Publications by authors named "Agnieszka Zakrzewska"

37 Publications

Age-Dependent Impairment in Endothelial Function and Arterial Stiffness in Former High Class Male Athletes Is No Different to That in Men With No History of Physical Training.

J Am Heart Assoc 2019 09 12;8(18):e012670. Epub 2019 Sep 12.

Department of Muscle Physiology Chair of Physiology and Biochemistry Faculty of Rehabilitation University School of Physical Education Krakow Poland.

Background Physical activity is generally considered to exert positive effects on the cardiovascular system in humans. However, surprisingly little is known about the delayed effect of professional physical training performed at a young age on endothelial function and arterial stiffness in aging athletes. The present study aimed to assess the impact of long-lasting professional physical training (endurance and sprint) performed at a young age on the endothelial function and arterial stiffness reported in older age in relation to glycocalyx injury, prostacyclin and nitric oxide production, inflammation, basal blood lipid profile, and glucose homeostasis. Methods and Results This study involved 94 male subjects with varied training backgrounds, including young athletes (mean age ∼25 years), older former high class athletes (mean age ∼60 years), and aged-matched untrained control groups. Aging increased arterial stiffness, as reflected by an enhancement in pulse wave velocity, augmentation index, and stiffness index (<10), as well as decreased endothelial function, as judged by the attenuation of flow-mediated vasodilation (FMD) in the brachial artery (=0.03). Surprisingly, no effect of the training performed at a young age on endothelial function and arterial stiffness was observed in the former athletes. Moreover, no effect of training performed at a young age (>0.05) on blood lipid profile, markers of inflammation, and glycocalyx shedding were observed in the former athletes. Conclusions Our study clearly shows that aging, but not physical training history, represents the main contributing factor responsible for decline in endothelial function and increase in arterial stiffness in former athletes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/JAHA.119.012670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817998PMC
September 2019

Keap1 controls protein S-nitrosation and apoptosis-senescence switch in endothelial cells.

Redox Biol 2020 01 22;28:101304. Epub 2019 Aug 22.

Department of Medical Biotechnology, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, 30-387, Krakow, Poland. Electronic address:

Premature senescence, a death escaping pathway for cells experiencing stress, is conducive to aging and cardiovascular diseases. The molecular switch between senescent and apoptotic fate remains, however, poorly recognized. Nrf2 is an important transcription factor orchestrating adaptive response to cellular stress. Here, we show that both human primary endothelial cells (ECs) and murine aortas lacking Nrf2 signaling are senescent but unexpectedly do not encounter damaging oxidative stress. Instead, they exhibit markedly increased S-nitrosation of proteins. A functional role of S-nitrosation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage and redirection of ECs to premature senescence. S-nitrosation and senescence are mediated by Keap1, a direct binding partner of Nrf2, which colocalizes and precipitates with nitric oxide synthase (NOS) and transnitrosating protein GAPDH in ECs devoid of Nrf2. We conclude that the overabundance of this "unrestrained" Keap1 determines the fate of ECs by regulation of S-nitrosation and propose that Keap1/GAPDH/NOS complex may serve as an enzymatic machinery for S-nitrosation in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731384PMC
January 2020

Proteomic characterization of early lung response to breast cancer metastasis in mice.

Exp Mol Pathol 2019 04 11;107:129-140. Epub 2019 Feb 11.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Department of Toxicology, Medyczna 9, 30-688 Krakow, Poland. Electronic address:

Introduction: The tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma.

Materials And Methods: The studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery).

Results: Palpable primary tumors were noted in the 2 week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1 week and progressed during the 2 week, accompanied by increased concentration of 2-OH-E, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1 week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2 week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz).

Conclusion: The analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexmp.2019.02.001DOI Listing
April 2019

Early and late endothelial response in breast cancer metastasis in mice: simultaneous quantification of endothelial biomarkers using a mass spectrometry-based method.

