Synthesis, Anticonvulsant, and Antinociceptive Activity of New 3-(2-Chlorophenyl)- and 3-(3-Chlorophenyl)-2,5-dioxo-pyrrolidin-1-yl-acetamides.

The new series of 3-(2-chlorophenyl)- and 3-(3-chlorophenyl)-pyrrolidine-2,5-dione-acetamide derivatives as potential anticonvulsant and analgesic agents was synthesized. The compounds obtained were evaluated in the following acute models of epilepsy: maximal electroshock (MES), psychomotor (6 Hz, 32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The most active substance-3-(2-chlorophenyl)-1-{2-[4-(4-fluorophenyl)piperazin-1-yl]-2-oxoethyl}-pyrrolidine-2,5-dione (6) showed more beneficial ED50 and protective index values than the reference drug-valproic acid (68.30 mg/kg vs. 252.74 mg/kg in the MES test and 28.20 mg/kg vs. 130.64 mg/kg in the 6 Hz (32 mA) test, respectively). Since anticonvulsant drugs are often effective in neuropathic pain management, the antinociceptive activity for two the promising compounds-namely, 6 and 19-was also investigated in the formalin model of tonic pain. Additionally, for the aforementioned compounds, the affinity for the voltage-gated sodium and calcium channels, as well as GABAA and TRPV1 receptors, was determined. As a result, the most probable molecular mechanism of action for the most active compound 6 relies on interaction with neuronal voltage-sensitive sodium (site 2) and L-type calcium channels. Compounds 6 and 19 were also tested for their neurotoxic and hepatotoxic properties and showed no significant cytotoxic effect.

The Influence of Trehalose on Atherosclerosis and Hepatic Steatosis in Apolipoprotein E Knockout Mice.

Atherosclerosis and nonalcoholic fatty liver disease (NAFLD) are frequent causes of death in the Western countries. Recently, it has been shown that autophagy dysfunction plays an important role in the pathogenesis of both atherosclerosis and NAFLD; thus, activators of autophagy might be useful for novel therapeutic interventions. Trehalose-a naturally occuring disaccharide present in plants, bacteria, fungi, insects, and certain types of shrimps-is a known inducer of autophagy. However, according to the literature, its anti-atherosclerotic and anti-steatotic potential seem to depend on the experimental setting. The aim of our study was to comprehensively describe the influence of a prolonged treatment with orally administered trehalose on the development of atherosclerotic lesions and hepatic steatosis in apolipoprotein E knockout (apoE-/-) mice in an experimental set up reflecting both moderate and severe proatherogenic conditions: male apoE-/- mice on a chow diet (CD) and female apoE-/- mice fed with a high-fat diet (HFD). We found that exogenous trehalose inhibited atherosclerosis and attenuated hepatic steatosis in apoE-/- mice. Such effects of trehalose were not associated with changes of plasma cholesterol, low-density lipoproteins (LDL), or high-density lipoproteins (HDL). Moreover, the anti-steatotic action of trehalose in the liver was associated with the induction of autophagy. The exact molecular mechanisms of both the anti-atherosclerotic action of trehalose and its inhibitory effect on liver steatosis require further clarification.

In vitro maturation of monocyte-derived dendritic cells results in two populations of cells with different surface marker expression, independently of applied concentration of interleukin-4.

Dendritic cells (DCs) play a crucial role in the development of adaptive immune response. Monocyte-derived dendritic cells (MDDCs) are generated in vitro to study DC biology and for use in immunotherapy. However, procedures to generate MDDCs vary and an impact this may have on their final phenotype is insufficiently studied. Monocytes isolated from healthy blood donors were cultured for 7 days with granulocyte-macrophage colony stimulating factor (50 ng/mL) and low (500 IU/mL, L-IL4) or high (1000 IU/mL, H-IL4) interleukin 4 (IL4), to obtain immature DCs and for the following 2 days with addition of soluble CD40 ligand (500 ng/mL) and prostaglandin E2 (1 µg/mL) to obtain mature DCs. We measured mean fluorescence activity and percentage of cells, positive for CD14, HLA-DR, CD80, CD83, CD86, CCR7, and CD1a or CD209 markers after 7 and 9 days of culture, in both IL4 concentrations. Percentage of positively staining mature MDDCs was higher than among immature cells, for all studied markers. Interestingly, varying IL4 concentrations had negligible impact on staining of mature MDDCs. However, immature L-IL4 cultured MDDCs were less intensely stained for HLA-DR and CD209 than H-IL4 immature DCs. Flow cytometry revealed presence of 2 populations of cells (dominant P1 and less prevalent P2), when either L-IL4 or H-IL4 was used. Among mature MDDCs, population P1 had higher percentage of positively staining cells than P2, for all studied markers except CCR7. In conclusion, both concentrations of IL4 produce in vitro heterogeneous populations of mature MDDCs with similar staining for cell surface markers.

