A cysteine-triggered fluorogenic donor base on native chemical ligation for tracking H2S delivery in vivo.

A hydrogen sulfide (H2S) donor is a fundamental molecular tool used as an exogenous source in biological studies and therapies. However, finding a controllable and visual fluorescent H2S donor is difficult. We report a new H2S donor, HSD560, the H2S release of which is triggered by cysteine. Importantly, the H2S generation is accompanied with enhanced green fluorescence, which could be utilized to track H2S release in cells using microscopy. H2S release from HSD560 undergoes a non-enzymatic native chemical ligation (NCL) process, which provides an accurate match with activated fluorescence and localization of H2S in zebrafish.

BaiJiu Increases Nitric Oxide Bioactivity of Chinese Herbs Used to Treat Coronary Artery Disease Through the NO3--NO2--NO Pathway.

BaiJiu (BJ) is a type of Chinese rice wine combined with the traditional Chinese herbs GuaLou (GL) and XieBai (XB), which have been used to treat and prevent coronary artery disease for nearly 2000 years in China. However, the mechanisms behind the compatibility of the components of this compound (GLXBBJ) have not been deeply investigated. In this study, the compatibility of the GLXBBJ compounds with nitric oxide (NO) bioactivity was evaluated in herbs, cells, and isolated aortic rings. Nitrate (NO3) and nitrite (NO2) concentrations were quantified by the Griess method. Nitric oxide (NO) was quantified by a multifunctional enzyme marker using a fluorescent probe. Qualitative analysis of L-arginine-endothelial NO synthase (eNOS) was performed by Western blotting. The tension of aortic rings was measured by multimyograph system. The ability of BJ to reduce NO3 to NO2 and NO2 to NO was strongest under hypoxic conditions and was not affected by temperature. BJ-containing serum significantly decreased the NO3 content and increased the NO2 content in hypoxic cells. Combining BJ with GL, XB, or GLXB resulted in stronger vasodilation effects. These results demonstrate that BJ effectively reduces NO3/NO2, although only a small amount of NO3 is present. Once combined with GL, XB, or GLXB, which are rich in NO3/NO2, robust NO bioactivity was generated through the NO3-NO2-NO pathway. Therefore, this study supports the potential of using traditional Chinese herbs for promoting medical innovation and for future drug development.

ß-arrestin1-biased ß1-adrenergic receptor signaling regulates microRNA processing.

RATIONALE: MicroRNAs (miRs) are small, noncoding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes after activation by a variety of signals such as those stimulated by ß-adrenergic receptors (ßARs). Initially discovered to desensitize ßAR signaling, ß-arrestins are now appreciated to transduce multiple effector pathways independent of G-protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the ß-arrestin-biased ßAR agonist, carvedilol, activates cellular pathways in the heart. OBJECTIVE: Here, we tested whether carvedilol could activate ß-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. METHODS AND RESULTS: In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs, but not their pri-miRs, in ß1AR-, G-protein-coupled receptor kinase 5/6-, and ß-arrestin1-dependent manner. Mechanistically, ß-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs. CONCLUSIONS: Our findings indicate a novel function for ß1AR-mediated ß-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.

MicroRNA-150 deletion in mice protects kidney from myocardial infarction-induced acute kidney injury.

Despite greater understanding of acute kidney injury (AKI) in animal models, many of the preclinical studies are not translatable. Most of the data were derived from a bilateral renal pedicle clamping model with warm ischemia. However, ischemic injury of the kidney in humans is distinctly different and does not involve clamping of renal vessel. Permanent ligation of the left anterior descending coronary artery model was used to test the role of microRNA (miR)-150 in AKI. Myocardial infarction in this model causes AKI which is similar to human cardiac bypass surgery. Moreover, the time course of serum creatinine and biomarker elevation were also similar to human ischemic injury. Deletion of miR-150 suppressed AKI which was associated with suppression of inflammation and interstitial cell apoptosis. Immunofluorescence staining with endothelial marker and marker of apoptosis suggested that dying cells are mostly endothelial cells with minimal epithelial cell apoptosis in this model. Interestingly, deletion of miR-150 also suppressed interstitial fibrosis. Consistent with protection, miR-150 deletion causes induction of its target gene insulin-like growth factor-1 receptor (IGF-1R) and overexpression of miR-150 in endothelial cells downregulated IGF-1R, suggesting miR-150 may mediate its detrimental effects through suppression of IGF-1R pathways.

