Publications by authors named "Bartosz Proniewski"

17 Publications

  • Page 1 of 1

Direct comparison of inorganic nitrite and nitrate on vascular dysfunction and oxidative damage in experimental arterial hypertension.

Nitric Oxide 2021 Jun 4;113-114:57-69. Epub 2021 Jun 4.

Department of Cardiology, Cardiology I, Laboratory of Molecular Cardiology, University Medical Center Mainz, Mainz, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Mainz, Germany. Electronic address:

Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO) and nitrate (NO) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO and NO co-therapy in mice with angiotensin-II (AT-II)-induced arterial hypertension. High-dose AT-II (1 mg/kg/d, 1w, s. c.) was used to induce arterial hypertension in male C57BL/6 mice. Additional inorganic nitrite (7.5 mg/kg/d, p. o.) or nitrate (150 mg/kg/d, p. o.) were administered via the drinking water. Blood pressure (tail-cuff method) and endothelial function (isometric tension) were determined. Oxidative stress and inflammation markers were quantified in aorta, heart, kidney and blood. Co-treatment with inorganic nitrite, but not with nitrate, normalized vascular function, oxidative stress markers and inflammatory pathways in AT-II treated mice. Of note, the highly beneficial effects of nitrite on all parameters and the less pronounced protection by nitrate, as seen by improvement of some parameters, were observed despite no significant increase in plasma nitrite levels by both therapies. Methemoglobin levels tended to be higher upon nitrite/nitrate treatment. Nutritional nitric oxide precursors represent a non-pharmacological treatment option for hypertension that could be applied to the general population (e.g. by eating certain vegetables). The more beneficial effects of inorganic nitrite may rely on superior NO bioactivation and stronger blood pressure lowering effects. Future large-scale clinical studies should investigate whether hypertension and cardiovascular outcome in general can be influenced by dietary inorganic nitrite therapy.
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http://dx.doi.org/10.1016/j.niox.2021.06.001DOI Listing
June 2021

In Vivo Magnetic Resonance Imaging-Based Detection of Heterogeneous Endothelial Response in Thoracic and Abdominal Aorta to Short-Term High-Fat Diet Ascribed to Differences in Perivascular Adipose Tissue in Mice.

J Am Heart Assoc 2020 11 19;9(21):e016929. Epub 2020 Oct 19.

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

Background Long-term feeding with a high-fat diet (HFD) induces endothelial dysfunction in mice, but early HFD-induced effects on endothelium have not been well characterized. Methods and Results Using an magnetic resonance imaging-based methodology that allows characterization of endothelial function in vivo, we demonstrated that short-term (2 weeks) feeding with a HFD to mice or to mice resulted in the impairment of acetylcholine-induced response in the abdominal aorta (AA), whereas, in the thoracic aorta (TA), the acetylcholine-induced response was largely preserved. Similarly, HFD resulted in arterial stiffness in the AA, but not in the TA. The difference in HFD-induced response was ascribed to distinct characteristics of perivascular adipose tissue in the TA and AA, related to brown- and white-like adipose tissue, respectively, as assessed by histology, immunohistochemistry, and Raman spectroscopy. In contrast, short-term HFD-induced endothelial dysfunction could not be linked to systemic insulin resistance, changes in plasma concentration of nitrite, or concentration of biomarkers of glycocalyx disruption (syndecan-1 and endocan), endothelial inflammation (soluble form of vascular cell adhesion molecule 1, soluble form of intercellular adhesion molecule 1 and soluble form of E-selectin), endothelial permeability (soluble form of fms-like tyrosine kinase 1 and angiopoietin 2), and hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). Conclusions Short-term feeding with a HFD induces endothelial dysfunction in the AA but not in the TA, which could be ascribed to a differential response of perivascular adipose tissue to a HFD in the AA versus TA. Importantly, early endothelial dysfunction in the AA is not linked to elevation of classical systemic biomarkers of endothelial dysfunction.
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http://dx.doi.org/10.1161/JAHA.120.016929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763398PMC
November 2020

Unexpected effects of long-term treatment with acetylsalicylic acid on late phase of pulmonary metastasis in murine model of orthotopic breast cancer.

