Publications by authors named "Ryszard Korbut"

45 Publications

Anti-atherosclerotic action of GW9508 - Free fatty acid receptors activator - In apoE-knockout mice.

Pharmacol Rep 2019 Aug 21;71(4):551-555. Epub 2019 Feb 21.

Chair of Pharmacology, Jagiellonian University Medical College, Kraków, Poland.

Background: In the past two decades, enhanced understanding of the biology of G-protein-coupled receptors (GPRs) has led to the identification of several such receptors as novel targets for free fatty acids (FFAs). Two GPRs, FFAR1 and FFAR4, have received special attention in the context of chronic inflammatory diseases, thanks to their anti-inflammatory activities.

Methods: The present study investigates the influence of prolonged treatment with GW9508 - agonist of FFAR1 and FFAR4 - on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods.

Results: GW9508 administration has led to the reduction of atheroscletoric plaque size in an apoE-knockout mice model. Moreover, a FFAR1/FFAR4 agonist reduced the content of macrophages by almost 20%, attributed by immunohistochemical phenotyping to the pro-inflammatory M1-like activation state macrophages.

Conclusions: Prolonged administration of GW9508 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR1/FFAR4 receptors holds promise for a new approach to the prevention or treatment of atherosclerosis.
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http://dx.doi.org/10.1016/j.pharep.2019.02.014DOI Listing
August 2019

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

Int J Mol Sci 2019 Mar 28;20(7). Epub 2019 Mar 28.

Chair of Pharmacology, Jagiellonian University Medical College, 31-531 Krakow, Poland.

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.
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http://dx.doi.org/10.3390/ijms20071552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479548PMC
March 2019

Vasodilatory Efficacy and Impact of Papaverine on Endothelium in Radial Artery Predilatation for CABG Surgery: in Search for Optimal Concentration.

Braz J Cardiovasc Surg 2018 Nov-Dec;33(6):553-558

Department of Cardiovascular Surgery and Transplantology, John Paul II Hospital, Jagiellonian University Medical College, Krakow, Poland.

Objective: The aim of this study was to compare the efficacy of two different papaverine concentrations (0.5 mg/ml and 2 mg/ml) for vasospasm prevention and their impact on endothelium integrity.

Methods: We have studied distal segments of radial arteries obtained by no-touch technique from coronary artery bypass graft (CABG) patients (n=10). The vasodilatory effect of papaverine (concentrations of 0.5 mg/ml and 2 mg/ml) was assessed in vitro, in isometric tension studies using ex vivo myography (organ bath technique) and arterial rings precontracted with potassium chloride (KCl) and phenylephrine. The impact of papaverine on endothelial integrity was studied by measurement of the percentage of vessel's circumference revealing CD34 endothelial marker.

Results: 2 mg/ml papaverine concentration showed stronger vasodilatatory effect than 0.5 mg/ml, but it caused significantly higher endothelial damage. Response to KCl was 7.35±3.33 mN for vessels protected with papaverine 0.5 mg/ml and 2.66±1.96 mN when papaverine in concentration of 2 mg/ml was used. The histological examination revealed a significant difference in the presence of undamaged endothelium between vessels incubated in papaverine 0.5 mg/ml (72.86±9.3%) and 2 mg/ml (50.23±13.42%), P=0.002.

Conclusion: Papaverine 2 mg/ml caused the higher endothelial damage. Concentration of 0.5 mg/ml caused better preservation of the endothelial lining.
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http://dx.doi.org/10.21470/1678-9741-2018-0139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326436PMC
March 2019

Differences in plasma fibrin clot composition in patients with thrombotic antiphospholipid syndrome compared with venous thromboembolism.

Sci Rep 2018 11 23;8(1):17301. Epub 2018 Nov 23.

Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland.

The prothrombotic fibrin clot phenotype has been reported in patients with thrombotic antiphospholipid syndrome (APS) and venous thromboembolism (VTE). Protein composition of plasma fibrin clots in APS has not been studied. We evaluated 23 patients with thrombotic APS, 19 with VTE alone, and 20 well-matched controls. A proteomic analysis of fibrin clots generated from citrated plasma was based on liquid chromatography-mass spectrometry. Plasma levels of thrombospondin-1 (TSP1), apolipoprotein(a), A-I, and B-100, complement components (C)3a, C5b-C9, histidine-rich glycoprotein (HRG), and prothrombin were evaluated using immunoenzymatic tests. In plasma fibrin clots of APS patients, compared with VTE subjects and controls, we identified decreased amounts of (pro)thrombin, antithrombin-III, apolipoprotein A-I, and HRG with no differences in plasma levels of antithrombin, prothrombin, along with lower plasma HRG and apolipoprotein A-I. In APS patients, plasma HRG positively correlated with amounts of clot-bound HRG, while apolipoprotein A-I was inversely associated with clot-bound levels of this protein. The most predominant proteins within the clots of APS patients were bone marrow proteoglycan, C5-C9, immunoglobulins, apolipoprotein B-100, platelet-derived proteins, and TSP1. Our study is the first to demonstrate differences in the protein composition of fibrin clots generated from plasma of thrombotic APS patients versus those with VTE alone.
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http://dx.doi.org/10.1038/s41598-018-35034-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251889PMC
November 2018