Nitric-oxide supplementation for treatment of long-term complications in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is required for the synthesis and channeling of L-arginine to nitric oxide synthase (NOS) for nitric oxide (NO) production. Congenital ASL deficiency causes argininosuccinic aciduria (ASA), the second most common urea-cycle disorder, and leads to deficiency of both ureagenesis and NO production. Subjects with ASA have been reported to develop long-term complications such as hypertension and neurocognitive deficits despite early initiation of therapy and the absence of documented hyperammonemia. In order to distinguish the relative contributions of the hepatic urea-cycle defect from those of the NO deficiency to the phenotype, we performed liver-directed gene therapy in a mouse model of ASA. Whereas the gene therapy corrected the ureagenesis defect, the systemic hypertension in mice could be corrected by treatment with an exogenous NO source. In an ASA subject with severe hypertension refractory to antihypertensive medications, monotherapy with NO supplements resulted in the long-term control of hypertension and a decrease in cardiac hypertrophy. In addition, the NO therapy was associated with an improvement in some neuropsychological parameters pertaining to verbal memory and nonverbal problem solving. Our data show that ASA, in addition to being a classical urea-cycle disorder, is also a model of congenital human NO deficiency and that ASA subjects could potentially benefit from NO supplementation. Hence, NO supplementation should be investigated for the long-term treatment of this condition.

Concentration- and stage-specific effects of nitrite on colon cancer cell lines.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM-100 µM and inhibits stage 3 HCT15 proliferation at 100 nM-1 µM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 µM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 µM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 µM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.

Nitrite and nitrate: cardiovascular risk-benefit and metabolic effect.

PURPOSE OF REVIEW: To review the most recent published literature on the biological effects of nitrite and nitrate in order to establish the context for potential health benefits vs. potential risks or adverse effects. Nitrite and nitrate are indigenous to our diet and are formed naturally within our body from the oxidation of nitric oxide. Emerging health benefits from dietary sources of nitrite and nitrate contradict decades of epidemiological research that have suggested an association of nitrite and nitrate in foods, primarily cured and processed meat, with certain cancers. RECENT FINDINGS: The major source of exposure of nitrite and nitrate comes from the consumption of nitrate-enriched vegetables. The preponderance of epidemiological studies shows a very weak association with consumption of meats and certain cancers, which contain very little nitrite and nitrate. Nitrite and nitrate in certain foods and diets can be metabolized to nitric oxide and promote cardiovascular benefits and cytoprotection. SUMMARY: The cardiovascular benefits of nitrite and nitrate are beginning to be translated in humans by the increasing number of clinical trials using nitrite and nitrate. The collective body of evidence suggests that foods enriched in nitrite and nitrate provide significant health benefits with very little risk.

Photo-controlled and photo-calibrated nanoparticle enabled nitric oxide release for anti-bacterial and anti-biofilm applications.

After numerous efforts to elucidate the biological role of nitric oxide (NO), NO treatments have become a hotspot at the forefront of medicine. NO-releasing substances are constantly needed, while the direct use of NO gas is unattainable in bio-systems. An ideal NO donor should possess controllable and visible NO-release capability. The reported NO donating nanoparticles, prepared via encapsulating a hydrophobic NO-releasing compound into DSPE-PEG2000, meet the criteria mentioned previously. The localization and flux of NO released from these nanoparticles could be manipulated by UV or blue light. Meanwhile, NOD-NPs emit a dose-dependent fluorescence intensity to calibrate the generation of NO. While the good biocompatibility of NOD-NPs has been validated, the NO from our nanoparticles demonstrates efficient anti-bacterial and anti-biofilm effects toward Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Therefore, the NOD-NPs developed in this work have potential application in evaluating the regulation of microbes by NO.

Molecular mechanism of Chuanxiong Rhizoma in treating coronary artery diseases.

Objective: Most of the studies on the herb Chuanxiong Rhizoma (CR) have focused on the l-arginine-nitric oxide (NO) pathway, but the nitrate-nitrite-NO (NO3 --NO2 --NO) pathway was rarely investigated. Therefore, the aim of this study was to evaluate the effects and mechanisms of action of CR in coronary artery disease (CAD). Methods: The NO3 -, NO2 - and NO levels were examined in the NO3 --NO2 --NO pathway. High-performance ion chromatography was used to quantify NO3 - and NO2 - levels. Then, NO was quantified using a multifunctional enzyme marker with a fluorescent probe. The tension of aortic rings was measured using a multi myograph system. Results: High content of NO3 - and low content of NO2 - was found in CR, and which could potently convert NO3 - to NO2 - in the presence of endogenous reductase enzyme. Incubating human coronary artery endothelial cells (HCAECs) with CR-containing serum showed that CR significantly decreased the NO3 - content and increased the levels of NO2 - and NO in the cells under hypoxic conditions. In addition, CR significantly relaxed isolated aortic rings when the l-arginine -NO pathway was blocked. The optimal concentration of CR for relaxation was 200 mg/mL. Conclusion: CR supplements large amounts of NO in cells and vessels to achieve relaxation via the NO3 --NO2 --NO pathway, thereby making up for the deficiency caused by the lack of NO after the l-arginine-NO pathway is suppressed. This study also supports the potential use of a traditional Chinese herb for future drug development.

