalyzed the oxidation of OPD. On the other hand, the introduced H2O2 reacted with Cu + ion to produce hydroxyl radicals (·OH) and Cu2+ ion through a Cu + -mediated Fenton-like reaction. The produced ·OH and recycled Cu2+ ion could take turns oxidizing OPD to generate more photoactive DAP, which triggering a self-sustaining chemical redox-cycling reaction and leading to a remarkable fluorescent improvement. It was worth mentioning that the cascade reaction did not stop until OPD molecules were completely consumed. Based on the H2O2-triggered cascade signal amplification, the strategy was exploited for the construction of split-type fluorescent immunoassay by taking interleukin-6 (IL-6) as the model target. It was realized for the ultrasensitive determination of IL-6 in a linear ranging from 20 fg/mL to 10 pg/mL with a limit of detection of 5 fg/mL. The study validated the practicability of the cascade signal amplification on the fluorescent bioanalysis and the superior performance in fluorescent immunoassay. It is expected that the strategy would offer new opportunities to develop ultrasensitive fluorescent methods for biosensor and bioanalysis.

Skeletal muscle-derived FSTL1 starting up angiogenesis by regulating endothelial junction via activating Src pathway can be upregulated by hydrogen sulfide.

Hydrogen sulfide (H2S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H2S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H2S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H2S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H2S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H2S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway.NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H2S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Hydrogen sulfide functions as a micro-modulator bound at the copper active site of Cu/Zn-SOD to regulate the catalytic activity of the enzyme.

The present study examines whether there is a mechanism beyond the current concept of post-translational modifications to regulate the function of a protein. A small gas molecule, hydrogen sulfide (H2S), was found to bind at active-site copper of Cu/Zn-SOD using a series of methods including radiolabeled binding assay, X-ray absorption near-edge structure (XANES), and crystallography. Such an H2S binding enhanced the electrostatic forces to guide the negatively charged substrate superoxide radicals to the catalytic copper ion, changed the geometry and energy of the frontier molecular orbitals of the active site, and subsequently facilitated the transfer of an electron from the superoxide radical to the catalytic copper ion and the breakage of the copper-His61 bridge. The physiological relevance of such an H2S effect was also examined in both in vitro and in vivo models where the cardioprotective effects of H2S were dependent on Cu/Zn-SOD.

Glutathione-stabilized copper nanoclusters mediated-inner filter effect for sensitive and selective determination of p-nitrophenol and alkaline phosphatase activity.

A simple and highly selective fluorescence biosensor has been exploited for p-nitrophenol (p-NP) and alkaline phosphatase (ALP) activity detection based on the glutathione-stabilized copper nanoclusters (GSH-CuNCs) mediated-inner filter effect (IFE). The GSH-CuNCs were prepared by employing GSH as stabilizer and ascorbic acid (AA) as reductant. The obtained GSH-CuNCs exhibited a strong blue fluorescence emission at 420 nm with an excitation wavelength of 365 nm, which overlapped largely with the absorption spectra of p-nitrophenol (p-NP). Therefore, the luminescence of GSH-CuNCs could be quenched by p-NP through inner filter effect. In addition, ALP catalyzed the substrate p-nitrophenyl phosphate (p-NPP) to form p-nitrophenol (p-NP), which also leading to the fluorescence quenching of GSH-CuNCs. The fluorescent strategy was realized for the sensitive determination of p-NP and ALP activity with the promising limit of detection of 20 nM (for p-NP) and 0.003 mU⋅mL-1 (for ALP). Furthermore, the method could be applied to detect the p-NP content in river water samples and ALP activity in human serum samples.

YB-1 Recruits Drosha to Promote Splicing of pri-miR-192 to Mediate the Proangiogenic Effects of H2S.