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.

Int Immunopharmacol 2018 Apr 28;57:165-171. Epub 2018 Feb 28.

Jagiellonian University Medical College, Faculty of Medicine, Department of Pharmacology, Ul. Grzegórzecka 16, 31-531 Kraków, Poland.

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 E (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.
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http://dx.doi.org/10.1016/j.intimp.2018.02.015DOI Listing
April 2018

Quantitative proteomics reveals decreased expression of major urinary proteins in the liver of apoE/eNOS-DKO mice.

Clin Exp Pharmacol Physiol 2018 07 23;45(7):711-719. Epub 2018 Mar 23.

Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays an important role, not only in endothelium-dependent vasodilation but also in lipid and glucose homeostasis in the liver and exerts beneficial effects on mitochondrial biogenesis and respiration. Thus, the aim of our study was to use iTRAQ-based quantitative proteomics to investigate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of the double (apolipoprotein E (apoE) and eNOS) knockout (apoE/eNOS-DKO) mice as compared to apoE KO mice (apoE ) - an animal model of atherosclerosis and hepatic steatosis. Collectively, the deficiency of eNOS resulted in increased expression of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the decreased expression of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, one of the most downregulated proteins were major urinary proteins (MUPs), which are abundantly expressed in the liver and were shown to be involved in the regulation of lipid and glucose metabolism. The exact functional consequences of the revealed alterations require further investigation.
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http://dx.doi.org/10.1111/1440-1681.12927DOI Listing
July 2018

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

Clin Proteomics 2017 28;14:38. Epub 2017 Nov 28.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried, 82152 Planegg, Germany.

Background: It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot.

Methods: The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results.

Results: Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS.

Conclusions: The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.
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http://dx.doi.org/10.1186/s12014-017-9173-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706328PMC
November 2017

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

Int J Mol Sci 2017 Aug 4;18(8). Epub 2017 Aug 4.

Department of Pharmacology, Jagiellonian University Medical College, 31-531 Krakow, Poland.

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.
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http://dx.doi.org/10.3390/ijms18081706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578096PMC
August 2017

The influence of AICAR - direct activator of AMP-activated protein kinase (AMPK) - on liver proteome in apoE-knockout mice.

Eur J Pharm Sci 2017 Jun 26;104:406-416. Epub 2017 Apr 26.

Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland.

There is a growing body of evidence that altered functioning of apoE may aggravate cellular energy homeostasis and stress response, leading to oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and inflammation, leading to hypercholesterolemia, dyslipidemia, liver steatosis and neurodegeneration. One of the key cellular responses to mitochondria and ER-stress related processes and cellular energy imbalance is AMP-activated protein kinase (AMPK), considered as a cellular master energy sensor and critical regulator of mitochondrial homeostasis. The aim of our study was to use differential proteomics and transcriptomics approach to elucidate the effect of direct AMPK activator AICAR on liver proteome in apoE mice - experimental model of atherosclerosis and moderate nonalcoholic steatosis. We applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling and two-dimensional chromatography coupled with mass spectrometry (2DLC-MS/MS) MudPIT strategy, as well as RT-PCR to investigate the changes in mitochondrial and cytosolic proteins and transcripts expression in 6-month old AICAR-treated apoE. AICAR elicited induction of proteins related to mitochondrial β-oxidation, protein degradation and energy producing pathways (i.a. tricarboxylic acid cycle members and mitochondrial adenylate kinase 2). On the other hand, AICAR repressed inflammatory and pro-apoptotic markers in the apoE mice liver, alongside reduction in several peroxisomal proteins, possibly suggesting induction of anti-oxidative pexophagy.
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http://dx.doi.org/10.1016/j.ejps.2017.04.021DOI Listing
June 2017

Recent Advances in the Gastric Mucosal Protection Against Stress-induced Gastric Lesions. Importance of Renin-angiotensin Vasoactive Metabolites, Gaseous Mediators and Appetite Peptides.