Anti-Atherosclerotic Action of Agmatine in ApoE-Knockout Mice.

Atherosclerosis is an inflammatory disease in which dysfunction of mitochondria play an important role, and disorders of lipid management intensify this process. Agmatine, an endogenous polyamine formed by decarboxylation of arginine, exerts a protective effect on mitochondria and modulates fatty acid metabolism. We investigated the effect of exogenous agmatine on the development of atherosclerosis and changes in lipid profile in apolipoprotein E knockout (apoE-/-) mice. Agmatine caused an approximate 40% decrease of atherosclerotic lesions, as estimated by en face and cross-section methods with an influence on macrophage but not on smooth muscle content in the plaques. Agmatine treatment did not changed gelatinase activity within the plaque area. What is more, the action of agmatine was associated with an increase in the number of high density lipoproteins (HDL) in blood. Real-Time PCR analysis showed that agmatine modulates liver mRNA levels of many factors involved in oxidation of fatty acid and cholesterol biosynthesis. Two-dimensional electrophoresis coupled with mass spectrometry identified 27 differentially expressed mitochondrial proteins upon agmatine treatment in the liver of apoE-/- mice, mostly proteins related to metabolism and apoptosis. In conclusion, prolonged administration of agmatine inhibits atherosclerosis in apoE-/- mice; however, the exact mechanisms linking observed changes and elevations of HDL plasma require further investigation.

Influence of atorvastatin on angiotensin I metabolism in resting and TNF-α -activated rat vascular smooth muscle cells.

INTRODUCTION: Vascular smooth muscle cells (VSMCs) are essential for maintaining vasculature homeostasis and function. By influence on its growth and activation both proinflammatory cytokines and peptides of the renin-angiotensin system (RAS) are potent regulators of VSMCs. Interestingly, angiotensin (Ang) II and Ang-(1-7) elicit opposite effects on VSMC activation, differentiation and proliferation. It has been suggested that statins, besides anti-inflammatory effects, may also modulate VSMC activation by their influence on the RAS. METHODS: The effect of atorvastatin on Ang I metabolism in a culture of explanted rat VSMCs was examined by liquid chromatography-mass spectrometry (LC-MS); expression of mRNA of the main RAS enzymes in VSMC was assessed by real-time polymerase chain reaction (PCR). RESULTS: In VSMC culture Ang-(1-7) was identified as a major product of Ang I metabolism. In this setting, TNF-α (1 ng/ml) caused a decrease in the conversion of Ang I to Ang-(1-7). This effect was accompanied by a decrease of mRNA expression of neutral endopeptidase (NEP) and angiotensin converting enzyme 2 (ACE2) and increase of mRNA of ACE. Interestingly, atorvastatin (3 µM) attenuated the effects of TNF-α on Ang-(1-7) production as well as reversed the influence of TNF-α on ACE and ACE2 expression. CONCLUSIONS: Enhancement by atorvastatin of the ACE2/Ang-(1-7) axis in VSMCs could represent a new and beneficial mechanism on cardiovascular action of this widely used drug.

Metabolism of bradykinin in aorta of hypertensive rats.

Alterations in the formation and metabolism of bradykinin (Bk) are hypothesized to play a role in the pathophysiology of hypertension, atherosclerosis and vascular complications of diabetes. However, despite its prominent role in cardiovascular regulation, studies on bradykinin have been limited by various difficulties in accurate measurements of this peptide in biological samples. In this study, using the LC-ESI-MS method we estimated the conversion of exogenous Bk to its main metabolites - Bk-(1-5) and Bk-(1-7) - in endothelial cell culture and in fragments of aorta of normotensive (WKY) and hypertensive rats (SHR). The effects of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors were more pronounced in SHR: perindoprilat inhibited Bk-(1-5) formation by 49 % and 76 % in WKY and SHR rats, respectively, and tiorphan tended to decrease formation of Bk-(1-5) in both groups of animals. The degradation of bradykinin and generation of both metabolites were significantly higher in the aorta of SHR rats than in WKY controls. Our results show that even in relatively early hypertension (in 4-month old SHR rats) inactivation of Bk by aorta wall is enhanced.

Application of magnetic resonance imaging in vivo for the assessment of the progression of systolic and diastolic dysfunction in a mouse model of dilated cardiomyopathy.