Aims: The genes targeted by miRNAs have been well studied. However, little is known about the feedback mechanisms to control the biosynthesis of miRNAs that are essential for the miRNA feedback networks in the cells. In this present study, we aimed at examining how hydrogen sulfide (H2S) promotes angiogenesis by regulating miR-192 biosynthesis. Results: H2S promoted in vitro angiogenesis and angiogenesis in Matrigel plugs embedded in mice by upregulating miR-192. Knockdown of the H2S-generating enzyme cystathionine γ-lyase (CSE) suppressed in vitro angiogenesis, and this suppression was rescued by exogenous H2S donor NaHS. Plakophilin 4 (PKP4) served as a target gene of miR-192. H2S up-regulated miR-192 via the VEGFR2/Akt pathway to promote the splicing of primary miR-192 (pri-miR-192), and it resulted in an increase in both the precursor- and mature forms of miR-192. H2S translocated YB-1 into the nuclei to recruit Drosha to bind with pri-miR-192 and promoted its splicing. NaHS treatment promoted angiogenesis in the hindlimb ischemia mouse model and the skin-wound-healing model in diabetic mice, with upregulated miR-192 and downregulated PKP4 on NaHS treatment. In human atherosclerotic plaques, miR-192 levels were positively correlated with the plasma H2S concentrations. Innovation and Conclusion: Our data reveal a role of YB-1 in recruiting Drosha to splice pri-miR-192 to mediate the proangiogenic effect of H2S. CSE/H2S/YB-1/Drosha/miR-192 is a potential therapeutic target pathway for treating diseases, including organ ischemia and diabetic complications. Antioxid. Redox Signal. 36, 760-783. The Clinical Trial Registration number is 2016-224.

A Common Molecular Switch for H2S to Regulate Multiple Protein Targets.

Hydrogen sulfide, a small molecule, produced by endogenous enzymes, such as CTH, CBS, and MPST using L-cysteine as substrates, has been reported to have numerous protective effects. However, the key problem that the target of H2S and how it can affect the structure and activity of biological molecules is still unknown. Till now, there are two main theories of its working mechanism. One is that H2S can modify the free thiol in cysteine to produce the persulfide state of the thiol and the sulfhydration of cysteine can significantly change the structure and activity of target proteins. The other theory is that H2S, as an antioxidant molecule, can directly break the disulfide bond in target proteins, and the persulfide state of thiol can be an intermediate product during the reaction. Both phenomena exit for no doubt since they are both supported by large amounts of experiments. Here, we will summarize both theories and try to discuss which one is the more effective or direct mechanism for H2S and what is the relationship between them. Therefore, we will discover more protein targets of H2S with the mechanism and understand more about the effect of this small molecule.

L-Histidine-DNA interaction: a strategy for the improvement of the fluorescence signal of poly(adenine) DNA-templated gold nanoclusters.

An interesting phenomenon is described that the fluorescence signal of poly(adenine) (A) DNA-templated gold nanoclusters (AuNCs) is greatly improved in the presence of L-histidine by means of L-histidine-DNA interaction. The modified nanoclusters display strong fluorescence emission with excitation/emission maxima at 290/475 nm. The fluorescence quantum yield (QY) is improved from 1.9 to 6.5%. Fluorescence enhancement is mainly ascribed to the L-histidine-DNA interaction leading to conformational changes of the poly(A) DNA template, which offer a better microenvironment to protect AuNCs. The assay enables L-histidine to be determined with good sensitivity and a linear response that covers the 1 to 50 nM L-histidine concentration range with a 0.3 nM limit of detection. The proposed method has been applied to the determination of imidazole-containing drugs in pharmaceutical samples. A turn-on fluorescent method has been designed for the sensitive detection of L-histidine as well as imidazole-containing drugs on the basis of the L-histidine-DNA interaction.

Fatty-acid receptor CD36 functions as a hydrogen sulfide-targeted receptor with its Cys333-Cys272 disulfide bond serving as a specific molecular switch to accelerate gastric cancer metastasis.