Curr Pharm Des 2017 ;23(27):3910-3922

Department of Pharmacology Jagiellonian University Medical College, Cracow. Poland.

Stress is known to cause severe adverse effects in the human gastrointestinal tract including mucosal microbleedings and erosions or even gastric ulceration but the mechanism of these complications has not been fully elucidated. The pathogenesis of stress-induced gastric damage involves the fall in Gastric Blood Flow (GBF), an increase in gastric acid secretion and gastric motility, enhanced adrenergic and cholinergic nerve activity and the rise in gastric mucosal generation of reactive oxygen species. The gastric mucosal defense mechanisms against the deleterious effect of stress include the activation of the hypothalamic-pituitary-adrenal axis which has been linked with glucocorticoids release capable of counteracting of stress-induced gastric lesions. Here we summarize the novel gastroprotective mechanisms against stress damage exhibited by angiotensin-(1-7), the newly discovered metabolite of Renin-Angiotensin System (RAS), the gaseous mediators such as nitric oxide (NO), hydrogen sulfide (H2S) or Carbon Monoxide (CO), and the food intake controlling peptides ghrelin, nesfatin- 1 and apelin possibly acting via brain-gut axis. These bioactive molecules such as RAS vasoactive metabolite angiotensin-(1-7) and appetite peptides have been shown to afford gastroprotective effect against stressinduced gastric lesions mainly mediated by an increase in gastric microcirculation. Gaseous mediators protect the gastric mucosa against stress lesions by mechanism involving the activation of PG/COX and CO/HO-1 biosynthetic pathways, and their anti-inflammatory and anti-oxidizing properties. Thus, these new components add new mechanistic aspects to the common cooperation of NO/NO-synthase, PG/COX systems and vasoactive sensory neuropeptides including CGRP but their gastroprotective efficacy against experimental stress ulcerogenesis requires the confirmation in human clinical trials.
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http://dx.doi.org/10.2174/1381612823666170220160222DOI Listing
July 2018

Proteomic Analysis of Mitochondria-Enriched Fraction Isolated from the Frontal Cortex and Hippocampus of Apolipoprotein E Knockout Mice Treated with Alda-1, an Activator of Mitochondrial Aldehyde Dehydrogenase (ALDH2).

Int J Mol Sci 2017 Feb 17;18(2). Epub 2017 Feb 17.

Chair of Pharmacology, Jagiellonian University Medical College, 31-531 Krakow, Poland.

The role of different genotypes of apolipoprotein E (apoE) in the etiology of Alzheimer's disease is widely recognized. It has been shown that altered functioning of apoE may promote 4-hydroxynonenal modification of mitochondrial proteins, which may result in mitochondrial dysfunction, aggravation of oxidative stress, and neurodegeneration. Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme considered to perform protective function in mitochondria by the detoxification of the end products of lipid peroxidation, such as 4-hydroxynonenal and other reactive aldehydes. The goal of our study was to apply a differential proteomics approach in concert with molecular and morphological techniques to elucidate the changes in the frontal cortex and hippocampus of apolipoprotein E knockout (apoE) mice upon treatment with Alda-1-a small molecular weight activator of ALDH2. Despite the lack of significant morphological changes in the brain of apoE mice as compared to age-matched wild type animals, the proteomic and molecular approach revealed many changes in the expression of genes and proteins, indicating the impairment of energy metabolism, neuroplasticity, and neurogenesis in brains of apoE mice. Importantly, prolonged treatment of apoE mice with Alda-1 led to the beneficial changes in the expression of genes and proteins related to neuroplasticity and mitochondrial function. The pattern of alterations implies mitoprotective action of Alda-1, however, the accurate functional consequences of the revealed changes require further research.
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http://dx.doi.org/10.3390/ijms18020435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343969PMC
February 2017

Influence of metformin on mitochondrial subproteome in the brain of apoE knockout mice.

Eur J Pharmacol 2016 Feb 19;772:99-107. Epub 2015 Dec 19.

Chair of Pharmacology, Jagiellonian University Medical College, 16 Grzegorzecka Street, 31 531 Krakow, Poland.

