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Supplemental Digital Content is available in the text. Background: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. Methods: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E–deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. Results: The endothelial cell–specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1β. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R–target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l -cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. Conclusions: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme. [ABSTRACT FROM AUTHOR]

Background: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions.Methods: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material.Results: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1β. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease.Conclusions: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme. [ABSTRACT FROM AUTHOR]

Background: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury., Methods: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo., Results: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence., Conclusions: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE., Competing Interests: Disclosures None.

Myocarditis is a challenging inflammatory disease of the heart, and better understanding of its pathogenesis is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by CYP (cytochrome P450) enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by sEH (soluble epoxide hydrolase), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and in the murine heart and correlated with the increased expression and activity of sEH after coxsackie B3 virus infection. Administration of a sEH inhibitor prevented coxsackie B3 virus-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated viral infection or improved viral resistance by activating type I IFN (interferon) signaling. At the molecular level, EETs enhanced the interaction between GSK3β (glycogen synthase kinase-3 beta) and TBK1 (TANK-binding kinase 1) to promote IFN-β production. Our findings revealed that EETs and sEH inhibitors prevent the progress of coxsackie B3 virus-induced myocarditis, particularly by promoting viral resistance by increasing IFN production., Competing Interests: Disclosures None.

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

Invasive fungal infection (IFI) remains as a significant cause of morbidity and mortality in patients with acute myelogenous leukemia (AML). Here, we report the subgroup analysis of China Assessment of Antifungal Therapy in Haematological Disease (CAESAR) study to evaluate the risk of IFI in patients with AML in 1st remission receiving high-dose cytarabine (HiDAC) as consolidation. A total of 638 patients with AML in 1st complete remission were selected from the database. Among them, 130 patients received HiDAC alone with total dose of 2-3 g/m(2) × 6 while 508 patients received multiple-agent combination chemotherapy (multiagent chemo group). The patients' characteristics were generally not different but more patients in HiDAC group had peripherally inserted central catheter (61.5% vs 44.5%, P = 0.002). The median duration of neutropenia was 8.0 days in both HiDAC (2-20) and multiagent chemo group (2-28). Number of patients with prolonged neutropenia (>14 days) tended to be more in multiagent chemo group but not significant different (16.3% vs 8.8%, respectively). There was no significant difference between 2 groups in persistent neutropenic fever (40.8% vs 33.1%), antifungal treatment (11.5% vs 11.4%), and incidence of proven/probable IFI (4 probable in HiDAC vs 1 proven/4 probable in multiagent chemo, P = 0.35) or possible IFI. As to the clinical outcome in terms of duration of hospitalization and death in remission, there was a trend of shorter duration of hospitalization in HiDAC (19 days, 3-70) compare to multiagent chemo group (21 days, 1-367, P = 0.057) while no death documented in HiDAC group and only 2 patients died in the multiagent chemo group (0.4%). As to risk factors associated with IFI in all 638 patients, there was a trend of more IFI in patients with severe neutropenia (3.0%, P = 0.089) and previous history of IFI (3.85%, P = 0.086) while the antifungal prophylaxis was not associated significantly reduced IFI. Overall, our data support the perception that HiDAC alone as consolidation in first remission AML patients was well tolerated and not associated with increased hematological toxicity and IFI than conventional combination chemotherapy. Antifungal prophylaxis may not necessary except for patients with previous history of IFI., Competing Interests: The authors have no funding conflicts of interest to disclose.

Bloodstream infection (BSI) is an important cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). To evaluate the causative bacteria and identify risk factors for BSI associated mortality in febrile neutropenia patients undergoing HSCT, we collected the clinical and microbiological data from patients underwent HSCT between 2008 and 2014 and performed a retrospective analysis. Throughout the study period, among 348 episodes of neutropenic fever in patients underwent HSCT, 89 episodes in 85 patients had microbiological defined BSI with a total of 108 isolates. Gram-negative bacteria (GNB) were the most common isolates (76, 70.3%) followed by gram-positive bacteria (GPB, 29, 26.9%) and fungus (3, 2.8%). As to the drug resistance, 26 multiple drug resistance (MDR) isolates were identified. Resistant isolates (n = 23) were more common documented in GNB, mostly Escherichia coli (9/36, 25%) and Klebsiella pneumonia (6/24, 25%). A total of 12 isolated were resistant to carbapenem including 4 K pneumoniae (4/24, 16.7%), 3 Stenotrophomonas maltophilia, and 1 Pseudomonas aeruginosa and other 4 GNB isolates (Citrobacter freumdii, Pseudomonas stutzeri, Acinetobacter baumanii, and Chryseobacterium indologenes). As to the GPB, only 3 resistant isolates were documented including 2 methicillin-resistant isolates (Staphylococcus hominis and Arcanobacterium hemolysis) and 1 vancomycin-resistant Enterococcus faecium. Among these 85 patients with documented BSI, 11 patients died of BSI as primary or associated cause with a BSI-related mortality of 13.1 ± 3.7% and 90-day overall survival after transplantation at 80.0 ± 4.3%. Patients with high-risk disease undergoing allo-HSCT, prolonged neutropenia (≥15 days) and infection with carbapenem-resistant GNB were associated with BSI associated mortality in univariate and multivariate analyses. Our report revealed a prevalence of GNB in BSI of neutropenic patients undergoing HSCT. Patients with high-risk diseases with prolonged neutropenia and carbapenem-resistant GNB were independent risk factors for BSI-related mortality.

Rationale: MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus., Objective: This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function., Methods and Results: Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins., Conclusions: Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus., (© 2015 American Heart Association, Inc.)

Objective: Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers important for endothelial gene expression and contributing to angiogenesis., Approach and Results: To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation., Conclusions: JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylase-dependent manner., (© 2015 American Heart Association, Inc.)

Arsenic, a natural substance that has been used as a drug for over 2000 years, has been revived because of its remarkable therapeutic efficacy in patients with acute promyelocytic leukemia (APL). Arsenic exerts a dose-dependent dual effect: it causes differentiation at low concentrations and apoptosis at relatively high concentrations. Specific degradation of the leukemogenic PML-RARalpha fusion protein induced by arsenic leads to the differentiation of leukemia cells. The arsenic-induced apoptosis occurs through direct effects on mitochondria, causing the release of apoptotic proteins into the cytosol and the activation of caspases. Preliminary in vitro studies have also extended the potential anti-cancer effect of arsenic to non-APL leukemias, lymphoid malignancies and other cancers. In vitro and in vivo studies demonstrate that arsenic exerts a broad spectrum of anti-cancer effects by induction of apoptosis, inhibition of cell proliferation, anti-angiogenesis and possible immunomodulation. Phase I and II clinical trials are underway to evaluate the feasibility, safety and potential effect of arsenic in various cancer types.