Gastrodin alleviates NTG-induced migraine-like pain via inhibiting succinate/HIF-1α/TRPM2 signaling pathway in trigeminal ganglion.

BACKGROUND: Increasing evidence highlights the involvement of metabolic disorder and calcium influx mediated by transient receptor potential channels in migraine; however, the relationship between these factors in the pathophysiology of migraine remains unknown. Gastrodin is the major component of the traditional Chinese medicine Tianma, which is extensively used in migraine therapy. PURPOSE: Our work aimed to explore the analgesic action of gastrodin and its regulatory mechanisms from a metabolic perspective. METHODS/RESULTS: After being treated with gastrodin, the mice were given nitroglycerin (NTG) to induce migraine. Gastrodin treatment significantly raised the threshold of sensitivity in response to both mechanical and thermal stimulus evidenced by von Frey and hot plate tests, respectively, and decreased total contact numbers in orofacial operant behavioral assessment. We found that the expression of transient receptor potential melastatin 2 (TRPM2) channel was increased in the trigeminal ganglion (TG) of NTG-induced mice, resulting in a sustained Ca2+ influx to trigger migraine pain. The content of succinate, a metabolic biomarker, was elevated in blood samples of migraineurs, as well as in the serum and TG tissue from NTG-induced migraine mice. Calcium imaging assay indicated that succinate insult elevated TRPM2-mediated calcium flux signal in TG neurons. Mechanistically, accumulated succinate upregulated hypoxia inducible factor-1α (HIF-1α) expression and promoted its translocation into nucleus, where HIF-1α enhanced TRPM2 expression through transcriptional induction in TG neurons, evidenced by luciferase reporter measurement. Gastrodin treatment inhibited TRPM2 expression and TRPM2-dependent Ca2+ influx by attenuating succinate accumulation and downstream HIF-1α signaling, and thereby exhibited analgesic effect. CONCLUSION: This work revealed that succinate was a critical metabolic signaling molecule and the key mediator of migraine pain through triggering TRPM2-mediated calcium overload. Gastrodin alleviated NTG-induced migraine-like pain via inhibiting succinate/HIF-1α/TRPM2 signaling pathway in TG neurons. These findings uncovered the anti-migraine effect of gastrodin and its regulatory mechanisms from a metabolic perspective and provided a novel theoretical basis for the analgesic action of gastrodin.

Quality by design for sumatriptan loaded nano-ethosomal mucoadhesive gel for the therapeutic management of nitroglycerin induced migraine.

Migraine is a progressive neurological condition often accompanied by nausea and vomiting. Various drugs have recently been used in the treatment of migraine, including sumatriptan (SUT). However, SUT has poor pharmacological effects mainly due to its reduced permeability, blood brain barrier (BBB) effect, half-life and bioavailability. Herein, we developed SUT loaded nano-ethosomes (SUT-NEs) for intranasal (IN) delivery, after their incorporation into chitosan based mucoadhesive gel (SUT-NEsG). The observed mean particle size of SUT-NEs was 109.45 ± 4.03 nm with spherical morphology, mono dispersion (0.191 ± 0.001), negatively charged (-20.90 ± 1.98 mV) and with excellent entrapment efficiency (96.90 ± 1.85 %). Fourier-transform infrared (FTIR) spectra have depicted the compatibility of the components. Moreover, SUT-NEsG was homogeneous having suitable viscosity and mucoadhesive strength. In vitro release and ex vivo permeation analysis showed sustained release and improved permeation of the SUT-NEsG, respectively. Additionally, histopathological studies of nasal membrane affirmed the safety of SUT-NEsG after IN application. In vivo pharmacokinetic study demonstrated improved brain bioavailability of SUT-NEsG as compared to orally administered sumatriptan solution (SUT-SL). Furthermore, significantly enhanced pharmacological effect of SUT-NEsG was observed in behavioral and biochemical analysis, immunohistochemistry for NF-κB, and enzyme linked immuno assay (ELISA) for IL-1ß and TNF-α in Nitroglycerin (NTG) induced migraine model. It can be concluded that migraine may be successfully managed through IN application of SUT-NEsG owing to the direct targeted delivery to the brain.

The status of knowledge on migraines: The role of microglia.