Neurodegenerative diseases are the set of progressive, age-related brain disorders, characterized by an excessive accumulation of mutant proteins in the certain regions of the brain. Such changes, collectively identified as causal factors of neurodegeneration, all impact mitochondria, imminently leading to their dysfunction. These observations predestine mitochondria as an attractive drug target for counteracting degenerative brain damage. The aim of this study was to use a differential proteomic approach to comprehensively assess the changes in mitochondrial protein expression in the brain of apoE-knockout mice (apoE(-/-)) and to investigate the influence of prolonged treatment with metformin - an indirect activator of AMP-activated protein kinase (AMPK) on the brain mitoproteome in apoE(-/-) mice. The quantitative assessment of the brain mitoproteome in apoE(-/-) revealed the changes in 10 proteins expression as compared to healthy C57BL/6J mice and 25 proteins expression in metformin-treated apoE(-/-) mice. Identified proteins mainly included apoptosis regulators, metabolic enzymes and structural proteins. In summary, our study provided proteomic characteristics suggesting the decrease of antioxidant defense and structural disturbances in the brain mitochondria of apoE(-/-) mice as compared to healthy controls. In this setting, the use of metformin changed the expression of several proteins primarily involved in metabolic processes, the regulation of apoptosis and the structural maintenance of mitochondria, what could potentially restore their native functionalities.
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http://dx.doi.org/10.1016/j.ejphar.2015.12.036DOI Listing
February 2016

The impact of mitochondrial aldehyde dehydrogenase (ALDH2) activation by Alda-1 on the behavioral and biochemical disturbances in animal model of depression.

Brain Behav Immun 2016 Jan 4;51:144-153. Epub 2015 Aug 4.

Jagiellonian University Medical College, 31-531 Krakow, Poland.

The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation.
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http://dx.doi.org/10.1016/j.bbi.2015.08.004DOI Listing
January 2016

Shotgun analysis of plasma fibrin clot-bound proteins in patients with acute myocardial infarction.

Thromb Res 2015 Apr 10;135(4):754-9. Epub 2015 Feb 10.

Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland. Electronic address:

Introduction: The presence and amount of the proteins within a plasma clot may influence clot properties, like susceptibility to fibrinolysis, however, the clot proteome has not yet been extensively described. The aim of the study was to investigate the protein composition of clots of four patients with acute myocardial infarction (AMI) in two time points: in the acute ischemic phase and two months later during the standard therapy.

Materials And Methods: Shotgun proteomic method (2DLC-MS/MS) was used to investigate time-dependent protein composition changes of clots prepared ex vivo from citrated plasma of the peripheral blood of patients with AMI.

Results: Proteomic analysis revealed a total number of 62 proteins identified in all 8 samples grouping into several distinct functional clusters (e.g. cholesterol transporter activity, immunoglobulin binding and peptidase regulatory activity). The protein signatures of clots differed significantly depending on time after ACS, showing 30% greater variability in protein composition of the clots prepared in the plasma two months after the onset of AMI. Several proteins potentially involved in clot formation and resolution showed an interesting pattern of changes over time.

Conclusion: We provided the first qualitative analysis of proteomes of fibrin clots generated ex vivo in plasma taken from patients with AMI showing differences between clots generated in the acute ischemic phase and those prepared two months later. It might be hypothesized that differences involving proteins of potential influence on within-clot fibrinolysis and clot stability may partially explain time-dependent changes in the clots structure and firmness in patients with AMI.
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http://dx.doi.org/10.1016/j.thromres.2015.02.005DOI Listing
April 2015

Polymorphism rs7023923 and monocyte count in blood donors and coronary artery disease patients.

Kardiol Pol 2015 7;73(6):445-50. Epub 2015 Jan 7.

Uniwersytet Jagielloński Collegium Medicum.

Background: The importance of the role of monocytes in coronary artery disease (CAD) is well documented. An increased number of circulating monocytes is associated with higher incidence of CAD. Both environmental and genetic factors influence monocytosis. The latter have been extensively studied since the development of high-throughput genome-wide association studies. Several associations between polymorphisms and monocytosis were found among healthy individuals; the first example was rs7023923. The magnitude of the association of studied polymorphisms with the trait of interest is often confounded by environmental factors and may therefore differ between patient and healthy populations. It is very important to determine the magnitude of the association among patients to predict outcome of the disease, e.g. myocardial infarction.

Aim: To determine whether the magnitude of association of rs7023923 with monocytosis, previously reported among healthy volunteers, is similar in patients in whom diagnosis of CAD was determined during elective coronarography.

Methods And Results: Leucocytosis and neutrophilocytosis were higher among patients with CAD, while thrombocytosis was lower. Monocyte count did not differ among the studied groups (p = 0.25). We confirmed the association of rs7023923 with monocytosis among healthy blood donors (p = 0.0156) but not among patients admitted for elective coronarography (p = 0.61). Inclusion of the age and sex of patients in the statistical model did not modify the results.

