BACKGROUND:Coronary microvascular dysfunction (CMD) is increasingly recognized as a mechanistically relevant contributor to HFpEF, yet the temporal and causal relationship between early functional CMD, coronary microvascular endothelial barrier injury, and fibrotic remodeling remains incompletely defined. The primary aim was to test whether CMD causally contributes to HFpEF-like remodeling and functional impairment in an isoprenaline (ISO) mouse model. A secondary aim was to determine whether empagliflozin (SGLT2i) or resolvin D2 modulates inflammatory and endothelial mechanisms of CMD. METHODS:Mice received Control, ISO, ISO + empagliflozin, or ISO + resolvin D2 (n = 5-6/group). ISO (100 mg·kg-1·day-1, s.c.) was given for 5 days. On days 7, 14, and 21 we assessed LV structure/function by cine-MRI, CFR by Doppler, diastolic indices by Doppler, and endothelial permeability/NO-dependent function by dynamic contrast-enhanced MRI (gadolinium-albumin) and L-NAME T1 mapping. Treatments were given days 7-21. RESULTS:ISO caused hypertrophy with preserved ejection fraction, diastolic dysfunction, and reduced exercise capacity. Resting coronary flow increased by day 7 with preserved hyperemic flow, lowering CFR before fibrosis or endothelial barrier failure. By day 21, permeability rose with inflammatory activation, blunted NO-mediated response, and perivascular/interstitial fibrosis. Empagliflozin lowered resting flow, normalized CFR, reduced endothelial permeability and inflammatory signaling, limited fibrosis, and improved diastolic indices and LV strain. Resolvin D2 reduced inflammatory markers but did not consistently restore CFR, permeability, or diastolic indices. CONCLUSIONS:Short-term ISO causes progressive CMD, and empagliflozin reverses key CMD features, supporting direct coronary microvascular modulation as a mechanism of SGLT2i benefit in HFpEF in this animal model.

G protein-coupled receptor (GPR) 18 is the receptor for the specialized pro-resolving lipid mediator (SPM) RvD2, which has proven efficacy in preventing atherosclerosis in mice. The aim of the present study was to establish direct vascular effects of GPR18 signaling. GPR18 knockout (KO) and wildtype (WT) mice underwent magnetic resonance imaging (MRI) for in vivo determination of endothelial function, and continuous 24 h telemetry for in vivo blood pressure monitoring. Isolated vessels derived from GPR18 KO and wildtype mice were used for determinations of ex vivo vascular reactivity and immunofluorescent quantifications. GPR18 KO mice exhibited endothelial dysfunction in the femoral arterial segment measured by MRI in vivo and in isolated arteries ex vivo as impaired ACh-induced vasodilatation, whereas the sensitivity to exogenous NO was unchanged. Endothelial function was not significantly different in the thoracic aortic segment between GPR18 KO and WT mice, demonstrating vascular bed-specific endothelial dysfunction as a result of GPR18 deletion. A significantly reduced eNOS expression and a larger indomethacin-sensitive component in ACh-induced relaxations observed in femoral arteries derived from GPR18 KO compared with WT mice suggest that the dysfunctional eNOS-mediated femoral endothelial function may be partly compensated by increased relaxant prostanoid modulation. Finally, conscious mean arterial blood pressure recorded by telemetry was significantly higher daytime compared with wildtype mice at 24 h telemetry measures, whereas young mice did not exhibit any significant differences in day- and night-time blood pressure. These observations linking GPR18 to vascular bed-specific endothelial dysfunction and age-dependent hypertension point to beneficial pro-resolving cardiovascular effects through GPR18 during aging.

Small, gaseous molecules such as nitric oxide, carbon monoxide and hydrogen sulfide are produced as signalling molecules in mammalian cells. Here, we show that low concentrations of cyanide are generated endogenously in various mammalian tissues and cells. We detect cyanide in several cellular compartments of human cells and in various tissues and the blood of mice. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. When generated at a specific rate, cyanide exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation, but impairs cellular bioenergetics at high concentrations. Cyanide can modify cysteine residues via protein S-cyanylation, which is detectable basally in cells and mice, and increases in response to glycine. Low-dose cyanide supplementation exhibits cytoprotective effects in hypoxia and reoxygenation models in vitro and in vivo. Conversely, pathologically elevated cyanide production in nonketotic hyperglycinaemia is detrimental to cells. Our findings indicate that cyanide should be considered part of the same group of endogenous mammalian regulatory gasotransmitters as nitric oxide, carbon monoxide and hydrogen sulfide.