Dis Model Mech 2019 03 1;12(3). Epub 2019 Mar 1.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland

The endothelium plays an important role in cancer metastasis, but the mechanisms involved are still not clear. In the present work, we characterised the changes in endothelial function at early and late stages of breast cancer progression in an orthotopic model of murine mammary carcinoma (4T1 cells). Endothelial function was analysed based on simultaneous microflow liquid chromatography-tandem mass spectrometry using multiple reaction monitoring (microLC/MS-MRM) quantification of 12 endothelium-related biomarkers, including those reflecting glycocalyx disruption - syndecan-1 (SDC-1), endocan (ESM-1); endothelial inflammation - vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin (E-sel); endothelial permeability - fms-like tyrosine kinase 1 (FLT-1), angiopoietin 2 (Angpt-2); and haemostasis - von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), as well as those that are pathophysiologically linked to endothelial function - adrenomedullin (ADM) and adiponectin (ADN). The early phase of metastasis in mouse plasma was associated with glycocalyx disruption (increased SDC-1 and ESM-1), endothelial inflammation [increased soluble VCAM-1 (sVCAM-1)] and increased vascular permeability (Angpt-2). During the late phase of metastasis, additional alterations in haemostasis (increased PAI-1 and vWF), as well as a rise in ADM and substantial fall in ADN concentration, were observed. In conclusion, in a murine model of breast cancer metastasis, we identified glycocalyx disruption, endothelial inflammation and increased endothelial permeability as important events in early metastasis, while the late phase of metastasis was additionally characterised by alterations in haemostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.036269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451429PMC
March 2019

Quantitative measurement of selected protein biomarkers of endothelial dysfunction in plasma by micro-liquid chromatography-tandem mass spectrometry based on stable isotope dilution method.

Talanta 2019 Mar 23;194:1005-1016. Epub 2018 Oct 23.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688 Krakow, Poland. Electronic address:

The aim of this study was to develop and validate the novel microLC/MS-MRM method for the simultaneous quantification of six proteins: angiopoietin 2 (Angpt-2), soluble form of fms-like tyrosine kinase 1 (sFLT-1), plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (t-PA), endocan (ESM-1), soluble form of E-selectin (sE-sel), and one peptide: adrenomedullin (ADM) in mouse plasma. Two approaches were compared: a stable isotope dilution (SID) method- used as a reference and a modified SID (mSID) procedure. In SID strategy the calibration curves were used, whereas in mSID the ratio between the chromatogram peak area of endogenous tryptic peptides at unknown concentration to chromatogram peak area of exogenous, stable isotope-labelled internal standards (SISs) added to the sample at known concentration was calculated. The microLC/MS-MRM method in the SID approach was linear from 0.250 pmol/mL to 250 pmol/mL for Angpt-2; 5 pmol/mL to 5000 pmol/mL for sFLT-1; 2.5 pmol/mL to 5000 pmol/mL for PAI-1; 0.375 pmol/mL to 250 pmol/mL for t-PA; 0.375 pmol/mL to 187.5 pmol/mL for ESM-1; 2.5 pmol/mL to 5000 pmol/mL for sE-sel and 0.375 pmol/mL to 250 pmol/mL for ADM. LPS-induced changes in plasma assessed based on SID and mSID approaches gave comparable quantitative results and featured LPS-induced dysregulation of endothelial permeability (Angpt-2, sFLT-1), glycocalyx injury (SDC-1) accompanied by a pro-thrombotic response (PAI-1). In addition, we applied microLC/MS-MRM method with mSID strategy to analyze human plasma samples from patients with chronic myeloid leukemia (CML) and obstructive sleep apnoea (OSA) and demonstrated usefulness of the method to characterize endothelial function in humans. In conclusion, the microLC/MS-MRM method with mSID strategy applied for simultaneous quantification of protein biomarkers of endothelial function in plasma represents a novel targeted proteomic platform for the comprehensive evaluation of endothelial function in mice and humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.talanta.2018.10.067DOI Listing
March 2019

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics.

Breast Cancer Res 2018 12 4;20(1):148. Epub 2018 Dec 4.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348, Krakow, Poland.

Background: The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis.

Methods: Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics.

Results: Based on primary tumour growth and pulmonary metastases, 1-2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3-4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate ("Warburg effect"). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins.

Conclusions: The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-018-1075-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278167PMC
December 2018

Immuno-Spin Trapping-Based Detection of Oxidative Modifications in Cardiomyocytes and Coronary Endothelium in the Progression of Heart Failure in Tgαq*44 Mice.

Front Immunol 2018 7;9:938. Epub 2018 May 7.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF). However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST) technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO and analyzed for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative modifications in cardiomyocytes and coronary endothelium in the heart. In Tgαq*44 mice with slowly developing HF, driven by cardiomyocyte-specific overexpression of Gαq* protein, an increase in superoxide production, despite compensatory activation of antioxidative mechanisms, results in the development of oxidative modifications not only in cardiomyocytes but also in coronary endothelium, at the transition phase of HF, before the end-stage disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.00938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949515PMC
July 2019

Dual antiplatelet therapy with clopidogrel and aspirin increases mortality in 4T1 metastatic breast cancer-bearing mice by inducing vascular mimicry in primary tumour.