Conclusions: Our data suggest that translation of the results of genetic association with the studied traits from healthy to patient population should be implemented with caution. It is possible that numerous environmental factors, which discriminate healthy volunteers from CAD patients, confound the magnitude of genetic associations and make interpretation of the data in patients less clear.
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http://dx.doi.org/10.5603/KP.a2014.0238DOI Listing
February 2017

Mitochondrial aldehyde dehydrogenase activation by Alda-1 inhibits atherosclerosis and attenuates hepatic steatosis in apolipoprotein E-knockout mice.

J Am Heart Assoc 2014 Nov 12;3(6):e001329. Epub 2014 Nov 12.

Department of Pharmacology, Jagiellonian University Medical College, Krakow, Poland (A.S., R.O., M.S., A.W., J.T., M., J.J., R.K.).

Background: Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE(-/-)) mice.

Methods And Results: Alda-1 caused decrease of atherosclerotic lesions approximately 25% as estimated by "en face" and "cross-section" methods without influence on plasma lipid profile, atherosclerosis-related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda-1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda-1-treated apoE(-/-) mice. Alda-1 attenuated formation of 4-hydroxy-2-nonenal (4-HNE) protein adducts and decreased triglyceride content in liver tissue. Two-dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda-1 treatment in liver of apoE(-/-) mice, mostly proteins related to metabolism and oxidative stress. The most up-regulated were the proteins that participated in beta oxidation of fatty acids.

Conclusions: Collectively, Alda-1 inhibited atherosclerosis and attenuated NAFLD in apoE(-/-) mice. The pattern of changes suggests a beneficial effect of Alda-1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda-1 action require further investigation.
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http://dx.doi.org/10.1161/JAHA.114.001329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338726PMC
November 2014

GTP cyclohydrolase I gene polymorphisms are associated with endothelial dysfunction and oxidative stress in patients with type 2 diabetes mellitus.

PLoS One 2014 4;9(11):e108587. Epub 2014 Nov 4.

Department of Internal and Agricultural Medicine, Jagiellonian University Medical College, Krakow, Poland; Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom.

Background: The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients.

Methods: Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA).

Results: Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables.

Conclusions: Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108587PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219671PMC
July 2015

Inactivation of heparin by cationically modified chitosan.

Mar Drugs 2014 Jun 30;12(7):3953-69. Epub 2014 Jun 30.

Chair of Pharmacology, Jagiellonian University Medical College, 16 Grzegórzecka Str., Cracow 31-531, Poland.

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3-tri methylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.
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http://dx.doi.org/10.3390/md12073953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113808PMC
June 2014

Role of angiotensin-(1-7) in gastroprotection against stress-induced ulcerogenesis. The involvement of mas receptor, nitric oxide, prostaglandins, and sensory neuropeptides.

J Pharmacol Exp Ther 2013 Dec 18;347(3):717-26. Epub 2013 Sep 18.

Department of Physiology (M.M., K.J., M.P., G.K-M., A.P-B., S.K., T.B.) and Department of Pharmacology (R.O., R.K.), Jagiellonian University Medical College, Cracow, Poland.

Angiotensin-(1-7) [Ang-(1-7)] is a major vasoactive metabolite of angiotensin I (Ang I), both being important components of the renin-angiotensin system (RAS). Ang-(1-7) acting via Mas receptor was documented in kidneys, heart, brain, and gastrointestinal (GI)-tract. We studied the gastroprotective activity of exogenous Ang-(1-7) in rats exposed to water immersion and restraint stress (WRS) without or with A-779 [d-Ala7-Ang-(1-7), an antagonist of Ang-(1-7) Mas receptors], AVE 0991 (5-formyl-4-methoxy-2-phenyl-1[[4-[2-(ethylaminocarbonylsulfonamido)-5-isobutyl-3-thienyl]-phenyl]-methyl]-imidazole), the agonist of Ang-(1-7) receptor, as well as the inhibition of nitric-oxide (NO) synthase, the suppression of cyclo-oxygenase (COX)-1 (indomethacin, SC-560 [5-(4-chloro-phenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl-pyrazole]), the activity COX-2 (rofecoxib), and denervation with capsaicin. The mRNA expression of constitutively expressed nitric-oxide synthase (cNOS), inducible nitric-oxide synthase (iNOS), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α was analyzed by reverse transcription polymerase chain reaction. The WRS lesions were dose-dependently reduced by pretreatment with Ang-(1-7), which also caused an increase in gastric blood flow (GBF) and luminal content of NO. COX-1 and COX-2 inhibitors or L-NNA (N5-[imino(nitroamino)methyl]-L-ornithine) reversed the reduction in lesion number and the rise in GBF evoked by Ang-(1-7). Ang II augmented the WRS lesions, decreased GBF and increased the plasma IL-1β and TNF-α levels. Capsaicin denervation attenuated the reduction of Ang-(1-7)-induced gastric lesions and the rise in GBF; these effects were restored by supplementation with calcitonin gene-related peptide (CGRP). The cNOS mRNA was upregulated while iNOS, IL-1β and TNF-α mRNAs were downregulated in Ang-(1-7)-pretreated rats. We conclude that Ang-(1-7), in contrast to Ang II, which worsened WRS ulcerogenesis, affords potent gastroprotection against WRS ulcerogenesis via an increase in GBF mediated by NO, endogenous prostaglandins, sensory neuropeptides, and anti-inflammatory action involving the inhibition of proinflammatory markers iNOS, IL-1β, and TNF-α.
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http://dx.doi.org/10.1124/jpet.113.207233DOI Listing
December 2013