BACKGROUND: The impairment of cardiac diastolic function is essential for the development and progression of heart failure, regardless of the systolic performance of the heart. Novel methods of diagnosis of diastolic dysfunction in experimental animals are needed in order to validate the effectiveness of novel heart failure treatment. AIM: The in vivo characterisation of diastolic and systolic function of the heart during heart failure progression in Tgalphaq*44 mice using magnetic resonance imaging (MRI) and original image analysis. METHODS: Cardiac function in vivo in both Tgalphaq*44 and FVB mice was analysed using MRI at 4.7 T. Magnetic resonance imaging was performed using an ECG triggered fast gradient echo (cine-like flow compensated FLASH) sequence. For the assessment of left ventricle (LV) dynamics at least 20 images per cardiac cycle were acquired in the midventricular short-axis projection at the level of papillary muscles. End-systolic (ESA) and end-diastolic (EDA) areas were estimated from the minimum and maximum values found in the area-time plot. Fractional area change (FAC) defined as (EDA-ESA)/EDA, ejection (ER) and filling (FR) rates defined as slope of the beginning part of the systolic and diastolic limbs were calculated. In addition, heart failure progression in Tgalphaq*44 mice was assessed by morphometric parameters (ventricular weight to body weight index and wet to dry lung weight index), level of BNP mRNA expression as well as survival. RESULTS: Systolic function assessed by FAC% and ER was stable but slightly impaired up to 10 months of age in Tgalphaq*44 mice as compared to the FVB mice. After 12 months of age of the Tgalphaq*44 mice there was a progressive deterioration of systolic function (ER at 10, 12, 14 months of age were 0.0188 +/- 0.00434, 0.0140 +/- 0.00474, 0.0115 +/- 0.00469 1/ms, respectively). Diastolic function of the Tgalphaq*44 hearts was preserved or even slightly augmented between 4 and 10 months of age, then at the age of 12 months and later profoundly impaired (FR at 10, 12, 14 months of age were 0.0280 +/- 0.01031, 0.0196 +/- 0.01050, 0.0158 +/- 0.00833 1/ms, respectively). CONCLUSIONS: The MRI allows reliable in vivo assessment of the systolic and diastolic function in Tgalphaq*44 mice. In Tgalphaq*44 mice after few months of stable and compensated phase of the heart failure decompensation develops that involves impairment of both systolic and diastolic and leads to the fully symptomatic dilated cardiomyopathy. The precise molecular mechanisms of the systolic and diastolic dysfunction and their relative contribution to the heart failure progression in Tgalphaq*44 mice remain to be established.

NO and PGI(2) in coronary endothelial dysfunction in transgenic mice with dilated cardiomyopathy.

OBJECTIVE: The aim of the present work was to analyze coronary endothelial function in the transgenic mouse model of dilated cardiomyopathy (Tgalphaq*44 mice). METHODS: Coronary vasodilatation, both NO-dependent (induced by bradykinin) and PGI(2)-dependent (induced by acetylcholine), was assessed in the isolated hearts of Tgalphaq*44 and FVB mice. Cardiac function was analyzed in vivo (MRI). RESULTS: In Tgalphaq*44 mice at the age of 2-4 months cardiac function was preserved and there were no alterations in endothelial function. By contrast, in Tgalphaq*44 mice at the age of 14-16 months cardiac function was significantly impaired and NO, but not PGI(2)-dependent coronary function was altered. Interestingly, the basal level of PGI(2) in coronary circulation increased fourfold as compared to FVB mice. Cardiac O(2) (-) production increased 1.5-fold and 3-fold in Tgalphaq*44 vs. FVB mice at the age of 2-6 and 14-16 months, respectively, and was inhibited by apocynin. Interestingly, inhibition of NADPH oxidase or NOS-3 normalized augmented PGI(2) production in Tgalphaq*44 mice. There was also an increased expression of gp91phox in Tgalphaq*44 vs. FVB hearts, without evident alterations in the expression of COX-1, COX-2, NOS-3 and PGI(2)-synthase. CONCLUSIONS: In the mouse model of dilated cardiomyopathy, endothelial dysfunction in coronary circulation is present in the late but not the early stage of heart failure pathology and is characterized by a decrease in NO bioavailability and a compensatory increase in PGI(2). Both the decrease in NO activity and the increase in PGI(2) activity may result from excessive O(2) (-) production by cardiac NADPH oxidase in Tgalphaq*44 hearts.

The role of haem oxygenase-1 in the decrease of endothelial intercellular adhesion molecule-1 expression by curcumin.