BACKGROUND: Hydrogen Sulfide (H2S), a third member of gasotransmitter family along with nitric oxide (NO) and carbon monoxide (CO), exerts a wide range of cellular and molecular actions in our body. There is a large body of evidence suggesting that H2S plays an important role in cancer metastasis; however, the molecular mechanisms of H2S-mediated acceleration of cancer metastasis remain unknown. METHODS: We examined the promote effects of H2S on phenotype of gastric cancer (GC) cells (including those of express wild type CD36 and mutant CD36) in vitro and in vivo. GC patients' samples were used for clinical translational significance evaluation. FINDINGS: H2S triggered lipid metabolism reprogramming by significantly up-regulating the expression of the fatty-acid receptor CD36 (CD36) and directly activating CD36 in GC cells. Mechanistically, a disulfide bond located between cysteine (Cys)333 and Cys272 within the CD36 protein structure that was labile to H2S-mediated modification. The long chain-fatty acid (LC-FA) binding pocket was capped by a turn in the CD36 protein, located between helical and sheet structures that were stabilized by the Cys333-Cys272. This limited the secondary binding between LC-FAs and lysine (Lys)334. Breaking the Cys333-Cys272 disulfide bond restored the second LC-FA binding conformation of CD36. Targeting CD36 in vivo blocked H2S-promoted metastasis and improved animal survival. INTERPRETATION: These findings identify that the Cys333-Cys272 disulfide bond disrupted the integrity of the second LC-FA binding conformation of CD36. Therefore, CD36 can directly activate LC-FA access to the cytoplasm by acting as a direct target molecule for H2S.

3-Mercaptopyruvate Sulfurtransferase, Not Cystathionine ß-Synthase Nor Cystathionine γ-Lyase, Mediates Hypoxia-Induced Migration of Vascular Endothelial Cells.

Hypoxia-induced angiogenesis is a common phenomenon in many physiological and patho-physiological processes. However, the potential differential roles of three hydrogen sulfide producing systems cystathionine γ-lyase (CSE)/H2S, cystathionine ß-synthase (CBS)/H2S, and 3-mercaptopyruvate sulfurtransferase (MPST)/H2S in hypoxia-induced angiogenesis are still unknown. We found that minor hypoxia (10% oxygen) significantly increased the migration of vascular endothelial cells while hypoxia (8% oxygen) significantly inhibited cell migration. The present study was performed using cells cultured in 10% oxygen. RNA interference was used to block the endogenous generation of hydrogen sulfide by CSE, CBS, or MPST in a vascular endothelial cell migration model in both normoxia and hypoxia. The results showed that CBS had a promoting effect on the migration of vascular endothelial cells cultured in both normoxic and hypoxic conditions. In contrast, CSE had an inhibitory effect on cell migration. MPST had a promoting effect on the migration of vascular endothelial cells cultured in hypoxia; however, it had no effect on the cells cultured in normoxia. Importantly, it was found that the hypoxia-induced increase in vascular endothelial cell migration was mediated by MPST, but not CSE or CBS. The western blot analyses showed that hypoxia significantly increased MPST protein levels, decreased CSE protein levels and did not change CBS levels, suggesting that these three hydrogen sulfide-producing systems respond differently to hypoxic conditions. Interestingly, MPST protein levels were elevated by hypoxia in a bi-phasic manner and MPST mRNA levels increased later than the first stage elevation of the protein levels, implying that the expression of MPST induced by hypoxia was also regulated at a post-transcriptional level. RNA pull-down assay showed that some candidate RNA binding proteins, such as nucleolin and Annexin A2, were dissociated from the 3'-UTR of MPST mRNA in hypoxia which implied their involvement in MPST mRNA regulation.

H2S regulates endothelial nitric oxide synthase protein stability by promoting microRNA-455-3p expression.