PLoS One 2020 6;15(4):e0230520. Epub 2020 Apr 6.

Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Krakow, Poland.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135281PMC
July 2020

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

Vitamin K-MK-7 improves nitric oxide-dependent endothelial function in ApoE/LDLR mice.

Vascul Pharmacol 2019 Nov - Dec;122-123:106581. Epub 2019 Aug 14.

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

Although, vitamin K displays vasoprotective effects, it is still not known whether K treatment improves endothelial function. In ApoE/LDLR mice at the stage prior to atherosclerosis development, four-week treatment with K-MK-7, given at a low dose (0.05 mg/kg), improved acetylcholine- and flow-induced, endothelium-dependent vasodilation in aorta or in femoral artery, as assessed by MRI in vivo. This effect was associated with an increased NO production, as evidenced by EPR measurements in ex vivo aorta. Treatment with higher doses of K-MK-7 (0.5; 5 mg/kg) resulted in a dose-dependent increase in plasma K-MK-7 and K-MK-4 concentration, without further improvement in endothelial function. In ApoE/LDLR mice with developed atherosclerotic plaques, treatment with a low (0.03 mg/kg) or high (10 mg/kg) dose of K-MK-7 resulted in a similar degree of endothelium-dependent vasodilation improvement and increase in plasma nitrate concentration, what was not associated with changes in thrombin generation as measured by CAT. Both doses of K-MK-7 also reduced media thickness in the brachiocephalic artery, but did not modify atherosclerotic plaque size. In conclusion, K-MK-7 improves NO-dependent endothelial function in ApoE/LDLR mice. This study, identifies the endothelial profile of the pharmacological activity of vitamin K, which has not been previously described.
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http://dx.doi.org/10.1016/j.vph.2019.106581DOI Listing
May 2020

Multiorgan Development of Oxidative and Nitrosative Stress in LPS-Induced Endotoxemia in C57Bl/6 Mice: DHE-Based Approach.

Oxid Med Cell Longev 2019 22;2019:7838406. Epub 2019 May 22.

Jagiellonian University, Jagiellonian Centre for Experimetal Therapeutics (JCET), Bobrzynskiego 14, 30-348 Krakow, Poland.

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O) abundance by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E), as well as ethidium (E) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E varied in the liver, lung, and kidney; however, the ratios of 2-OH-E/E and 2-OH-E/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O production . Furthermore, the ratio of 2-OH-E/E oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O-mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.
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http://dx.doi.org/10.1155/2019/7838406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556324PMC
December 2019

Degradation of Glycocalyx and Multiple Manifestations of Endothelial Dysfunction Coincide in the Early Phase of Endothelial Dysfunction Before Atherosclerotic Plaque Development in Apolipoprotein E/Low-Density Lipoprotein Receptor-Deficient Mice.

J Am Heart Assoc 2019 03;8(6):e011171

1 Jagiellonian University Jagiellonian Centre for Experimental Therapeutics Krakow Poland.

Background The impairment of endothelium-dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide. Methods and Results Herein, we demonstrate that in 4- to 8-week-old apolipoprotein E/low-density lipoprotein receptor-deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine-induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow-mediated dilation in the femoral artery was fully preserved. In 4-week-old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28-week-old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine-induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow-mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability. Conclusions In conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.
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http://dx.doi.org/10.1161/JAHA.118.011171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475045PMC
March 2019

Combined Raman- and AFM-based detection of biochemical and nanomechanical features of endothelial dysfunction in aorta isolated from ApoE/LDLR-/- mice.

Nanomedicine 2019 02 11;16:97-105. Epub 2018 Dec 11.