Oncotarget 2018 Apr 3;9(25):17810-17824. Epub 2018 Apr 3.

Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland.

Platelet inhibition has been considered an effective strategy for combating cancer metastasis and compromising disease malignancy although recent clinical data provided evidence that long-term platelet inhibition might increase incidence of cancer deaths in initially cancer-free patients. In the present study we demonstrated that dual anti-platelet therapy based on aspirin and clopidogrel (ASA+Cl), a routine regiment in cardiovascular patients, when given to cancer-bearing mice injected orthotopically with 4T1 breast cancer cells, promoted progression of the disease and reduced mice survival in association with induction of vascular mimicry (VM) in primary tumour. In contrast, treatment with ASA+Cl or platelet depletion did reduce pulmonary metastasis in mice, if 4T1 cells were injected intravenously. In conclusion, distinct platelet-dependent mechanisms inhibited by ASA+Cl treatment promoted cancer malignancy and VM in the presence of primary tumour and afforded protection against pulmonary metastasis in the absence of primary tumour. In view of our data, long-term inhibition of platelet function by dual anti-platelet therapy (ASA+Cl) might pose a hazard when applied to a patient with undiagnosed and untreated malignant cancer prone to undergo VM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.24891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915156PMC
April 2018

Vascular Nitric Oxide-Superoxide Balance and Thrombus Formation after Acute Exercise.

Med Sci Sports Exerc 2018 07;50(7):1405-1412

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, POLAND.

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 (O2) 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-PGF1α.

Results: An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 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-PGF1α concentrations.

Conclusion: An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1249/MSS.0000000000001589DOI Listing
July 2018

Type 1 diabetic patients have better endothelial function after simultaneous pancreas-kidney transplantation than after kidney transplantation with continued insulin therapy.

Diab Vasc Dis Res 2018 03 13;15(2):122-130. Epub 2017 Dec 13.

1 Department of General, Vascular and Transplant Surgery, Medical University of Silesia, Katowice, Poland.

The purpose of this study was to analyse the influence of simultaneous pancreas-kidney or kidney transplantation on endothelial function and systemic inflammation in type 1 diabetic patients with end-stage renal disease. In 39 simultaneous pancreas-kidney, 39 type 1 diabetic kidney and 52 non-diabetic kidney recipients, flow-mediated dilatation was measured. Additionally, blood glycated haemoglobin, serum creatinine and lipids, plasma nitrites [Formula: see text] and nitrates, asymmetric dimethylarginine, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin, high-sensitivity C-reactive protein, tumour necrosis factor-α, interleukin 1β and interleukin 6 concentrations were assessed. During 58 ± 31 months follow-up period, flow-mediated dilatation and [Formula: see text] were greater in simultaneous pancreas-kidney than in type 1 diabetic kidney recipients [10.4% ± 4.7% vs 7.7% ± 4.2%, p < 0.05 and 0.94 (0.74-1.34) vs 0.24 (0.20-0.43) μmol/L, p < 0.01, respectively]. In type 1 diabetic patients after simultaneous pancreas-kidney or kidney transplantation, [Formula: see text] correlated with flow-mediated dilatation (r = 0.306, p < 0.05) and with blood glycated haemoglobin (r = -0.570, p < 0.001). The difference in [Formula: see text] was linked to blood glycated haemoglobin and estimated glomerular filtration rate, whereas the difference in flow-mediated dilatation was linked to [Formula: see text]. The levels of inflammatory markers (except soluble vascular cell adhesion molecule-1) were similar in simultaneous pancreas-kidney and type 1 diabetic kidney recipients. Improved endothelial function in type 1 diabetic patients with end-stage renal disease after simultaneous pancreas-kidney compared to kidney transplantation is associated with normalisation of glucose metabolism but not with improvement in plasma pro-inflammatory cytokines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1479164117744423DOI Listing
March 2018

Development, validation and application of a micro-liquid chromatography-tandem mass spectrometry based method for simultaneous quantification of selected protein biomarkers of endothelial dysfunction in murine plasma.