Intercellular adhesion molecule-1 (ICAM-1) is involved in neutrophil transmigration across endothelium during sepsis-induced acute lung injury and anti-ICAM-1 interventions may represent new strategy of pulmonary protection. Haem oxygenase-1 (HO-1) has been demonstrated to exert anti-inflammatory actions via decrease of expression of adhesion molecules. We investigated the role of HO-1 in the action of curcumin, a naturally occurring yellow pigment isolated from plant Curcuma longa L., on ICAM-1 expression in tumour necrosis factor-alpha-stimulated EA.hy926 cells and lungs of lipopolysaccharide-treated mice. Both, in vitro and in vivo curcumin induced HO-1 and curcumin-elicited induction of HO-1 was associated with inhibition ICAM-1 expression. Moreover, curcumin significantly inhibited pulmonary sequestration of leucocytes in response to lipopolysaccharide as evidenced by decrease of myeloperoxidase activity in lung tissue. Both in vitro and in vivo effects of curcumin were reversed by an inhibitor of HO activity, chromium (III) mesoporphyrin IX chloride. We conclude that induction of HO-1, via decrease of endothelial ICAM-1, plays a pivotal role in curcumin-dependent prevention of pulmonary sequestration of neutrophils in a mouse model of endotoxaemia.

[NOS-2 induction in rat lungs after Escherichia coli lupopolisaccharide administration]. / Indukcja NOS-2 w plucach szczurów po podaniu lipopolisacharydu Escherichia coli.

Endotoxin-induced acute lung injury may be differently modulated by nitric oxide, dependently on the phase of endotoxemia as well as on the amounts and cellular source of this free radical. We investigated rapid (within 1 hr) increase of NOS-2 mRNA and activity in rat lungs following endotoxin treatment. The increase of lung NOS-2 correlated with LPS-induced pulmonary neutrophil sequestration. Sequestrated leukocytes seem to be responsible for rapid NOS-2 induction in lung tissue after LPS treatment. The pathophysiological role of this phenomenon requires further clarification.

Evaluation of Apolipoprotein M Serum Concentration as a Biomarker of HNF-1alpha MODY.

Apolipoprotein M (apoM) is a 26-kDa protein expressed mainly in the liver and kidneys. It is present predominantly in high-density lipoproteins (HDL). ApoM expression is influenced by the hepatocyte nuclear factor-1alpha (HNF-1alpha), which is a transcription factor associated with the pathogenesis of MODY. Some earlier data suggested that apoM levels were lower in the serum of HNF-1alpha MODY subjects, than in that of other diabetics and healthy controls. The aim of this study was to evaluate apoM as a biomarker for HNF-1alpha MODY. We included in this study 48 HNF-1alpha mutation carriers (40 diabetic patients and 8 subjects with normal glucose levels in the fasted state) from the Polish Nationwide Registry of MODY. In addition, we examined 55 T2DM patients and 55 apparently healthy volunteers who had normal fasting glucose levels. ApoM was measured by the sandwich dot-blot technique with recombinant apoM (Abnova) as a protein standard, mouse anti-human apoM monoclonal primary antibody and rat anti-mouse HRP-conjugated secondary antibody (BD Biosciences). Mean apoM level in the MODY group was 13.6 mug/ml, SD 1.9 (13.5 mug/ml, SD 1.7 in diabetic subjects and 13.9 mug/ml, SD 2.0 in non-diabetic mutation carriers respectively). In the T2DM group, mean apoM level was 13.7 mug/ml, SD 2.1, while it reached 13.8 mug/ml, SD 2.0 in healthy controls. There was no difference between apoM serum concentrations in all the study groups. In summary, our study showed no association between HNF-1alpha mutations resulting in MODY phenotype and apoM levels. Thus, we cannot confirm the clinical usefulness of apoM as a biomarker of HNF-1alpha MODY.

Compensation of endothelium-dependent responses in coronary circulation of eNOS-deficient mice.

Nitric oxide plays a fundamental role in the regulation of blood flow. Here we analyzed compensatory mechanisms for the genetic eNOS deficiency in aorta and in coronary circulation. Vasodilation induced by acetylcholine, bradykinin, adenosine, and ADP as well as by S-nitroso-penicillamine (SNAP) was assessed in isolated aorta and in isolated mouse hearts from eNOS-/- and age-matched eNOS+/+ mice. In aorta from eNOS+/+ mice acetylcholine-induced vasodilation was entirely dependent on NO, and this response was absent in aorta from eNOS-/- mice. In eNOS+/+ mouse hearts responses induced by bradykinin, adenosine and ADP were partially dependent on NO, but not on PGI2, cytochrome P450-dependent metabolites, or H2O2. On the other hand, vasodilation induced by acetylcholine involved NO, but not PGI2, in its immediate, short-lasting phase, whereas PGI2 and NO mediated delayed, longer-lasting phase of this response. In eNOS-/- mouse hearts coronary vasodilator function was compensated. Responses induced by acetylcholine and adenosine, but not by bradykinin or ADP, were in part compensated by NO, most likely derived from nNOS. However, the major mechanisms compensating for the loss of eNOS in the coronary circulation did not rely on NO, PGI2, cytochrome P450-derived metabolites of arachidonic acid or on H2O2. Deficiency of eNOS is largely compensated in coronary circulation but not in aorta.