Institute of Physiology II, University of Münster, Robert-Koch Str. 27b, 48149 Münster, Germany; Institute of Physiology, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany. Electronic address:

Endothelial dysfunction is recognized as a critical condition in the development of cardiovascular disorders. This multifactorial process involves changes in the biochemical and mechanical properties of endothelial cells leading to disturbed release of vasoprotective mediators. Hypercholesterolemia and increased stiffness of the endothelial cortex are independently shown to result in reduced release of nitric oxide and thus endothelial dysfunction. However, direct evidence linking these parameters to each other is missing. Here, a novel method combining Raman spectroscopy for biochemical analysis and Atomic Force Microscopy (AFM) for analyzing the endothelial nanomechanics was established. Using this dual approach, the same areas of native ex vivo aortas were investigated, either derived from mice with endothelial dysfunction (ApoE/LDLR-/-) or wild type mice. In particular an increased intracellular lipid content and elevated cortical stiffness/elasticity were shown in ApoE/LDLR-/- aortas, demonstrating a direct link between endothelial dysfunction, the biochemical composition and the nanomechanical properties of endothelial cells.
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http://dx.doi.org/10.1016/j.nano.2018.11.014DOI Listing
February 2019

Spectroscopy-based characterization of Hb-NO adducts in human red blood cells exposed to NO-donor and endothelium-derived NO.

Analyst 2018 Sep;143(18):4335-4346

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland.

The work presents the complementary approach to characterize the formation of various Hb species inside isolated human RBCs exposed to NO, with a focus on the formed Hb-NO adducts. This work presents a complementary approach based on Resonance Raman Spectroscopy (RRS) supported by Blood Gas Analysis, Electron Paramagnetic Resonance Spectroscopy, UV-Vis Absorption Spectroscopy and Mössbauer Spectroscopy to characterize the formation of various Hb species, with a focus on the Hb-NO adducts formed inside isolated human RBCs exposed to NO, under the experimental conditions of low and high levels of oxygen Hb saturation. In the present work, we induced Hb-NO adducts using PAPA-NONOate, a NO-donor with known chemistry and kinetics of NO release, and confirmed the formation of Hb-NO adducts in RBCs incubated with Human Aortic Endothelial Cells (HAECs) stimulated to produce NO. Our results provide a new insight into the formation of Hb-NO adducts after the exposure of RBCs with high oxyHb content to exogenous NO with special attention to the formation of LSHbIIINO in addition to LSHbIINO and metHb (HS/LSHbIIIH2O). We also point out that reliable characterization of Hb-NO adducts requires complementary techniques. Among them, RRS, as a label-free and non-destructive tool, appears to be an important discrimination technique in the studies of Hb-NO adducts inside intact RBCs.
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http://dx.doi.org/10.1039/c8an00302eDOI Listing
September 2018

Nitric oxide deficiency and endothelial-mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice.

Breast Cancer Res 2018 08 3;20(1):86. Epub 2018 Aug 3.

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

Background: Mesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial-mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice.

Methods: NO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1-5 weeks after 4T1 cancer cell inoculation in Balb/c mice.

Results: Phosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation.

Conclusions: Decreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis.
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http://dx.doi.org/10.1186/s13058-018-1013-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091065PMC
August 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.
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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.
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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.
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http://dx.doi.org/10.1249/MSS.0000000000001589DOI Listing
July 2018

CORM-401 induces calcium signalling, NO increase and activation of pentose phosphate pathway in endothelial cells.

FEBS J 2018 04 8;285(7):1346-1358. Epub 2018 Mar 8.