Small, gaseous molecules, known as gasotransmitters (NO, CO, H2S), are produced endogenously in mammalian cells and serve important biological roles. Hydrogen cyanide, traditionally considered a cytotoxic molecule in mammals, serves as an endogenous mediator in several plants and bacterial species. Here we show that low concentrations of cyanide are generated endogenously in mouse liver and human hepatocytes. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. Cyanide, in turn, is detectable in several cellular compartments. Cyanide is also detectable basally in the blood of mice; its levels increase after treatment of the animals with glycine. Rhodanese activity regulates endogenous cyanide levels. Cyanide, when generated endogenously at an optimal level, exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation. Dysregulation of endogenous cyanide, either below or above optimal levels, impairs cellular bioenergetics. The regulatory effects of cyanide are in part mediated by posttranslational modification of cysteine residues via protein cyanylation; cyanylated protein residues can be detected basally, and increase after treatment with glycine. Controlled low-dose cyanide supplementation exhibits cytoprotective effects, as demonstrated in hypoxia and reoxygenation models in vitro and in vivo . However, pathologically elevated cyanide production, as demonstrated in nonketotic hyperglycinemia – an autosomal recessive disease of glycine metabolism – is deleterious to the cells. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES:Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1.CONCLUSIONS:The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.

Although, vitamin K-2 displays vasoprotective effects, it is still not known whether K-2 treatment improves endothelial function. In ApoE/LDLR-/- mice at the stage prior to atherosclerosis development, four-week treatment with K-2-MK-7, given at a low dose (0.05 mg/kg), improved acetylcholine- and flow-induced, endothelium-dependent vasodilation in aorta or in femoral artery, as assessed by MRI in vivo. This effect was associated with an increased NO production, as evidenced by EPR measurements in ex vivo aorta. Treatment with higher doses of K-2-MK-7 (0.5; 5 mg/kg) resulted in a dose-dependent increase in plasma K-2-MK-7 and K-2-MK-4 concentration, without further improvement in endothelial function. In ApoE/LDLR-/- mice with developed atherosclerotic plaques, treatment with a low (0.03 mg/kg) or high (10 mg/kg) dose of K-2-MK-7 resulted in a similar degree of endothelium-dependent vasodilation improvement and increase in plasma nitrate concentration, what was not associated with changes in thrombin generation as measured by CAT. Both doses of K-2-MK-7 also reduced media thickness in the brachiocephalic artery, but did not modify atherosclerotic plaque size. In conclusion, K-2-MK-7 improves NO-dependent endothelial function in ApoE/LDLR-/- mice. This study, identifies the endothelial profile of the pharmacological activity of vitamin K-2, which has not been previously described.

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O2∙-) abundancein vivoby quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E+), as well as ethidium (E+) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (∙NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E+varied in the liver, lung, and kidney; however, the ratios of 2-OH-E+/E+and 2-OH-E+/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of∙NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of∙NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased∙NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E2). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O2∙-productionin vivo. Furthermore, the ratio of 2-OH-E+/E+oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O2∙--mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2− generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression.

ABSTRACT Introduction An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)–dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity. Methods At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide ( • O 2 − ) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography–based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF 1α . Results An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of • O 2 − in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF 1α concentrations. Conclusion An acute bout of strenuous exercise in mice reduced NO and in parallel increased • O 2 − production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased • O 2 − production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.

The objective of this study was to develop and validate the method based on micro-liquid chromatography-tandem mass spectrometry (microLC/MS-MRM) for simultaneous determination of adiponectin (ADN), von Willebrand factor (vWF), soluble form of vascular cell adhesion molecule 1 (sVCAM-1), soluble form of intercellular adhesion molecule 1 (sICAM-1) and syndecan-1 (SDC-1) in mouse plasma. The calibration range was established from 2.5pmol/mL to 5000pmol/mL for ADN; 5pmol/mL to 5000pmol/mL for vWF; 0.375pmol/mL to 250pmol/mL for sVCAM-1 and sICAM-1; and 0.25pmol/mL to 250pmol/mL for SDC-1. The method was applied to measure the plasma concentration of selected proteins in mice fed high-fat diet (HFD), and revealed the pro-thrombotic status by increased concentration of vWF (1.31±0.17 nmol/mL (Control) vs 1.98±0.09 nmol/mL (HFD), p <0.05) and the dysregulation of adipose tissue metabolism by decreased concentration of ADN (0.62±0.08 nmol/mL (Control) vs 0.37±0.06 nmol/mL (HFD), p <0.05). In conclusion, the microLC/MS-MRM-based method allows for reliable measurements of selected protein biomarkers of endothelial dysfunction in mouse plasma.