J Pharm Biomed Anal 2018 Feb 10;149:465-474. Epub 2017 Nov 10.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348, Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688, Krakow, Poland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2017.11.023DOI Listing
February 2018

AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes.

Sci Rep 2017 11 21;7(1):15951. Epub 2017 Nov 21.

Center for Nanometer-scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Łojasiewicza 11, 30-348, Krakow, Poland.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-16179-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698475PMC
November 2017

Breast cancer pulmonary metastasis is increased in mice undertaking spontaneous physical training in the running wheel; a call for revising beneficial effects of exercise on cancer progression.

Am J Cancer Res 2017 1;7(9):1926-1936. Epub 2017 Sep 1.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian UniversityBobrzynskiego 14, 30-348, Krakow, Poland.

It has been repeatedly shown that regular aerobic exercise exerts beneficial effects on incidence and progression of cancer. However, the data regarding effects of exercise on metastatic dissemination remain conflicting. Therefore, in the present study the possible preventive effects of voluntary wheel running on primary tumor growth and metastases formation in the model of spontaneous pulmonary metastasis were analyzed after orthotopic injection of 4T1 breast cancer cells into mammary fat pads of female Balb/C mice. This study identified that in the mice injected with 4T1 breast cancer cells and running on the wheels (4T1 ex) the volume and size of the primary tumor were not affected, but the number of secondary nodules formed in the lungs was significantly increased compared to their sedentary counterparts (4T1 sed). This effect was associated with decreased NO production in the isolated aorta of exercising mice (4T1 ex), suggesting deterioration of endothelial function that was associated with lower platelet count without their overactivation. This was evidenced by comparable selectin P, active GPIIb/IIIa expression, fibrinogen and vWF binding on the platelet surface. In conclusion, voluntary wheel running appeared to impair, rather than improve endothelial function, and to promote, but not decrease metastasis in the murine orthotopic model of metastatic breast cancer. These results call for revising the notion of the persistent beneficial effects of voluntary exercise on breast cancer progression, though further studies are needed to elucidate mechanisms involved in pro-metastatic effects of voluntary exercise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622226PMC
September 2017

Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

Cardiovasc Res 2016 Nov;112(2):590-605

Department of Biochemistry, Medical University of Gdansk, 1 Debinki St., 80-211 Gdansk, Poland.

Aims: 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.

Methods And Results: 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.

Conclusions: 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvw203DOI Listing
November 2016

Differential effects of liver steatosis on pharmacokinetic profile of two closely related hepatoselective NO-donors; V-PYRRO/NO and V-PROLI/NO.

Pharmacol Rep 2017 May 2;69(3):560-565. Epub 2017 Feb 2.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Kraków, Poland.

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 (0.03 mmol/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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pharep.2017.01.031DOI Listing
May 2017

Differential effects of liver steatosis on pharmacokinetic profile of two closely related hepatoselective NO-donors; V-PYRRO/NO and V-PROLI/NO.

Pharmacol Rep 2017 Jun 2;69(3):560-565. Epub 2017 Feb 2.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Kraków, Poland; Chair of Pharmacology, Jagiellonian University Medical College, Kraków, Poland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pharep.2017.01.031DOI Listing
June 2017

Pharmacokinetic Profile of 1-Methylnicotinamide Nitrate in Rats.

J Pharm Sci 2017 05 30;106(5):1412-1418. Epub 2017 Jan 30.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland; Chair of Pharmacology, Jagiellonian University Medical College, Grzegorzecka 16, Krakow 31-531, Poland. Electronic address:

Treatment with 1-methylnicotinamide (MNA), a major metabolite of nicotinamide, exerts antithrombotic, anti-inflammatory, and vasoprotective effects. Yet, pharmacokinetic (PK) profile of MNA has not been fully characterized. In the present work, we analyze the PK profile of the MNA given as a nitrate (MNANO) in comparison to nitrite (MNANO) or chloride (MNACl) in rats. The bioavailability of MNA administered as MNANO equaled 22.4% as compared to MNANO or MNACl (9.2% and 9.1%, respectively). Moreover, in single-pass intestinal perfusion experiments, effective permeability of MNA given as MNANO was higher as compared to MNA administered as MNANO or MNACl. In turn, t was the shortest and C the highest (0.22 h and 56.65μM) for intragastrically administered MNANO comparing to MNANO (1.92 h, 21.74μM) or MNACl (0.63 h, 16.13μM). Transfer constant between central and peripheral compartments (k) and volume of distribution (V) for MNANO (0.33 h and 1.96 L/kg) were higher as compared to MNANO or MNACl (0.11 h, 0.08 h for k and 1.05 L/kg, 0.76 L/kg for V, respectively). In conclusion, we characterized PK profile of MNA and demonstrated that nitrate ion augmented bioavailability and favorably modified PK profile of MNA. Furthermore, given vasoprotective properties of MNA as well as nitrate, MNANO represents a bifunctional compound.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2017.01.022DOI Listing
May 2017

Carbon monoxide shifts energetic metabolism from glycolysis to oxidative phosphorylation in endothelial cells.