The influence of angiotensin-(1-7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice.

Biochim Biophys Acta 2013 Dec 27;1834(12):2463-9. Epub 2013 Aug 27.

Chair of Pharmacology, Jagiellonian University Medical College, 16 Grzegorzecka Str, 31 531, Krakow, Poland.

Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1-7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1-7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE(-/-) mice). Proteins changed in apoE(-/-) mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE(-/-) mice.
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http://dx.doi.org/10.1016/j.bbapap.2013.08.008DOI Listing
December 2013

Mechanisms of oxidative stress in human aortic aneurysms--association with clinical risk factors for atherosclerosis and disease severity.

Int J Cardiol 2013 Oct 15;168(3):2389-96. Epub 2013 Mar 15.

Translational Medicine Laboratory, Department of Internal and Agricultural Medicine, Jagiellonian University School of Medicine, Cracow, Poland; Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University School of Medicine, Cracow, Poland.

Unlabelled: Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2(-)) production in human AAA.

Methods And Results: AAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n=16) than in risk factor matched controls (n=16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2(-) in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size.

Conclusions: Increased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.
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http://dx.doi.org/10.1016/j.ijcard.2013.01.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819986PMC
October 2013

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

J Renin Angiotensin Aldosterone Syst 2014 Dec 6;15(4):378-83. Epub 2013 Feb 6.

School of Medicine, Jagiellonian University, Poland.

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.
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http://dx.doi.org/10.1177/1470320313475907DOI Listing
December 2014

Assessment of angiotensin I metabolism in the human placenta using an LC/MS method.

Folia Med Cracov 2013 ;53(1):31-9

Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland.

The local renin-angiotensin system (RAS) in the placenta plays a very important role in placental development. It is well known that during normal pregnancy most of the circulating and local RAS components are over-expressed and any disruption of this new balance may cause pregnancy complications. The aim of this study was to assess the metabolism of Ang I in placentas from normal pregnancy, in an ex vivo model, using an LC/MS method. The obtained results suggest that placental tissue is able to produce many angiotensin peptides but the main metabolite is Ang-(1-7).
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July 2014

[Endothelial dysfunction related to oxidative stress and inflammation in perivascular adipose tissue].

Postepy Biochem 2012 ;58(2):186-94

Laboratorium Medycyny Translacyjnej, Katedra i Klinika Chorób Wewnetrznych i Medycyny Wsi UJ, CM, Katedra Farmakologii UJ CM, Kraków.

Endothelial dysfunction plays an important role in the pathogenesis of many common diseases, like atherosclerosis and hypertension. The key role of the interaction between oxidative stress and inflammation in causal mechanisms of these diseases is widely accepted. Until recently, perivascular adipose tissue was not taken into account while looking at mechanisms of these disorders. However, it has recently been demonstrated that most processes involved in endothelial dysfunction development are taking place in this tissue. Adipocytes are an important source of free radicals and pro-inflammatory cytokines. These molecules lead to further enhancement of oxidative stress, through uncoupling of endothelial nitric oxide synthase (eNOS) and production of peroxynitrite radical instead of nitric oxide which further disrupts eNOS function. In addition, macrophages and T lymphocytes infiltrate adipose tissue as a result of chemotactic proteins release, upon oxidative stress activation, which further enhances inflammation. Thus, the chronic inflammation, which develops in this compartment of adipose tissue in patients with obesity, is the first step in the development of atherosclerotic plaque or hypertension. That is why comprehensive understanding of ongoing processes within perivascular adipocytes is so important.
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January 2013

The effect of AVE 0991, nebivolol and doxycycline on inflammatory mediators in an apoE-knockout mouse model of atherosclerosis.