Migraines are a considerable social problem and economic burden worldwide. Current acute treatments are based on inhibiting meningeal neurogenic inflammation which has poor results in some patients, whereas the site of action of prophylactic medicines are unknown; therefore, exploring new treatment mechanisms and methods is increasingly needed. Recent evidence suggests that microglia and microglia-mediated neuroinflammation are important in migraine pathogenesis. In the cortical spreading depression (CSD) migraine model, microglia were activated after multiple CSD stimulations, suggesting that microglial activation may be associated with recurrent attacks of migraine with aura. In the nitroglycerin-induced chronic migraine model, the microglial response to extracellular stimuli leads to the activation of surface purine receptors P2X4、P2X7、P2Y12, which mediate signal transduction through intracellular signalling cascades, such as the BDNF/TrkB, NLRP3/IL-1ß and RhoA/ROCK signalling pathways, and release inflammatory mediators and cytokines that enhance pain by increasing the excitability of nearby neurons. Inhibition of the expression or function of these microglial receptors and pathways inhibits the abnormal excitability of TNC (trigeminal nucleus caudalis) neurons and intracranial as well as extracranial hyperalgesia in migraine animal models. These findings suggest that microglia may be central in migraine recurrent attacks and a potential target for the treatment of chronic headaches.

Endocrine disruption: Reproductive toxicity of glyceryl trinitrate and isosorbide mononitrate in male Wistar rats.

Glyceryl trinitrate (GTN) and isosorbide mononitrate (IM) are organic nitrates which release nitric oxide upon metabolism with potential to adversely affect male reproductive function. Therefore, this study was designed to evaluate the sub-chronic effect of these organic nitrates on reproductive system in male rats. Wistar rats were separately treated with GTN and IM at 2.5, 5 and 7.5 mg/kg/day by oral gavage for 45 days. At the end of treatment, serum blood samples were taken from anaesthetized rats for assessment of hormonal profile. Epididymis was removed to analyse sperm parameters. Rat testes were dissected to perform histopathological evaluation and oxidative stress biomarkers. The GTN and IM treated groups showed a significant decrease in sperm parameters (count, motility and viability) and serum testosterone in comparison to normal control group. The GTN and IM treatment also altered sperm morphology such as bent tail and head deformities as compared to control. A significant decrease in catalase activity and, increase in nitric oxide and malondialdehyde were observed in high dose drug treated groups. Moreover, a significant increase in follicle stimulating hormone and decrease in testosterone levels were evident in all drug treated groups. The level of luteinizing hormone was raised in rats treated with medium doses of drugs while it decreased at the highest dose of both drugs. Histological study showed vacuolization and degeneration of seminiferous tubules. It is concluded that GTN and IM treatment adversely affected the male reproductive function by altering sperm parameters and disrupting the reproductive hormone profile which may be attributed to the increased level of nitric oxide and oxidative stress.

Biotransformation of organic nitrates by glutathione S-transferases and other enzymes: An appraisal of the pioneering work by William B. Jakoby.

Organic nitrates (R-ONO2; R, organic residue) such as nitroglycerin are used as drugs in part for more than a century. Their pharmacological use is associated with clinically relevant tolerance which is reportedly known since 1888. The underlying mechanisms of both, the mechanisms of action and the main pharmacological effect, which is vasodilatation and reduction of blood pressure, and the development of tolerance, which means increasing need of drug amount in sustained long-term therapy, are still incompletely understood. William B. Jakoby and associates were the first to report the biotransformation of organic nitrates, notably including nitroglycerin (i.e., glycerol trinitrate; GTN), by glutathione S-transferase (GST)-catalyzed conjugation of glutathione (GSH) to the nitrogen atom of one of the three nitrate groups of GTN to generate glutathione sulfenyl nitrite (glutathione thionitrate, S-nitroglutathione; GSNO2). Jakoby's group was also the first to suggest that GSNO2 reacts with a second GSH molecule to produce inorganic nitrite (ONO-) and glutathione disulfide (GSSG) without the catalytic involvement of GST. This mechanism has been adopted by others to the biotransformation of GTN by mitochondrial aldehyde dehydrogenase (mtALDH-(CysSH)2) which does not require GSH as a substrate. The main difference between these reactions is that mtALDH forms an internal thionitrate (mtALDH-(CysSH)-CysSNO2) which releases inorganic nitrite upon intra-molecular reaction to form mtALDH disulfide (mtALDH-(CysS)2). Subsequently, ONO- and GSNO2 are reduced by several proteins and enzymes to nitric oxide (NO) which is a very potent activator of soluble guanylyl cyclase to finally relax the smooth muscles thus dilating the vasculature. GSNO2 is considered to rearrange to GSONO which undergoes further reactions including GSNO and GSSG formation. The present article is an appraisal of the pioneering work of William B. Jakoby in the area of the biotransformation of organic nitrates by GST. The two above mentioned enzymatic reactions are discussed in the context of tolerance development to organic nitrates, still a clinically relevant pharmacological concern.

