Benjamin L. Predmore

A Fluorescent Probe for Fast and Quantitative Detection of Hydrogen Sulfide in Blood

Hydrogen sulfide (H2S), well known for its unpleasant rotten egg smell, was traditionally considered as a toxic gas. However, recent studies have demonstrated that hydrogen sulfide is an endogenously produced gaseous signaling compound (gasotransmitter) with importance on par with that of the other two known endogenous gasotransmitters, nitric oxide (NO)[1] and carbon monoxide (CO).[2] H2S has been recognized for mediating a wide range of physiological effects. Studies have shown that H2S can have an effect on the cardiovascular system[3] by acting as a K-ATP channel opener.[4] Several studies have shown the protective roles of H2S, in situations such as myocardial ischemia, most likely through a combination of antioxidant and anti-apoptotic signaling.[5] Further studies also showed that H2S may be a therapeutic benefit for the treatment of ischemia-induced heart failure.[6-7] It is also a modulator in the central nervous system,[8-10] respiratory system, gastrointestinal system, and endocrine system.[11] It seems that hydrogen sulfide exhibits almost all the beneficial effects of NO without generating the toxic reactive oxygen species (ROS). In contrast, it also acts as an anti-oxidant or scavenger of ROS. Furthermore, research has indicated that hydrogen sulfide level is related to diseases such as Down syndrome[12] and Alzheimer’s disease.[13] Therefore, recent years have seen a steady increase in the interest in understanding hydrogen sulfide ’s physiological and pathological functions.[11, 14-15] One significant limiting factor in studying hydrogen sulfide is the lack of sensors and agents that allow for its rapid and accurate detection. There have been literature methods using colorimetric,[16-18] electrochemical analysis[19-21] and gas chromatography.[22-23] However, hydrogen sulfide catabolism is known to be fast, which could result in continuous fluctuation in its concentration, leading to difficulties in accurate analysis of this important molecule. Current methods do not allow for fast, accurate, and real-time determinations. Literature reported endogenous sulfide concentrations vary substantially among publications with most publications suggesting that sulfide concentration in blood is in the 10-100 μM range.[24-29] There are other studies suggesting sulfide concentration being much lower than this.[30-31] Therefore, there is an urgent need for the development of new methods for the efficient detection of sulfide in biological systems.

Hydrogen Sulfide in Biochemistry and Medicine

SIGNIFICANCE An abundance of experimental evidence suggests that hydrogen sulfide (H(2)S) plays a prominent role in physiology and pathophysiology. Many targets exist for H(2)S therapy. The molecular targets of H(2)S include proteins, enzymes, transcription factors, and membrane ion channels. RECENT ADVANCES Novel H(2)S precursors are being synthesized and discovered that are capable of releasing H(2)S in a slow and sustained manner. This presents a novel and advantageous approach to H(2)S therapy for treatment of chronic conditions associated with a decline in endogenous H(2)S, such as diabetes and cardiovascular disease. CRITICAL ISSUES While H(2)S is cytoprotective at physiological concentrations, it is not universally cytoprotective, as it appears to have pro-apoptotic actions in cancer cells and is well known to be toxic at supraphysiological concentrations. Many of the pleiotropic effects of H(2)S on health are associated with the inhibition of inflammation and upregulation of prosurvival pathways. The powerful anti-inflammatory, cytoprotective, immunomodulating, and trophic effects of H(2)S on the vast majority of normal cells seem to be mediated mainly by its actions as an extremely versatile direct and indirect antioxidant and free radical scavenger. While the overall effects of H(2)S on transformed (i.e., malignant) cells can be characterized as pro-oxidant and pro-apoptotic, they contrast sharply with the cytoprotective effects on most normal cells. FUTURE DIRECTIONS H(2)S has become a molecule of great interest, and several slow-releasing H(2)S prodrugs are currently under development. We believe that additional agents regulating H(2)S bioavailability will be developed during the next 10 years.

H2S Protects Against Pressure Overload Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase (eNOS)

Background— Cystathionine &ggr;-lyase (CSE) produces H2S via enzymatic conversion of L-cysteine and plays a critical role in cardiovascular homeostasis. We investigated the effects of genetic modulation of CSE and exogenous H2S therapy in the setting of pressure overload–induced heart failure. Methods and Results— Transverse aortic constriction was performed in wild-type, CSE knockout, and cardiac-specific CSE transgenic mice. In addition, C57BL/6J or CSE knockout mice received a novel H2S donor (SG-1002). Mice were followed up for 12 weeks with echocardiography. We observed a >60% reduction in myocardial and circulating H2S levels after transverse aortic constriction. CSE knockout mice exhibited significantly greater cardiac dilatation and dysfunction than wild-type mice after transverse aortic constriction, and cardiac-specific CSE transgenic mice maintained cardiac structure and function after transverse aortic constriction. H2S therapy with SG-1002 resulted in cardioprotection during transverse aortic constriction via upregulation of the vascular endothelial growth factor–Akt–endothelial nitric oxide synthase–nitric oxide–cGMP pathway with preserved mitochondrial function, attenuated oxidative stress, and increased myocardial vascular density. Conclusions— Our results demonstrate that H2S levels are decreased in mice in the setting of heart failure. Moreover, CSE plays a critical role in the preservation of cardiac function in heart failure, and oral H2S therapy prevents the transition from compensated to decompensated heart failure in part via upregulation of endothelial nitric oxide synthase and increased nitric oxide bioavailability.