FEBS Lett 2016 Oct 11;590(20):3469-3480. Epub 2016 Oct 11.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Carbon monoxide (CO) modulates mitochondrial respiration, but the mechanisms involved are not completely understood. The aim of the present study was to investigate the acute effects of CO on bioenergetics and metabolism in intact EA.hy926 endothelial cells using live cell imaging techniques. Our findings indicate that CORM-401, a compound that liberates CO, reduces ATP production from glycolysis, and induces a mild mitochondrial depolarization. In addition, CO from CORM-401 increases mitochondrial calcium and activates complexes I and II. The subsequent increase in mitochondrial respiration leads to ATP production through oxidative phosphorylation. Thus, our results show that nonactivated endothelial cells rely primarily on glycolysis, but in the presence of CO, mitochondrial Ca increases and activates respiration that shifts the metabolism of endothelial cells from glycolysis- to oxidative phosphorylation-dependent ATP production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12434DOI Listing
October 2016

Moderate-intensity endurance training improves endothelial glycocalyx layer integrity in healthy young men.

Exp Physiol 2017 01;102(1):70-85

Department of Muscle Physiology, Faculty of Rehabilitation, University School of Physical Education, Krakow, Poland.

New Findings: What is the central question of this study? The main aim of the present study was to determine the effect of prolonged moderate-intensity endurance training on the endothelial glycocalyx layer integrity in relationship to the training-induced changes in oxidative stress and antioxidant defence in humans. What is the main finding and its importance? We have shown, for the first time, a protective effect of prolonged moderate-intensity endurance training on endothelial glycocalyx layer integrity, as judged by significantly lower basal and end-exercise serum concentrations of glycocalyx damage markers, i.e. syndecan-1 and heparan sulfate, accompanied by attenuation of oxidative stress and enhancement of antioxidant defence after training in previously untrained healthy young men. In this study, we evaluated the effect of 20 weeks of moderate-intensity endurance training (ET) on the endothelial glycocalyx layer integrity in relationship to the training-induced changes in antioxidant defence. Eleven healthy young, untrained men performed an incremental cycling exercise bout until exhaustion before and after 20 weeks of ET. Endurance training consisted of 40 min sessions, mainly of moderate intensity (∼50% of maximal oxygen uptake), performed four times per week. Venous blood samples were taken at rest and at the end of the maximal exercise test. Muscle biopsies from vastus lateralis were taken before and after the training. Endurance training resulted in a significant increase in physical capacity (P < 0.05) as reflected by an increase in power output reached at the lactate threshold and at maximal oxygen uptake. Training led to a decrease (P < 0.05) in basal and end-exercise concentrations of blood markers of glycocalyx damage (syndecan-1 and heparan sulfate). The lowering of glycocalyx shedding after the ET was accompanied by an attenuation of oxidative stress, as evidenced by a decrease in the basal plasma concentration of isoprostanes, and by an increase in antioxidant defence, reflected by an enhancement in superoxide dismutase 2 protein content in vastus lateralis (P < 0.05). In contrast, training did not induce a significant increase in basal nitrite/nitrate plasma concentration (P > 0.05). Moderate-intensity ET exerts a pronounced protective effect on endothelial glycocalyx integrity at rest and during exercise, probably through an improvement of antioxidant defence that may represent the vasoprotective mechanisms highly responsive to moderate-intensity endurance training.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/EP085887DOI Listing
January 2017

Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension.

Respir Res 2016 08 31;17(1):108. Epub 2016 Aug 31.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzyńskiego 14, Krakow, Poland.

Background: Pulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans.

Methods: PAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured.

Results: MCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration-findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls.