Effects of Nitroglycerine on Renal Ischemia-Reperfusion Injury in Adult Male Rats.

BACKGROUND: Ischemia-reperfusion (I-R) leads to acute kidney injury (AKI). The present study investigated the effects of nitroglycerine (NG) on improving renal dysfunctions caused by I-R in rats. METHODOLOGY: Twenty-four rats were equally divided into four groups: (1) the control group, (2) the sham group, (3) the I-R group, and (4) NG-treated groups.NG (50 µg/kg) was injected intraperitoneally after induction of IR. I-R was induced through clamping of the bilateral renal artery and vein of both kidneys for 20 min followed by 24 h of reperfusion. RESULTS: NG significantly increased the creatinine clearance levels and renal blood flow rate (which was reduced by I-R). NG also significantly improved serum electrolytes (sodium and potassium) that were disordered by I-R. In addition, NG significantly offset impaired antioxidant defense mechanism and inhibited lipid peroxidation. CONCLUSIONS: The results show NG has a protective effect on renal tissue against AKI caused by I-R. These protective effects mediated through antioxidant activity and decrease of lipid peroxidation.

Role of Mitochondrial Aldehyde Dehydrogenase in Nitroglycerin-Mediated Vasodilation: Observations Concerning the Dose-Response Relationship.

The mechanism of the bioactivation of nitroglycerin has long been controversial, with a number of suggested enzymatic pathways. More recently, aldehyde dehydrogenase-2 (ALDH-2) has been reported as the important enzyme involved in the bioactivation of nitroglycerin at therapeutically relevant concentrations. Other previously described enzyme systems can also bioactivate nitroglycerin, but only at concentrations, which are significantly higher than achieved in clinical practice. This study investigated the vascular response to nitroglycerin given over a wide range of concentrations in subjects with and without the ALDH-2 Glu504Lys polymorphism, a common genetic variant that greatly reduces the activity of ALDH-2 (n = 10 in both groups). Forearm blood flow (FBF) responses to a brachial artery infusion of nitroglycerin were assessed using venous occlusion plethysmography. Intra-arterial infusion of nitroglycerin caused a significant increase in FBF beginning at 0.464 µg/min with increasing responses seen in both groups at all infusion rates. However, there were no differences in the FBF responses to nitroglycerin in those with and without the ALDH-2 polymorphism, suggesting that ALDH-2 is not solely responsible for the bioactivation of nitroglycerin at either low (therapeutically relevant) or high concentrations of nitroglycerin.

Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

Methane Emission, Rumen Fermentation, and Microbial Community Response to a Nitrooxy Compound in Low-Quality Forage Fed Hu Sheep.

The effects of nitroglycerine (NG) on the rumen methane emission, fermentation, and microbial community of Hu sheep were investigated. Eight sheep were fed NG (100 mg/head/day); another eight sheep served as controls. NG decreased methane emission of Hu sheep by ~ 19.3% (P < 0.05) without adversely affecting the production performance or rumen fermentation (P > 0.05). The alpha and beta diversity indexes of the bacterial and archaeal community showed no significant differences (P > 0.05). The dominant methanogenic species was the Methanobrevibacter gottschalkii clade, accounting for ~ 60%, followed by the Methanobrevibacter boviskoreani and Methanobrevibacter ruminantium clades. Prevotella 1 was the most dominant bacterial genus, accounting for ~ 42%, followed by the Rikenellaceae RC9 and Bacteroidales BS11 gut groups. In addition, pearson correlation analysis showed a few Methanomassiliicoccales species significantly correlated with several bacterial genera (P < 0.05).

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase.

BACKGROUND: Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. METHODS: Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. RESULTS: In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. CONCLUSIONS: Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

Elevated Monocyte to High-Density Lipoprotein Cholesterol Ratio and Endothelial Dysfunction in Behçet Disease.