The Polysulfide, Diallyl trisulfide, Protects the Ischemic Myocardium by Preservation of Endogenous Hydrogen Sulfide and Increasing Nitric Oxide Bioavailability

Diallyl trisulfide (DATS), a polysulfide constituent found in garlic oil, is capable of the release of hydrogen sulfide (H(2)S). H(2)S is a known cardioprotective agent that protects the heart via antioxidant, antiapoptotic, anti-inflammatory, and mitochondrial actions. Here, we investigated DATS as a stable donor of H(2)S during myocardial ischemia-reperfusion (MI/R) injury in vivo. We investigated endogenous H(2)S levels, infarct size, postischemic left ventricular function, mitochondrial respiration and coupling, endothelial nitric oxide (NO) synthase (eNOS) activation, and nuclear E2-related factor (Nrf2) translocation after DATS treatment. Mice were anesthetized and subjected to a surgical model of MI/R injury with and without DATS treatment (200 μg/kg). Both circulating and myocardial H(2)S levels were determined using chemiluminescent gas chromatography. Infarct size was measured after 45 min of ischemia and 24 h of reperfusion. Troponin I release was measured at 2, 4, and 24 h after reperfusion. Cardiac function was measured at baseline and 72 h after reperfusion by echocardiography. Cardiac mitochondria were isolated after MI/R, and mitochondrial respiration was investigated. NO metabolites, eNOS phosphorylation, and Nrf2 translocation were determined 30 min and 2 h after DATS administration. Myocardial H(2)S levels markedly decreased after I/R injury but were rescued by DATS treatment (P < 0.05). DATS administration significantly reduced infarct size per area at risk and per left ventricular area compared with control (P < 0.001) as well as circulating troponin I levels at 4 and 24 h (P < 0.05). Myocardial contractile function was significantly better in DATS-treated hearts compared with vehicle treatment (P < 0.05) 72 h after reperfusion. DATS reduced mitochondrial respiration in a concentration-dependent manner and significantly improved mitochondrial coupling after reperfusion (P < 0.01). DATS activated eNOS (P < 0.05) and increased NO metabolites (P < 0.05). DATS did not appear to significantly induce the Nrf2 pathway. Taken together, these data suggest that DATS is a donor of H(2)S that can be used as a cardioprotective agent to treat MI/R injury.

Beta3-Adrenoreceptor Stimulation Ameliorates Myocardial Ischemia-Reperfusion Injury Via Endothelial Nitric Oxide Synthase and Neuronal Nitric Oxide Synthase Activation

OBJECTIVES This paper examined whether nebivolol protects the heart via nitric oxide (NO) synthase and NO-dependent signaling in an in vivo model of acute myocardial infarction. BACKGROUND Beta(3)-adrenergic receptor (AR) activation promotes endothelial nitric oxide synthase (eNOS) activity and NO bioavailability. We hypothesized that specific beta(3)-AR agonists would attenuate myocardial ischemia-reperfusion (MI/R) injury via eNOS activation and increased NO bioavailability. METHODS Mice were subjected to 45 min of myocardial ischemia in vivo followed by 24 h of reperfusion (R). Nebivolol (500 ng/kg), CL 316243 (1 μg/kg), BRL-37344 (1 μg/kg), or vehicle (VEH) was administered at the time of R. Myocardial area-at-risk (AAR) and infarct size (INF)/AAR was measured at 24 h of R. Cardiac tissue and plasma were collected to evaluate eNOS phosphorylation, neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase expression, and nitrite and nitrosothiol levels. RESULTS Nebivolol (500 ng/kg) reduced INF/AAR by 37% (p < 0.001 vs. VEH) and serum troponin-I levels from 41 ± 4 ng/ml to 25 ± 4 ng/ml (p < 0.05 vs. VEH). CL 316243 and BRL-37344 reduced INF by 39% and 42%, respectively (p < 0.001 vs. VEH). Nebivolol and CL 316243 increased eNOS phosphorylation at Ser-1177 (p < 0.05 vs. VEH) and increased nitrite and total nitrosylated protein levels. Nebivolol and CL 316243 significantly increased myocardial nNOS expression. Nebivolol failed to reduce INF after MI/R in beta(3)-AR (-/-), eNOS(-/-), and in nNOS(-/-) mice. CONCLUSIONS Our results indicate that beta(3)-AR agonists protect against MI/R injury. Furthermore, the cardioprotective effects of beta(3)-AR agonists are mediated by rapid eNOS and nNOS activation and increased NO bioavailability.