The aims of the present study are to determine whether hydrogen sulfide (H2S) is involved in the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, and to identify the role of microRNA-455-3p (miR-455-3p) during those processes. In cultured human umbilical vein endothelial cells (HUVECs), the expression of miR-455-3p, eNOS protein and the NO production was detected after administration with 50 µM NaHS. The results indicated that H2S could augment the expression of miR-455-3p and eNOS protein, leading to the increase of NO level. We also found that overexpression of miR-455-3p in HUVECs increased the protein levels of eNOS whereas inhibition of miR-455-3p decreased it. Moreover, H2S and miR-455-3p could no longer increase the protein level of eNOS in the presence of proteasome inhibitor, MG-132. In vivo, miR-455-3p and eNOS expression were considerably increased in C57BL/6 mouse aorta, muscle and heart after administration with 50 µmol/kg/day NaHS for 7 days. We also identified that H2S levels and miR-455-3p expression increased in human atherosclerosis plaque while H2S levels decreased in plasma of atherosclerosis patients. Our data suggest that the stability of eNOS protein and the NO production could be regulated by H2S through miR-455-3p.

[The Molecular Switches and Atomic Biological Mechanisms Underlying the Physiological and Pathophysio-logical Effects of Hydrogen Sulfide to Regulate Its "Receptors"].

Hydrogen sulfide (H2S)has emerged as pivotal signaling molecules since it is recognized as the third gasotransmitter together with nitric oxide and carbon monoxide. The development of detecting technologies contributed to the research in H2S biology.H2S plays significant roles in human body systems, such as the cardiovascular system, nervous system, respiratory system etc.. Alterations of H2S concentrations have been connected with many diseases. Hypertension, atherosclerosis, neurodegenerative disorder, asthma and many other diseases are found to be related with abnormal H2S metabolism. It has become a potential drug for therapeutic purposes. Understanding the mechanism of H2S biology, including a molecular switch contained in its "receptor", has deepened the research on how small molecules regulate big molecules, as well as providing new strategy for the therapeutic approaches for varies of diseases.

Hydrogen sulfide promotes angiogenesis by downregulating miR-640 via the VEGFR2/mTOR pathway.

We previously found hydrogen sulfide (H2S) to be a new proangiogenic factor. However, the mechanisms underlying the cardiovascular effect of this small gas molecule remain largely unknown. The aim of the present study was to identify the essential microRNAs (miRNAs) involved in the transduction of H2S signals in vascular endothelial cells (ECs). The expression of miR-640 and its signaling elements, vascular endothelial growth factor receptor 2 (VEGFR2), hypoxia inducible factor 1-α (HIF1A), and mammalian target of rapamycin (mTOR), was measured using quantitative PCR and Western blotting. Overexpression and inhibition of miR-640 were performed to clarify their roles in mediating the effect of H2S. In addition, knockdown of VEGFR2, HIF1A, and mTOR was performed using siRNAs, dominant negative mutants, or inhibitors to examine their roles in the transduction of the H2S signals. miR-640 levels decreased in vascular ECs that were treated with H2S, whereas overexpression of miR-640 blunted the proangiogenic effect of H2S. Knockdown of either VEGFR2 or mTOR blunted the downregulation of miR-640 and the proangiogenic effect induced by H2S. In addition, miR-640 bound to the 3'-UTR of HIF1A mRNA and then inhibited the expression of HIF1A. The inhibition could be recovered by treating cells with H2S. Thus we concluded that miR-640 plays a pivotal role in mediating the proangiogenic effect of H2S; H2S acts through downregulation of the expression of miR-640 and increasing the levels of HIF1A through the VEGFR2-mTOR pathway.

[Effect of the treatment acceptance on the perinatal outcomes in women with subclinical hypothyroidism, positive thyroid gland peroxidase antibody in early pregnancy].