Behçet disease (BD) is a multisystemic disorder characterized by endothelial dysfunction and inflammation. Monocyte to high-density lipoprotein cholesterol ratio (MHR) is a recently emerged indicator of inflammation and oxidative stress. Sixty patients with BD and 50 control individuals were included to investigate the relationship between MHR and endothelial dysfunction. Endothelial function was assessed by flow- and nitroglycerin-mediated dilatation technique (FMD and NMD, respectively). Serum high-sensitivity C-reactive protein (hsCRP) levels were measured in all study participants. The MHR and hsCRP levels were significantly higher in patients with active BD than in controls. Brachial artery FMD was significantly lower in patients with active BD than in controls. Brachial artery NMD was similar between groups. There was a strong inverse correlation between MHR and FMD and a strong positive correlation between MHR and serum hsCRP levels. Thus, elevated MHR may be a useful marker reflecting impaired endothelial function and systemic inflammation in patients with BD.

The Role of Nitroglycerin and Other Nitrogen Oxides in Cardiovascular Therapeutics.

The use of nitroglycerin in the treatment of angina pectoris began not long after its original synthesis in 1847. Since then, the discovery of nitric oxide as a biological effector and better understanding of its roles in vasodilation, cell permeability, platelet function, inflammation, and other vascular processes have advanced our knowledge of the hemodynamic (mostly mediated through vasodilation of capacitance and conductance arteries) and nonhemodynamic effects of organic nitrate therapy, via both nitric oxide-dependent and -independent mechanisms. Nitrates are rapidly absorbed from mucous membranes, the gastrointestinal tract, and the skin; thus, nitroglycerin is available in a number of preparations for delivery via several routes: oral tablets, sublingual tablets, buccal tablets, sublingual spray, transdermal ointment, and transdermal patch, as well as intravenous formulations. Organic nitrates are commonly used in the treatment of cardiovascular disease, but clinical data limit their use mostly to the treatment of angina. They are also used in the treatment of subsets of patients with heart failure and pulmonary hypertension. One major limitation of the use of nitrates is the development of tolerance. Although several agents have been studied for use in the prevention of nitrate tolerance, none are currently recommended owing to a paucity of supportive clinical data. Only 1 method of preventing nitrate tolerance remains widely accepted: the use of a dosing strategy that provides an interval of no or low nitrate exposure during each 24-h period. Nitric oxide's important role in several cardiovascular disease mechanisms continues to drive research toward finding novel ways to affect both endogenous and exogenous sources of this key molecular mediator.

Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction: targeting nitric oxide and endothelin.

Hemoglobin-based oxygen carrier (HBOC)-201 is a cell-free modified hemoglobin solution potentially facilitating oxygen uptake and delivery in cardiovascular disorders and hemorrhagic shock. Clinical use has been hampered by vasoconstriction in the systemic and pulmonary beds. Therefore, we aimed to 1) determine the possibility of counteracting HBOC-201-induced pressor effects with either adenosine (ADO) or nitroglycerin (NTG); 2) assess the potential roles of nitric oxide (NO) scavenging, reactive oxygen species (ROS), and endothelin (ET) in mediating the observed vasoconstriction; and 3) compare these effects in resting and exercising swine. Chronically instrumented swine were studied at rest and during exercise after administration of HBOC-201 alone or in combination with ADO. The role of NO was assessed by supplementation with NTG or administration of the eNOS inhibitor Nω-nitro-l-arginine. Alternative vasoactive pathways were investigated via intravenous administration of the ETA/ETB receptor blocker tezosentan or a mixture of ROS scavengers. The systemic and to a lesser extent the pulmonary pressor effects of HBOC-201 could be counteracted by ADO; however, dosage titration was very important to avoid systemic hypotension. Similarly, supplementation of NO with NTG negated the pressor effects but also required titration of the dose. The pressor response to HBOC-201 was reduced after eNOS inhibition and abolished by simultaneous ETA/ETB receptor blockade, while ROS scavenging had no effect. In conclusion, the pressor response to HBOC-201 is mediated by vasoconstriction due to NO scavenging and production of ET. Further research should explore the effect of longer-acting ET receptor blockers to counteract the side effect of hemoglobin-based oxygen carriers.NEW & NOTEWORTHY Hemoglobin-based oxygen carrier (HBOC)-201 can disrupt hemodynamic homeostasis, mimicking some aspects of endothelial dysfunction, resulting in elevated systemic and pulmonary blood pressures. HBOC-201-induced vasoconstriction is mediated by scavenging nitric oxide (NO) and by upregulating endothelin (ET) production. Pressor effects can be prevented by adjuvant treatment with NO donors or direct vasodilators, such as nitroglycerin or adenosine, but dosages must be carefully monitored to avoid hypotension. However, hemodynamic normalization is more easily achieved via administration of an ET receptor blocker.