Conclusions: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12931-016-0423-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007701PMC
August 2016

QUANTIFICATION AND PHARMACOKINETICS OF 1 -METHYLPYRIDINIUM AND 1,4-DIMETHYLPYRIDINIUM IN RATS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY. TISSUE DISTRIBUTION OF 1,4-DIMETHYLPYRIDINIUM IN RATS.

Acta Pol Pharm 2016 Sep;73(5):1111-1121

A sensitive and specific liquid chromatography tandem mass spectrometry method for quantification of 1-methylpyridinium (1-MP) and 1,4-dimethylpyridinium (1,4-DMP) in rat plasma and tissues homogenates was developed. Chromatographic separation was performed on an Aquasil C18 analytical column with an isocratic elution of acetonitrile and water, both with an addition of formic acid (0.1%, v/v). Detection was achieved by triple quadrupole mass spectrometer TSQ Quantum Ultra equipped with a heated electrospray ionization source (HESI). The limit of quantification for both compounds was 0.05 pg/mL in plasma and 0.25 μg/g in studied tissues. The method was applied to pharmacokinetics and bioavailability of both 1-MP and 1,4-DMP with tissue distribution of 1,4-DMP in rats. Pharmacokinetic studies of 1-MP and 1,4-DMP were carried out following their intravenous or intragastric administration to male Wistar rats at the dose of 100 mg/kg. The terminal half-lives of I-MP and 1,4-DMP after their intravenous administration were 55.3 and 70.8 min, respectively. The absolute bioavailability was 51 and 31% for t-MP and 1,4-DMP, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
September 2016

Simultaneous quantification of PGI2 and TXA2 metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method.

J Pharm Biomed Anal 2016 Sep 28;129:148-154. Epub 2016 Jun 28.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland; Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, 31-531 Krakow, Poland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2016.06.050DOI Listing
September 2016

Antiatherosclerotic Effects of 1-Methylnicotinamide in Apolipoprotein E/Low-Density Lipoprotein Receptor-Deficient Mice: A Comparison with Nicotinic Acid.

J Pharmacol Exp Ther 2016 Feb 2;356(2):514-24. Epub 2015 Dec 2.

Jagiellonian Centre for Experimental Therapeutics (Ł.M., A.J., E.M., M.G.-G., M.B., B.S., A.Z., A.K., M.W., S.C.) and Faculty of Chemistry (M.B.), Jagiellonian University, Krakow, Poland; Department of Toxicology, Faculty of Pharmacy (A.K., M.W.) and Chair of Pharmacology (S.C.), Jagiellonian University Medical College, Krakow, Poland; and Department of Human Nutrition, Faculty of Food Technology, University of Agriculture, Krakow, Poland (R.K.)

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 F1 α 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.115.228643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047228PMC
February 2016

Towards a comprehensive endothelial biomarkers profiling and endothelium-guided pharmacotherapy.

Pharmacol Rep 2015 Aug 29;67(4):771-7. Epub 2015 Jun 29.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Kraków, Poland; Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical College, Kraków, Poland. Electronic address:

Endothelial dysfunction has prognostic, diagnostic and therapeutic significance in cardiovascular disease, but the endothelial phenotype is still not measured routinely to stratify the cardiovascular risk and tailor therapy. Flow-mediated dilation (FMD), the gold-standard technique for the functional assessment of endothelial function that is increasingly used in clinical settings measures the nitric oxide (NO)-dependent function only. However, the endothelial dysfunction involves a plethora of pathophysiologically important biochemical changes beyond alterations in the NO bioavailability. Still, in many diseases, some plasma protein biomarkers reflecting the pro-thrombotic and the pro-inflammatory endothelial phenotypes have poor selectivity, specificity, and a weak predictive value if they are used alone. Therefore, a multi biomarker strategy seems to be a reasonable and promising alternative. Here, we propose a multi-biomarker strategy to diagnose the endothelial dysfunction and to monitor the efficacy of an endothelium-targeted therapy. This strategy is based on the panel of endothelial biomarkers, reflecting various aspects and mechanisms of dysfunctional endothelium. The potential of an advanced analytical platform like the ultra-high performance liquid chromatography (UHPLC) coupled to mass spectrometry-based multiple reaction monitoring for simultaneous quantification of multiple endothelial biomakers is also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pharep.2015.06.008DOI Listing
August 2015

Effects of 1-Methylnicotinamide (MNA) on Exercise Capacity and Endothelial Response in Diabetic Mice.