Med Sci Monit 2012 Oct;18(10):BR389-93

Jagiellonian University School of Medicine, Cracow, Poland.

Background: The aim of this study was to investigate whether the 3 different substances that can decrease the development of atherosclerosis--nebivolol, AVE 0991 and doxycycline--could at the same time diminish the level of inflammatory indicators interleukin-6 (IL-6), interleukin-12 (IL-12), serum amyloid A (SAA), and monocyte chemotactic protein-1 (MCP-1).

Material/methods: Forty 8-week-old female apoE-knockout mice on the C57BL/6J background were divided into 4 groups and put on chow diet for 4 months. Three experimental groups received the same diet as a control group, mixed with AVE 0991 at a dose 0.58 µmol per kg of body weight per day, nebivolol at a dose 2.0 µmol per kg of body weight per day, and doxycycline at a dose 1.5 mg per kg of body weight per day. At the age of 6 months, the mice were sacrificed.

Results: All inflammatory indicators (MCP-1, IL-6, IL-12 and SAA) were diminished by AVE 0991. There was also a tendency to lower MCP-1, IL-6, IL-12 and SAA levels by nebivolol and doxycycline; however, it did not reach statistical significance.

Conclusions: Of the 3 presented substances, only AVE 0991 was able to diminish the rise of inflammatory markers. Therefore, drug manipulations in the renin-angiotensin-aldosterone axis seem to be the most promising in the future treatment of atherogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560549PMC
http://dx.doi.org/10.12659/msm.883478DOI Listing
October 2012

Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin.

J Proteomics 2012 Dec 30;77:167-75. Epub 2012 Aug 30.

Chair of Pharmacology, Jagiellonian University Medical College, Poland.

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. Metformin, a widely known anti-diabetic drug, used for patients with type 2 diabetes mellitus, is also claimed to be useful in treatment of NAFLD. However, both the clinical efficacy and the putative mechanisms underlying the clinical effects of metformin in treating NAFLD are unclear. Adenosine monophosphate-activated protein kinase (AMPK), the primary molecular target for metformin, is a known regulator of mitochondrial function. Thus, we used a proteomic approach to investigate the effect of metformin on liver mitochondria of apolipoprotein E knockout (apoE(-/-)) mice, an animal model of NAFLD. Two-dimensional electrophoresis coupled with mass spectrometry was applied to study the changes in liver mitochondrial protein expression in 6-month old metformin-treated apoE(-/-) mice as compared to non-treated animals. Collectively, 25 differentially expressed proteins were indentified upon metformin treatment including proteins related to metabolism, oxidative stress and cellular respiration. The most up-regulated protein was glycine N-methyltransferase (GNMT) - an enzyme, whose deficiency was shown to be directly related to the development of NAFLD. Our results clearly point to the strong mitochondrial action of metformin in NAFLD. Up-regulation of GNMT may represent an important mechanism of beneficial action of metformin in NAFLD treatment.
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http://dx.doi.org/10.1016/j.jprot.2012.08.015DOI Listing
December 2012

Local inflammation is associated with aortic thrombus formation in abdominal aortic aneurysms. Relationship to clinical risk factors.

Thromb Haemost 2012 Nov 5;108(5):812-23. Epub 2012 Sep 5.

Translational Medicine Laboratory, Department of Internal and Agricultural Medicine, Jagiellonian University School of Medicine, J Dietl Hospital, Ul Skarbowa 1, 31-121 Cracow, Poland.

Intraluminal thrombus formation in aortic abdominal aneurysms (AAA) is associated with adverse clinical prognosis. Interplay between coagulation and inflammation, characterised by leukocyte infiltration and cytokine production, has been implicated in AAA thrombus formation. We studied leukocyte (CD45+) content by flow cytometry in AAA thrombi from 27 patients undergoing surgical repair. Luminal parts of thrombi were leukocyte-rich, while abluminal segments showed low leukocyte content. CD66b+ granulocytes were the most prevalent, but their content was similar to blood. Monocytes (CD14+) and T cells (CD3+) were also abundant, while content of B lymphocytes (CD19+) and NK cells (CD56+CD16+) were low. Thrombi showed comparable content of CD14highCD16- monocytes and lower CD14highCD16+ and CD14dimCD16+, than blood. Monocytes were activated with high CD11b, CD11c and HLA-DR expression. Total T cell content was decreased in AAA thrombus compared to peripheral blood but CD8 and CD3+CD4-CD8- (double negative T cell) contents were increased in thrombi. CD4+ cells were lower but highly activated (high CD69, CD25 and HLA-DR). No differences in T regulatory (CD4+CD25+FoxP3+) cell or pro-atherogenic CD4+CD28null lymphocyte content were observed between thrombi and blood. Thrombus T cells expressed high levels of CCR5 receptor for chemokine RANTES, commonly released from activated platelets. Leukocyte or T cell content in thrombi was not correlated with aneurysm size. However, CD3+ content was significantly associated with smoking in multivariate analysis taking into account major risk factors for atherosclerosis. In conclusion, intraluminal AAA thrombi are highly inflamed, predominantly with granulocytes, CD14highCD16- monocytes and activated T lymphocytes. Smoking is associated with T cell infiltration in AAA intraluminal thrombi.
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http://dx.doi.org/10.1160/TH12-05-0339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281161PMC
November 2012