OBJECTIVE: To investigate if women with subclinical hypothyroidism (SCH), positive thyroid gland peroxidase antibody (TPOAb) in early pregnancy accepted treatment or not had effect on perinatal outcomes. METHODS: 15 000 pregnant women who delivered in Women and Infants Hospital of Zhengzhou from January 1, 2013 to June 30, 2014 were recruited retrospectively. Among them, 2 042 women had SCH in early pregnancy. The diagnostic standard of SCH was serum free thyroxine (FT4) between 12.91-22.35 pmol/L and TSH level between 5.22-10.00 mU/L. TPOAb level ≥ 34 U/L was defined as positive result. The 2 042 patients with SCH were divided into the treated group (1 236 cases) and the untreated group (806 cases), according to whether or not women accepted the levothyroxine treatment. Meanwhile, the 2 042 patients with SCH were divided into the TPOAb (+) treated group (1 021 cases), the TPOAb (+) untreated group (201 cases), the TPOAb (-) treated group (215 cases) and the TPOAb (-) untreated group (605 cases), according to the TPOAb result and acceptance the levothyroxine treatment. 2 000 pregnant women with normal thyroid function who delivered in the same period were selected as the control group. Perinatal outcomes were analyzed. RESULTS: (1) The incidence of SCH in early pregnancy was 13.61% (2 042/15 000). 60.53% (1 236/2 042) accepted levothyroxine treatment and 39.47% (806/2 042) did not. (2) The incidence of abortion (5.71%, 46/806), premature delivery (6.20%, 50/806), gestational hypertension disease (13.90%, 112/806), gestational diabetes mellitus (GDM; 6.58%, 53/806), fetal growth restriction (FGR; 12.28%, 99/806) and low birth weight infants (10.17%, 82/806) in the untreated group were higher than those in the treated group [3.96% (49/1 236), 4.21% (52/1 236), 10.76% (133/1 236), 4.13% (51/1 236), 8.90% (110/1 236), 7.52% (93/1 236), respectively] and the control group [3.60% (72/2 000), 4.00% (80/2 000), 10.70% (214/2 000), 3.80% (76/2 000), 9.60% (192/2 000), 7.50% (150/2 000), respectively]. The differences were statistically significant (P < 0.05). While there was no statistically significant difference in the incidence of placental abruption, anemia in pregnant women, or fetal distress among the three groups (P > 0.05). (3)The incidences of abortion (11.44%, 23/201), premature delivery (12.44%, 25/201), gestational hypertension disease (22.89%, 46/201), GDM (8.46%, 17/201), FGR (19.90%, 40/201) and low birth weight infants (16.42%, 33/201) in the TPOAb (+) untreated group were higher than those in TPOAb (+) treated group [4.02% (41/1 021), 4.21% (43/1 021), 10.77% (110/1 021), 4.11% (42/1 021), 8.72% (89/1 021), 7.35% (75/1 021), respectively] and the control group, with statistically significant differences (P < 0.05). The incidence of the pregnancy complications in the TPOAb (+) treated group was higher than those in the control group, but the differences were not statistically significant (P > 0.05). (4) There were no statistically significant difference (P > 0.05) in the incidence of abortion (3.72%, 8/215), premature delivery (4.19%, 9/215), gestational hypertension disease (10.70%, 23/215), GDM (4.19%, 9/215), FGR (9.77%, 21/215) or low birth weight infants (8.37%, 18/215) among the TPOAb (-) treated group, the TPOAb (-) untreated group [3.80% (23/605), 4.13% (25/605), 10.91% (66/605), 5.95% (36/605), 9.75% (59/605), 8.10% (49/605), respectively] and the control group. CONCLUSIONS: (1) The incidence of abortion, premature delivery, gestational hypertension disease, GDM, FGR and low birth weight infants could be increased in women with SCH in early pregnancy. (2) Thyroxine treatment could reduce the incidence of pregnancy complications in women with SCH in early pregnancy.

H2S Is a Promoter of Angiogenesis: Identification of H2S "Receptors" and Its Molecular Switches in Vascular Endothelial Cells.