[Progression of the mechanism study on experimental migraine treated with acupuncture in rat model].

In the paper, by taking acupuncture and migraine as the key words to retrieve CNKI and PubMed database, the literature analysis was done on the mechanism study on experimental migraine treated with acupuncture in rat model. The results showed that acupuncture mechanism study focused on the regulation and control of the relevant neurotransmitters/neuromodulators of migraine, such as calcitonin gene-related peptide (CGRP), serotonin (5-HT), nitric oxide (NO), etc. Moreover, in the paper, the review had been done on the neurotransmitters/neuromodulators involved in the study.

Tumor-Targeting Salmonella typhimurium A1-R: An Overview.

The present chapter reviews the development of the tumor-targeting amino-acid auxotrophic strain S. typhimurium A1 and the in vivo selection and characterization of the high-tumor-targeting strain S. typhimurium A1-R. Efficacy of S. typhimurium A1-R in nude-mouse models of prostate, breast, pancreatic, and ovarian cancer, as well as sarcoma and glioma in orthotopic mouse models is described. Also reviewed is efficacy of S. typhimurium A1-R targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma, breast-cancer brain metastasis, and experimental breast-cancer bone metastasis in orthotopic mouse models. The efficacy of S. typhimurium A1-R on pancreatic cancer stem cells, on pancreatic cancer in combination with anti-angiogenic agents, as well as on cervical cancer, soft-tissue sarcoma, and pancreatic cancer patient-derived orthotopic xenograft (PDOX) mouse models, is also described.

Enhancement of Tumor-Targeted Delivery of Bacteria with Nitroglycerin Involving Augmentation of the EPR Effect.

The use of bacteria, about 1 µm in size, is now becoming an attractive strategy for cancer treatment. Solid tumors exhibit the enhanced permeability and retention (EPR) effect for biocompatible macromolecules such as polymer-conjugated anticancer agents, liposomes, and micelles. This phenomenon permits tumor-selective delivery of such macromolecules. We report here that bacteria injected intravenously evidenced a property similar to that can of these macromolecules. Bacteria that can accumulate selectively in tumors may therefore be used in cancer treatment.Facultative or anaerobic bacteria will grow even under the hypoxic conditions present in solid tumors. We found earlier that nitric oxide (NO) was among the most important factors that facilitated the EPR effect via vasodilatation, opening of endothelial cell junction gaps, and increasing the blood flow of hypovascular tumors. Here, we describe the augmentation of the EPR effect by means of nitroglycerin (NG), a commonly used NO donor, using various macromolecular agents in different tumor models. More importantly, we report that NG significantly enhanced the delivery of Lactobacillus casei to tumors after intravenous injection of the bacteria, more than a tenfold increase in bacterial accumulation in tumors after NG treatment. This finding suggests that NG has a potential advantage to enhance bacterial therapy of cancer, and further investigations of this possibility are warranted.

Stable isotope-assisted LC-MS/MS monitoring of glyceryl trinitrate bioactivation in a cell culture model of nitrate tolerance.

The nitric oxide (NO) metabolites nitrite (NO2(-)) and nitrate (NO3(-)) can be quantified as an endpoint of endothelial function. We developed a LC-MS/MS method of measuring nitrite and nitrate isotopologues, which has a lower limit of quantification (LLOQ) of 1 nM. This method allows for isotopic labeling to differentiate newly formed nitrite and nitrate from nanomolar to micromolar background levels of nitrite and nitrate in biological matrices. This method utilizes 2,3-diaminonaphthalene (DAN) derivatization, which reacts with nitrite under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 µm 2.1 × 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of (14)N-NAT (m/z 170.1) and (15)N-NAT (m/z 171.1). Both nitrite and nitrate demonstrated a linear detector response (1 nM - 10 µM, 1 nM - 100 nM, respectively), and were unaffected by common interferences (Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phenol red, and NADPH). This method requires minimal sample preparation, making it ideal for most biological applications. We applied this method to develop a cell culture model to study the development of nitrate tolerance in human endothelial cells (EA.hy926).

Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels.

The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.