Machine learning identifies two autophagy-related genes as markers of recurrence in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is the most common cancer worldwide. Patient outcomes following recurrence of CRC are very poor. Therefore, identifying the risk of CRC recurrence at an early stage would improve patient care. Accumulating evidence shows that autophagy plays an active role in tumorigenesis, recurrence, and metastasis. METHODS: We used machine learning algorithms and two regression models, univariable Cox proportion and least absolute shrinkage and selection operator (LASSO), to identify 26 autophagy-related genes (ARGs) related to CRC recurrence. RESULTS: By functional annotation, these ARGs were shown to be enriched in necroptosis and apoptosis pathways. Protein-protein interactions identified SQSTM1, CASP8, HSP80AB1, FADD, and MAPK9 as core genes in CRC autophagy. Of 26 ARGs, BAX and PARP1 were regarded as having the most significant predictive ability of CRC recurrence, with prediction accuracy of 71.1%. CONCLUSION: These results shed light on prediction of CRC recurrence by ARGs. Stratification of patients into recurrence risk groups by testing ARGs would be a valuable tool for early detection of CRC recurrence.

To develop a novel animal model of myocardial infarction: A research imperative.

Although great progress has been made in therapeutic interventions for coronary artery disease (CAD), it is still the deadliest disease in the world. Currently animals that are similar to human beings in their cardiovascular pathophysiology are being used to explore the pathogenesis and therapy of CAD. There have been a series of developments in creating CAD animal models using mice, rats, rabbits, dogs, and pigs, but unfortunately there is still no acceptable model for human CAD. The ideal CAD animal model should satisfy several conditions as follows. First of all, it should have a pathophysiological process for CAD that is similar to humans. Second, it should be useable for assessing drug efficacy. The last and most important condition is that the model can be used to duplicate clinical therapeutic skills. The limitations of current methods for making animal models have meant that these models not only do not duplicate the actual pathogenesis, but also cannot be used to simulate clinical therapy, and do not support scientific evaluation of drug efficacy. Therefore, the development of a fit-for-purpose animal model for CAD is imperative for future research. Such a development will lead to rapid progress and greater efficiency in CAD research. This paper summarizes the present situation in the field of CAD animal models, and puts forwards ideas for developing a novel animal model of myocardial infarction.

Tongxinluo Improves Apolipoprotein E-Deficient Mouse Heart Function.

BACKGROUND: Our previous studies have shown that Tongxinluo (TXL), a compound Chinese medicine, can decrease myocardial ischemia-reperfusion injury, protect capillary endothelium function, and lessen cardiac ventricle reconstitution in animal models. The aim of this study was to illuminate whether TXL can improve hypercholesterolemia-impaired heart function by protecting artery endothelial function and increasing microvascular density (MVD) in heart. Furthermore, we will explore the underlying molecular mechanism of TXL cardiovascular protection. METHODS: After intragastric administration of TXL (0.1 ml/10 g body weight) to C57BL/6J wild-type mice (n = 8) and ApoE-/- mice (n = 8), total cholesterol, high-density lipoprotein-cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, triglyceride, and blood glucose levels in serum were measured. The parameters of heart rate (HR), left ventricular diastolic end diameter, and left ventricular systolic end diameter were harvested by ultrasonic cardiogram. The left ventricular ejection fraction, stroke volume, cardiac output, and left ventricular fractional shortening were calculated. Meanwhile, aorta peak systolic flow velocity (PSV), end diastolic flow velocity, and mean flow velocity (MFV) were measured. The pulsatility index (PI) and resistant index were calculated in order to evaluate the vascular elasticity and resistance. The endothelium-dependent vasodilatation was evaluated by relaxation of aortic rings in response to acetylcholine. Western blotting and real-time quantitative reverse transcription polymerase chain reaction were performed for protein and gene analyses of vascular endothelial growth factor (VEGF). Immunohistochemical detection was performed for myocardial CD34 expression. Data in this study were compared by one-way analysis of variance between groups. A value of P < 0.05 was considered statistically significant. RESULTS: Although there was no significant decrease of cholesterol level (F = 2.300, P = 0.240), TXL inhibited the level of triglyceride and VLDL (F = 9.209, P = 0.024 and F = 9.786, P = 0.020, respectively) in ApoE-/- mice. TXL improved heart function of ApoE-/- mice owing to the elevations of LVEF, SV, CO, and LVFS (all P < 0.05). TXL enhanced aortic PSV and MFV (F = 10.774, P = 0.024 and F = 11.354, P = 0.020, respectively) and reduced PI of ApoE-/- mice (1.41 ± 0.17 vs. 1.60 ± 0.17; P = 0.037). After incubation with 10 µmol/L acetylcholine, the ApoE-/- mice treated with TXL aortic segment relaxed by 44% ± 3%, significantly higher than control group mice (F = 9.280, P = 0.040). TXL also restrain the angiogenesis of ApoE-/- mice aorta (F = 21.223, P = 0.010). Compared with C57BL/6J mice, the MVD was decreased in heart tissue of untreated ApoE-/- mice (54.0 ± 3.0/mm2 vs. 75.0 ± 2.0/mm2; F = 16.054, P = 0.010). However, TXL could significantly enhance MVD (65.0 ± 5.0/mm2 vs. 54.0 ± 3.0/mm2; F = 11.929, P = 0.020) in treated ApoE-/- mice. In addition, TXL obviously increased the expression of VEGF protein determined by Western blot (F = 20.247, P = 0.004). CONCLUSIONS: TXL obviously improves the ApoE-/- mouse heart function from different pathways, including reduces blood fat to lessen atherosclerosis; enhances aortic impulsivity, blood supply capacity, and vessel elasticity; improves endothelium-dependent vasodilatation; restraines angiogenesis of aorta-contained plaque; and enhances MVD of heart. The molecular mechanism of MVD enhancement maybe relate with increased VEGF expression.