Angiogenesis is a physiological process in organ development and also a compensatory response in ischemia. When ischemia occurs, oxygen sensors in vascular endothelial cells sense the decrease in oxygen, thus activating downstream signaling pathways to promote the proliferation, migration, and tube formation of the endothelial cells. The new vasculatures are formed by sprouting from preexisting vessels, in order to maintain oxygen homeostasis in ischemic tissues (Folkman and Shing 1992). Collateral circulation is sometimes established under chronic ischemic conditions such as chronic myocardial ischemia (Banai et al. 1994). However, naturally occurring angiogenesis is usually not sufficient to compensate for ischemia in ischemic tissues. Proangiogenic drugs may be useful to promote angiogenesis in these diseases.

Cardiac H2S Generation Is Reduced in Ageing Diabetic Mice.

Aims. To examine whether hydrogen sulfide (H2S) generation changed in ageing diabetic mouse hearts. Results. Compared to mice that were fed tap water only, mice that were fed 30% fructose solution for 15 months exhibited typical characteristics of a severe diabetic phenotype with cardiac hypertrophy, fibrosis, and dysfunction. H2S levels in plasma, heart tissues, and urine were significantly reduced in these mice as compared to those in controls. The expression of the H2S-generating enzymes, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase, was significantly decreased in the hearts of fructose-fed mice, whereas cystathionine-ß-synthase levels were significantly increased. Conclusion. Our results suggest that this ageing diabetic mouse model developed diabetic cardiomyopathy and that H2S levels were reduced in the diabetic heart due to alterations in three H2S-producing enzymes, which may be involved in the pathogenesis of diabetic cardiomyopathy.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

VEGFR2 functions as an H2S-targeting receptor protein kinase with its novel Cys1045-Cys1024 disulfide bond serving as a specific molecular switch for hydrogen sulfide actions in vascular endothelial cells.

AIMS: The potential receptor for hydrogen sulfide (H2S) remains unknown. RESULTS: H2S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H2S-induced migration of human vascular endothelial cells. H2S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H2S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045-Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migration-promoting effect of H2S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045-Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H2S. The formation of the Cys1045-Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045-Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H2S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine γ-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia. INNOVATION AND CONCLUSION: The study provides the first piece of evidence of a molecular switch in H2S-targeting receptor protein kinase in H2S-induced angiogenesis and that may be applicable to additional kinases containing functionally important disulfide bonds in mediating various H2S actions.

PI3K p110α isoform-dependent Rho GTPase Rac1 activation mediates H2S-promoted endothelial cell migration via actin cytoskeleton reorganization.

Hydrogen sulfide (H(2)S) is now considered as the third gaseotransmitter, however, the signaling pathways that modulate the biomedical effect of H(2)S on endothelial cells are poorly defined. In the present study, we found in human endothelial cells that H(2)S increased cell migration rates and induced a marked reorganization of the actin cytoskeleton, which was prevented by depletion of Rac1. Pharmacologic inhibiting vascular endothelial growth factor receptor (VEGFR) and phosphoinositide 3-kinase (PI3K) both blunted the activation of Rac1 and the promotion of cell migration induced by H(2)S. Moreover, H(2)S-induced Rac1 activation was selectively dependent on the presence of the PI3K p110α isoform. Activated Rac1 by H(2)S thus in turn resulted in the phosphorylation of the F-actin polymerization modulator, cofilin. Additionally, inhibiting of extracellular signal-regulated kinase (ERK) decreased the augmented cell migration rate by H(2)S, but had no effect on Rac1 activation. These results indicate that Rac1 conveys the H(2)S signal to microfilaments inducing rearrangements of actin cytoskeleton that regulates cell migration. VEGFR-PI3K was found to be upstream pathway of Rac1, while cofilin acted as a downstream effector of Rac1. ERK was also shown to be involved in the action of H(2)S on endothelial cell migration, but independently of Rac1.