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11–19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

PURPOSE:To analyze the effect of liver steatosis and obesity on pharmacokinetic profile of two structurally-related liver-selective NO-donors - V-PYRRO/NO and V-PROLI/NO.METHODS:C57BL/6 mice were fed control or high-fat diet for 15 weeks to induced liver steatosis and obesity (HFD mice). Pharmacokinetics and renal elimination studies were conducted in vivo following iv dosing of V-PYRRO/NO and V-PROLI/NO (0.03mmol/kg). Hepatic clearance was evaluated ex vivo in the isolated perfused mice liver and in vitro with the use of liver microsomes.RESULTS:V-PYRRO/NO and V-PROLI/NO, despite similar structure, displayed different pharmacokinetic properties. V-PYRRO/NO was uptaken and metabolized by the liver, while V-PROLI/NO was eliminated unchanged with urine. In HFD mice, despite increased CYP450 metabolism of V-PYRRO/NO the elimination rate was slower most likely due to the impairment of hepatic microcirculation caused by liver fat accumulation. In turn, in HFD mice renal clearence of V-PROLI/NO was accelerated and volume of distribution was increased most likely due to additional intracellular water in HFD mice.CONCLUSIONS:The pharmacokinetics of V-PROLI/NO, the novel proline-based analog of V-PYRRO/NO with additional single carboxylic acid moiety, attached to the molecule of V-PYRRO/NO to improve the water solubility, was differently affected by liver steatosis and obesity as compared with the parent compound V-PYRRO/NO.

To assess alterations in perfusion and liver function in the concanavalin A (ConA)-induced mouse model of acute liver failure (ALF) using two magnetic resonance imaging (MRI)-based methods: dynamic contrast-enhanced MRI (DCE-MRI) with Gd-EOB-DTPA contrast agent and arterial spin labelling (ASL).

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.

Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis.Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular inflammation and improved endothelial function.This study highlights the importance of extracellular nucleotides and adenosine metabolism in the atherosclerotic vessel in both experimental and clinical setting. The increased eADA activity marks an early stage of atherosclerosis, contributes to its progression and could represent a novel target for therapy.

1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)–deficient mice. ApoE/LDLR−/− mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1α and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR−/− mice, an effect associated with an improvement in prostacyclin– and nitric oxide–dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.

The balance between vascular prostacyclin (PGI(2)) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A(2) (TXA(2)) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI(2) (6-keto-PGF(1 alpha), 2,3-dinor-6-keto-PGF(1 alpha)) and TXA(2) (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF(1 alpha), and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF(1 alpha), and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1to100 ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI(2) and TXA(2) generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF(1 alpha), concentration in plasma. Changes in 2,3-dinor-6-keto-PGF(1 alpha), and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI(2)/TXA(2) homeostasis than measurements in urine. (C) 2016 Elsevier B.V. All rights reserved.

V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney.

Background and purpose: There is an unmet medical need for novel NAFLD treatments. Here we have examined the effects of liver-selective NO donor (V-PYRRO/NO) as compared with metformin on hepatic steatosis and glucose tolerance in mice fed high fat diet.Material and methods: Effects of V-PYRRO/NO (5 mg kg(-1)) or metformin (616 mg kg(-1)) were examined in C57BL/6J mice fed high fat diet (HF, 60 kcal% fat). Quantitative determination of steatosis, liver fatty acid composition and western blot analysis of selected proteins involved in mitochondrial biogenesis, fatty acid de nova synthesis and oxidation, triacylglycerols and cholesterol transport from the liver were performed. Liver NO and nitrate concentration and blood biochemistry were also analyzed.Results: V-PYRRO/NO and metformin reduced liver steatosis with simultaneous reduction of total liver triacylglycerols, diacylglycerols and ceramides fraction and reversed HF-induced decrease in UFA/SFA ratio. V-PYRRO/NO substantially improved postprandial glucose tolerance, while the effect of metformin was modest and more pronounced on HOMA IR index. The anti-steatotic mechanism of V-PYRRO/NO was dependent on NO release, differed from that of metformin and involved improved glucose tolerance and inhibition of de novo fatty acid synthesis by Akt activation and ACC phosphorylation. In turn, major mechanism of metformin action involved increased expression of proteins implicated in mitochondrial biogenesis and metabolism (PGC-1 alpha, PPAR alpha, COX IV, cytochrome c, HADHSC).Conclusions: V-PYRRO/NO acts as a liver-specific NO donor prodrug affording pronounced anti-steatotic effects and may represent an efficient, mechanistically novel approach to prevent liver steatosis and insulin resistance. (C) 2015 Elsevier Inc. All rights reserved.