PLoS One 2015 26;10(6):e0130908. Epub 2015 Jun 26.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland; Department of Experimental Pharmacology, Jagiellonian University Medical College, Krakow, Poland.

1-Methylnicotinamide (MNA), which was initially considered to be a biologically inactive endogenous metabolite of nicotinamide, has emerged as an anti-thrombotic and anti-inflammatory agent with the capacity to release prostacyclin (PGI2). In the present study, we characterized the effects of MNA on exercise capacity and the endothelial response to exercise in diabetic mice. Eight-week-old db/db mice were untreated or treated with MNA for 4 weeks (100 mg·kg-1), and their exercise capacity as well as NO- and PGI2-dependent response to endurance running were subsequently assessed. MNA treatment of db/db mice resulted in four-fold and three-fold elevation of urine concentrations of MNA and its metabolites (Met-2PY + Met-4PY), respectively (P<0.01), but did not affect HbA1c concentration, fasting glucose concentration or lipid profile. However, insulin sensitivity was improved (P<0.01). In MNA-treated db/db mice, the time to fatigue for endurance exercise was significantly prolonged (P<0.05). Post-exercise Δ6-keto-PGF1α (difference between mean concentration in the sedentary and exercised groups) tended to increase, and post-exercise leukocytosis was substantially reduced in MNA-treated animals. In turn, the post-exercise fall in plasma concentration of nitrate was not affected by MNA. In conclusion, we demonstrated for the first time that MNA improves endurance exercise capacity in mice with diabetes, and may also decrease the cardiovascular risk of exercise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130908PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482656PMC
April 2016

Hepatoselective Nitric Oxide (NO) Donors, V-PYRRO/NO and V-PROLI/NO, in Nonalcoholic Fatty Liver Disease: A Comparison of Antisteatotic Effects with the Biotransformation and Pharmacokinetics.

Drug Metab Dispos 2015 Jul 13;43(7):1028-36. Epub 2015 Apr 13.

Jagiellonian Centre for Experimental Therapeutics (K.K., M.W., E.M., A.Z., A.G.-D., B.S., K.W., A.K., S.Ch.), Department of Pharmacokinetics and Physical Pharmacy, Medical College (K.K., M.W., A.G.-D., A.K.), and Department of Experimental Pharmacology, Chair of Pharmacology, Medical College (S.Ch.), Jagiellonian University, Krakow, Poland; Department of Physiology, Medical University of Bialystok, Bialystok, Poland (P.Z., A.Ch.); Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland (J.E.S.); and Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland (R.J.H., L.K.K.)

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/dmd.115.063388DOI Listing
July 2015

Treatment of hypertension in chronic kidney disease patients under specialized care: one-center cross-sectional analyses.

Blood Press 2015 Apr 26;24(2):79-85. Epub 2014 Dec 26.

Department of Nephrology, Transplantology and Internal Medicine, Medical University of Gdansk , Poland.

Aim: The study analyzed hypertension management and control rates among non-dialysis, non-transplanted hypertensive chronic kidney disease (CKD) patients under specialized care in Gdansk nephrology center in 1996-2011.

Patients And Methods: It was a retrospective, cross-sectional study analyzing data from medical records of 190, 490, 1799 and 1696 subjects with CKD, who received outpatient care in 1996, 2001, 2006 and 2011, and were included in four independent surveys, respectively.

Results: The average number of antihypertensive drugs per patient increased significantly (p < 0.01) as follows 1.74 ± 0.9 (1996), 2.08 ± 1.01 (2011), 2.5 ± 1.19 (2006) and 2.65 ± 1.18 (2011). The percentage of patients receiving diuretics, beta-blockers and drugs inhibiting renin-angiotensin-aldosterone increased significantly in subsequent years, while a frequency of therapy with calcium channel blockers decreased (p < 0.001). 16%, 30%, 42% and 54% of subjects had causal BP values < 140/90 mmHg (p < 0.001). When specific thresholds for CKD patients according to JNC recommendations were used, the control rate was worse but also showed significant improvement in the second, third and final surveys, i.e. 9%, 12%, 14% and 24% (p < 0.001). The subgroup analysis revealed that a better control rate was observed in following groups: < 65 years old; I-II stage of CKD; primary glomerulonephritis; without cardiovascular complications or diabetes.

Conclusion: The study may show an improvement in the effectiveness of antihypertensive treatment in CKD patients under specialized care in Gdansk Nephrology Centre in 1996-2011.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/08037051.2014.986931DOI Listing
April 2015

Detection of spreading depolarization with intraparenchymal electrodes in the injured human brain.