MicroRNA-150 protects the mouse heart from ischaemic injury by regulating cell death.

AIMS: Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the ß-arrestin-biased ß-blocker, carvedilol that ß-arrestin1-biased ß1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown. METHODS AND RESULTS: Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes. CONCLUSION: These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.

Requirement of argininosuccinate lyase for systemic nitric oxide production.

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Food sources of nitrates and nitrites: the physiologic context for potential health benefits.

The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Despite the physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites as healthful dietary components has received little attention. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, we found that nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds the World Health Organization's Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients.

Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications.

Traditional Chinese medicine (TCM) has been used for centuries to treat and prevent certain ailments and diseases. Although TCM has served as mainstream medical care throughout Asia for many generations, it is considered an alternative medical system in much of the Western world. Because many TCMs are used primarily for cardiovascular indications characterized by a nitric oxide (NO) insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. We tested a group of convenience samples of TCMs obtained in the United States for endogenous nitrite, nitrate, nitroso, and nitrite reductase activity as well as their ability to relax isolated aortic rings. The results from this study reveal that all of the TCMs tested reveal NO bioactivity through their inherent nitrite and nitrate content and their ability to reduce nitrite to NO. Many of the TCM extracts contain a nitrite reductase activity greater by 1000 times that of biological tissues. Repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs.

Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction.

The nitrite anion is an endogenous product of mammalian nitric oxide (NO) metabolism, a key intermediate in the nitrogen cycle in plants, and a constituent of many foods. Research over the past 6 years has revealed surprising biological and cytoprotective activity of this anion. Hypercholesterolemia causes a proinflammatory phenotype in the microcirculation. This phenotype appears to result from a decline in NO bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide, or both. Since nitrite has been shown to be potently cytoprotective and restore NO biochemical homeostasis, we investigated if supplemental nitrite could attenuate microvascular inflammation caused by a high cholesterol diet. C57Bl/6J mice were fed either a normal diet or a high cholesterol diet for 3 wk to induce microvascular inflammation. Mice on the high cholesterol diet received either nitrite-free drinking water or supplemental nitrite at 33 or 99 mg/l ad libitum in their drinking water. The results from this investigation reveal that mice fed a cholesterol-enriched diet exhibited significantly elevated leukocyte adhesion to and emigration through the venular endothelium as well as impaired endothelium-dependent relaxation in arterioles. Administration of nitrite in the drinking water inhibited the leukocyte adhesion and emigration and prevented the arteriolar dysfunction. This was associated with sparing of reduced tetrahydrobiopterin and decreased levels of C-reactive protein. These data reveal novel anti-inflammatory properties of nitrite and implicate the use of nitrite as a new natural therapy for microvascular inflammation and endothelial dysfunction associated with hypercholesterolemia.

Kinetic and cellular characterization of novel inhibitors of S-nitrosoglutathione reductase.

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of beta-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-kappaB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.