Acute Humanin Therapy Attenuates Myocardial Ischemia and Reperfusion Injury in Mice

Objective—Humanin (HN), an endogenous antiapoptotic peptide, has previously been shown to protect against Alzheimers disease and a variety of cellular insults. We evaluated the effects of a potent analog of HN (HNG) in an in vivo murine model of myocardial ischemia and reperfusion. Methods and Results—Male C57BL6/J mice (8 to 10 week old) were subjected to 45 minutes of left coronary artery occlusion followed by a 24-hour reperfusion. HNG or vehicle was administered IP 1 hour prior or at the time of reperfusion. The extent of myocardial infarction per area-at-risk was evaluated at 24 hours using Evans Blue dye and 2-3-5-triphenyl tetrazolium chloride staining. Left ventricular function was evaluated at 1 week after ischemia using high-resolution, 2D echocardiography (VisualSonics Vevo 770). Myocardial cell signaling pathways and apoptotic markers were assessed at various time points (0 to 24 hours) following reperfusion. Cardiomyocyte survival and apoptosis in response to HNG were assessed in vitro. HNG reduced infarct size relative to the area-at-risk in a dose-dependent fashion, with a maximal reduction at the dose of 2 mg/kg. HNG therapy enhanced left ventricular ejection fraction and preserved postischemic left ventricular dimensions (end-diastolic and end-systolic), resulting in improved cardiac function. Treatment with HNG significantly increased phosphorylation of AMPK and phosphorylation of endothelial nitric oxide synthase in the heart and attenuated Bcl-2-associated X protein and B-cell lymphoma-2 levels following myocardial ischemia and reperfusion. HNG improved cardiomyocyte survival and decreased apoptosis in response to daunorubicin in vitro. Conclusion—These data show that HNG provides cardioprotection in a mouse model of myocardial ischemia and reperfusion potentially through activation of AMPK-endothelial nitric oxide synthase-mediated signaling and regulation of apoptotic factors. HNG may represent a novel agent for the treatment of acute myocardial infarction.

Development of Hydrogen Sulfide-Based Therapeutics for Cardiovascular Disease

The physiological role of the gaseous signaling molecule hydrogen sulfide (H2S) was first realized in the mid-1990s with the work of Abe and Kimura. Since then, it has become evident that this endogenous gas is extremely important in the homeostasis of the cardiovascular system and the pathogenesis of cardiovascular disease. Several biotechnology companies have developed and are developing H2S-based therapeutic compounds, and there are ongoing clinical trials investigating the therapeutic potential of H2S. Several organic and chemical compounds that are known H2S donors have the potential to be developed into effective H2S-based therapeutic agents. This review will provide a historical and current perspective on the role(s) of H2S in the cardiovascular system and the current state of development and future outlook of H2S-based therapies for cardiovascular disease.

Red blood cells stored for increasing periods produce progressive impairments in nitric oxide–mediated vasodilation

Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)‐mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow.

Hydrogen sulfide-mediated myocardial pre- and post-conditioning

Coronary artery disease is a major cause of morbidity and mortality in the Western world. Acute myocardial infarction, resulting from coronary artery atherosclerosis, is a serious and often fatal consequence of coronary artery disease, resulting in cell death in the myocardium. Pre- and post-conditioning of the myocardium are two treatment strategies that reduce the amount of cell death significantly. Hydrogen sulfide has recently been identified as a potent cardioprotective signaling molecule, which is a highly effective pre- and post-conditioning agent. The cardioprotective signaling pathways involved in hydrogen sulfide-based pre- and post-conditioning will be explored in this article.

Single-dose pharmacokinetics of different oral sodium nitrite formulations in diabetes patients.

BACKGROUND Diabetic foot ulcers, although associated with macrovascular disease and neuropathy, have a microvascular disease causing ischemia not amenable to surgical intervention. Nitrite selectively releases nitric oxide in ischemic tissues, and diabetes subjects have low nitrite levels that do not increase with exercise. This study explores the safety and pharmacokinetics of a single dose of sodium nitrite in subjects with diabetic foot ulcers. SUBJECTS AND METHODS Using a blinded, randomized crossover study design, 12 subjects with diabetes mellitus and active or healed foot ulcers received a single dose of sodium nitrite on two occasions 7-28 days apart, once with an immediate release (IR) formulation and once with an enteric-coated (EC) formulation for delayed release. Serum nitrite, nitrate, methemoglobin, sulfhemoglobin, blood pressure, pulse rate, complete blood count, chemistry panel, electrocardiogram, and adverse events were followed for up to 6 h after each dose. The IR and EC nitrite levels were analyzed by one-way analysis of variance and by pharmacokinetic modeling. RESULTS The IR formulation elevated nitrite levels between 0.25 and 0.75 h (P<0.05). The EC formulation did not elevate nitrite levels significantly, but both formulations gave plasma nitrite levels previously suggested to be therapeutic (approximately 2-5 μM). The IR formulation gave an asymptomatic blood pressure drop of 10/6 mm Hg (P<0.003), and two subjects experienced mild flushing. There was no elevation of methemoglobin or other safety concerns. Pharmacokinetic modeling of plama nitrite levels gave r(2) values of 0.81 and 0.97 for the fits for IR and EC formulations, respectively. CONCLUSIONS Oral sodium nitrite administration is well tolerated in diabetes patients.