Beneficial effect of amantadine on postoperative pain reduction and consumption of morphine in patients subjected to elective spine surgery.

Pain Med 2012 Mar 7;13(3):459-65. Epub 2012 Feb 7.

Jagiellonian University Medical College, Krakow, Poland.

Objective: To analyze the effect of coadministration of morphine and amantadine on postoperative pain reduction and morphine consumption in patients after elective spine surgery.

Methods: In double-blinded study, 60 patients (ASA physical status I-II) were randomized into two groups. Group A was given oral amantadine 50 or 100 mg 1 hour before surgery and 8, 20, 32 hours after operation. Group P received placebo at identical times. Pain was assessed using numerical rating scale before first administration of morphine and in 2, 3, 4, 6, 24, and 48 hours after operation. The amounts of morphine consumed were recorded up to 48 hours after surgery. Blood samples were taken twice in 2 hours after surgery and plasma levels of morphine and its main metabolites were measured.

Results: As compared with placebo, amantadine significantly reduced intra-operative Fentanyl use and sensation of postoperative pain. Up to 48 hours after operation, the cumulative consumption of morphine was 25% lower in the amantadine group. Moreover, intensity of nausea and vomiting tended to be lower in A group. Starting from 12th hour after surgery, the level of postoperative sedation was lower in patients who received amantadine, as compared with placebo group. No significant differences in plasma levels of morphine ant its metabolites were observed between A and P groups.

Conclusions: Pre- and postoperative administration of amantadine significantly reduced fentanyl use during operation, as well as reduced the postoperative pain and decreased morphine consumption in young patients undergoing orthopedic surgery.
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http://dx.doi.org/10.1111/j.1526-4637.2011.01321.xDOI Listing
March 2012

Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists.

J Med Chem 2011 Oct 6;54(19):6586-96. Epub 2011 Sep 6.

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland.

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 μm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.
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http://dx.doi.org/10.1021/jm200380wDOI Listing
October 2011

Mechanisms of increased vascular superoxide production in human varicose veins.

Pol Arch Med Wewn 2011 Sep;121(9):279-86

Department of Internal and Agricultural Medicine, Jagiellonian University, Medical College, Kraków, Poland.

Introduction: Varicose vein disease is one of the most common morbidities in the developed countries. Recent studies have shown that oxidative stress is increased in varicose veins (VV) and venous insufficiency. However, the exact mechanisms of oxidative stress in VV remain unknown.

Objectives: The aim of the study was to measure superoxide anion production and analyze its enzymatic sources in VV in comparison with control human saphenous veins (HSV). Superoxide production was also compared between the proximal and distal segments of the veins.

Patients And Methods: Proximal and distal segments of varicose veins (14 patients, aged 52 ±3.5 years) and control veins (15 patients, aged 56 ±4 years) were obtained during VV removal or elective coronary artery bypass graft surgery, respectively. Subjects were matched for age, sex, and the major risk factors for atherosclerosis. Superoxide was measured by lucigenin-enhanced chemiluminescence (5 μmol/l) in the presence and absence of oxidase inhibitors.

Results: Superoxide production was increased in VV compared with control HSV. This increase was particularly evident in the distal segments of VV. There was a significant correlation between superoxide production in the proximal and distal segments of HSV but not of VV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and uncoupled nitric oxide synthase (NOS) were the major sources of superoxide in VV, because their inhibitors greatly attenuated superoxide production in VV.

Conclusions: NADPH oxidases and NOS could represent valuable drug targets for pharmacological treatment and prevention of varicose vein disease. Oxidative stress may provide a link between endothelial dysfunction, inflammation, and immune activation and the development of chronic venous dysfunction.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281159PMC
September 2011