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

Carbon monoxide-releasing molecules (CO-RMs) induce nitric oxide (NO) release (which requires NADPH), and Ca -dependent signalling; however, their contribution in mediating endothelial responses to CO-RMs is not clear. Here, we studied the effects of CO liberated from CORM-401 on NO production, calcium signalling and pentose phosphate pathway (PPP) activity in human endothelial cell line (EA.hy926). CORM-401 induced NO production and two types of calcium signalling: a peak-like calcium signal and a gradual increase in cytosolic calcium. CORM-401-induced peak-like calcium signal, originating from endoplasmic reticulum, was reduced by thapsigargin, a SERCA inhibitor, and by dantrolene, a ryanodine receptors (RyR) inhibitor. In contrast, the phospholipase C inhibitor U73122 did not significantly affect peak-like calcium signalling, but a slow and progressive CORM-401-induced increase in cytosolic calcium was dependent on store-operated calcium entrance. CORM-401 augmented coupling of endoplasmic reticulum and plasmalemmal store-operated calcium channels. Interestingly, in the presence of NO synthase inhibitor (l-NAME) CORM-401-induced increases in NO and cytosolic calcium were both abrogated. CORM-401-induced calcium signalling was also inhibited by superoxide dismutase (poly(ethylene glycol)-SOD). Furthermore, CORM-401 accelerated PPP, increased NADPH concentration and decreased the ratio of reduced to oxidized glutathione (GSH/GSSG). Importantly, CORM-401-induced NO increase was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN), but neither by dantrolene nor by an inhibitor of large-conductance calcium-regulated potassium ion channel (paxilline). The results identify the primary role of CO-induced NO increase in the regulation of endothelial calcium signalling, that may have important consequences in controlling endothelial function.
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http://dx.doi.org/10.1111/febs.14411DOI Listing
April 2018

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

SERCA, complex I of the respiratory chain and ATP-synthase inhibition are involved in pleiotropic effects of NS1619 on endothelial cells.

Eur J Pharmacol 2016 Sep 1;786:137-147. Epub 2016 Jun 1.

Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteura St., 02-093 Warsaw, Poland.

A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells. The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by L-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca(2+)]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase. In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca(2+) from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase.
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http://dx.doi.org/10.1016/j.ejphar.2016.05.039DOI Listing
September 2016

The liver-selective NO donor, V-PYRRO/NO, protects against liver steatosis and improves postprandial glucose tolerance in mice fed high fat diet.

Biochem Pharmacol 2015 Feb 19;93(3):389-400. Epub 2014 Dec 19.

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

Background And Purpose: There is an unmet medical need for novel NAFLD treatments. Here we have examined the effects of liver-selective NO donor (V-PYRRO/NO) as compared with metformin on hepatic steatosis and glucose tolerance in mice fed high fat diet.

Material And Methods: Effects of V-PYRRO/NO (5 mgkg(-1)) or metformin (616 mgkg(-1)) were examined in C57BL/6J mice fed high fat diet (HF, 60 kcal% fat). Quantitative determination of steatosis, liver fatty acid composition and western blot analysis of selected proteins involved in mitochondrial biogenesis, fatty acid de novo synthesis and oxidation, triacylglycerols and cholesterol transport from the liver were performed. Liver NOx and nitrate concentration and blood biochemistry were also analyzed.

Results: V-PYRRO/NO and metformin reduced liver steatosis with simultaneous reduction of total liver triacylglycerols, diacylglycerols and ceramides fraction and reversed HF-induced decrease in UFA/SFA ratio. V-PYRRO/NO substantially improved postprandial glucose tolerance, while the effect of metformin was modest and more pronounced on HOMA IR index. The anti-steatotic mechanism of V-PYRRO/NO was dependent on NO release, differed from that of metformin and involved improved glucose tolerance and inhibition of de novo fatty acid synthesis by Akt activation and ACC phosphorylation. In turn, major mechanism of metformin action involved increased expression of proteins implicated in mitochondrial biogenesis and metabolism (PGC-1α, PPARα, COX IV, cytochrome c, HADHSC).

Conclusions: V-PYRRO/NO acts as a liver-specific NO donor prodrug affording pronounced anti-steatotic effects and may represent an efficient, mechanistically novel approach to prevent liver steatosis and insulin resistance.
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http://dx.doi.org/10.1016/j.bcp.2014.12.004DOI Listing
February 2015