Neurocrit Care 2014 Feb;20(1):21-31

Department of Neurosurgery, King's College Hospital, London, UK.

Background: Spreading depolarization events following ischemic and traumatic brain injury are associated with poor patient outcome. Currently, monitoring these events is limited to patients in whom subdural electrodes can be placed at open craniotomy. This study examined whether these events can be detected using intra-cortical electrodes, opening the way for electrode insertion via burr hole.

Methods: Animal work was carried out on adult Sprague-Dawley rats in a laboratory setting to investigate the feasibility of recording depolarization events. Subsequently, 8 human patients requiring craniotomy for traumatic brain injury or aneurysmal subarachnoid hemorrhage were monitored for depolarization events in an intensive care setting with concurrent strip (subdural) and depth (intra-parenchymal) electrode recordings.

Results: (1) Depolarization events can be reliably detected from intra-cortically placed electrodes. (2) A reproducible slow potential change (SPC) waveform morphology was identified from intra-cortical electrodes on the depth array. (3) The depression of cortical activity known to follow depolarization events was identified consistently from both intra-cortical and sub-cortical electrodes on the depth array.

Conclusions: Intra-parenchymally sited electrodes can be used to consistently identify depolarization events in humans. This technique greatly extends the capability of monitoring for spreading depolarization events in injured patients, as electrodes can be sited without the need for craniotomy. The method provides a new investigative tool for the evaluation of the contribution of these events to secondary brain injury in human patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12028-013-9938-7DOI Listing
February 2014

Capillary electrophoresis/frontal analysis versus equilibrium dialysis in dexamethasone sodium phosphate-serum albumin binding studies.

Electrophoresis 2012 Nov 24;33(22):3323-30. Epub 2012 Oct 24.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Plasma protein binding of drugs may have significant effect on its pharmacodynamic, toxicological and pharmacokinetic properties, since only the free drug can pass across biological membrane and get to its specific site of action. Many drugs show a high affinity to albumin which is the most abundant plasma protein. In the present study capillary electrophoresis in the frontal analysis mode (CE/FA), as promising technique for assessment of drug-protein interaction was used. The free drug concentration was measured from height of the frontal peak and calculated based on the external drug standard in absence of protein. With a known concentration of total drug, the percentage of protein bound drug was determined. The binding parameters were also estimated based on the equilibrium dialysis experiment which is considered to be a reference method. This study was designed to examine the interaction of dexamethasone sodium phosphate (DXM) with BSA and HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate buffer, I = 0.17). Using fixed, at physiological level, HSA and BSA concentrations and increasing DXM concentrations, the number of binding sites (n) and binding constant (K(a) ) was calculated from both nonlinear regression fitting and Scatchard Plot. Despite some differences, it can be concluded that the CE/FA is comparable with equilibrium dialysis, but since the first one offers advantages such as low sample consumption, short analysis time, and high separation efficiency, it can be used in high-throughput screening of drug protein binding at the early stage of drug discovery. Interspecies differences in binding of a drug to albumins have been observed and it should be taken into account in interpretation of the results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/elps.201200166DOI Listing
November 2012

Liquid chromatography-mass spectrometry method for the analysis of 1,4-dimethylpyridinium in rat plasma--application to pharmacokinetic studies.

Biomed Chromatogr 2013 Jan 30;27(1):73-9. Epub 2012 Apr 30.

Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Krakow, Poland.

A sensitive and specific liquid chromatography electrospray ionization-mass spectrometry method for determination of 1,4-dimethylpyridinium (1,4-DMP) in rat plasma has been developed and validated. Chromatography was performed on an Aquasil C(18) analytical column (4.6 × 150 mm, 5 µm, Thermo Scientific, Rockford, IL, USA) with isocratic elution using a mobile phase containing acetonitrile and water with an addition of 0.1% of formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization was used for ion production. The limit of detection in the single ion monitoring mode was found to be 10 ng/mL. The limit of quantification was 50 ng/mL. The precision and accuracy for both within-day and between-day determination of 1,4-dimethylpyridinium was 2.4-7.56 and 90.93-111.48%. The results of this analytical method validation allow pharmacokinetic studies to be carried out in rats. The method was used for the pilot study of the pharmacokinetic behavior of 1,4-DMP in rats after intravenous administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bmc.2750DOI Listing
January 2013