Publications by authors named "Kamil Przyborowski"

14 Publications

  • Page 1 of 1

Distinct Pharmacological Properties of Gaseous CO and CO-Releasing Molecule in Human Platelets.

Int J Mol Sci 2021 Mar 30;22(7). Epub 2021 Mar 30.

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

Carbon monoxide (CO)-gaseous or released by CO-RMs-both possess antiplatelet properties; however, it remains uncertain whether the mechanisms involved are the same. Here, we characterise the involvement of soluble guanylate cyclase (sGC) in the effects of CO-delivered by gaseous CO-saturated buffer (CO) and generated by CORM-A1-on platelet aggregation and energy metabolism, as well as on vasodilatation in aorta, using light transmission aggregometry, Seahorse XFe technique, and wire myography, respectively. ODQ completely prevented the inhibitory effect of CO on platelet aggregation, but did not modify antiplatelet effect of CORM-A1. In turn, CO did not affect, whereas CORM-A1 substantially inhibited energy metabolism in platelets. Even though activation of sGC by BAY 41-2272 or BAY 58-2667 inhibited significantly platelet aggregation, their effects on energy metabolism in platelets were absent or weak and could not contribute to antiplatelet effects of sGC activation. In contrast, vasodilatation of murine aortic rings, induced either by CO or CORM-A1, was dependent on sGC. We conclude that the source (CO vs. CORM-A1) and kinetics (rapid vs. slow) of CO delivery represent key determinants of the mechanism of antiplatelet action of CO, involving either impairment of energy metabolism or activation of sGG.
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http://dx.doi.org/10.3390/ijms22073584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037872PMC
March 2021

Enhanced cardiac hypoxic injury in atherogenic dyslipidaemia results from alterations in the energy metabolism pattern.

Metabolism 2021 01 13;114:154400. Epub 2020 Oct 13.

Department of Biochemistry, Faculty of Medicine, Medical University of Gdansk, 1 Debinki St., 80-211 Gdansk, Poland. Electronic address:

Objective: Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury.

Methods: Dyslipidaemic apolipoprotein E and low-density lipoprotein receptor (ApoE/LDLR) double knockout (ApoE/LDLR) and wild-type C57BL/6 (WT) mice aged six months old were studied. Cardiac injury under reduced oxygen supply was evaluated by 5 min exposure to 5% oxygen in the breathing air under electrocardiogram (ECG) recording and with the assessment of troponin I release. To address the mechanisms LC/MS was used to analyse the cardiac proteome pattern or in vivo metabolism of stable isotope-labelled substrates and HPLC was applied to measure concentrations of cardiac high-energy phosphates. Furthermore, the effect of blocking fatty acid use with ranolazine on the substrate preference and cardiac hypoxic damage was studied in ApoE/LDLR mice.

Results: Hypoxia induced profound changes in ECG ST-segment and troponin I leakage in ApoE/LDLR mice but not in WT mice. The evaluation of the cardiac proteomic pattern revealed that ApoE/LDLR as compared with WT mice were characterised by coordinated increased expression of mitochondrial proteins, including enzymes of fatty acids' and branched-chain amino acids' oxidation, accompanied by decreased expression levels of glycolytic enzymes. These findings correlated with in vivo analysis, revealing a reduction in the entry of glucose and enhanced entry of leucine into the cardiac Krebs cycle, with the cardiac high-energy phosphates pool maintained. These changes were accompanied by the activation of molecular targets controlling mitochondrial metabolism. Ranolazine reversed the oxidative metabolic shift in ApoE/LDLR mice and reduced cardiac damage induced by hypoxia.

Conclusions: We suggest a novel mechanism for myocardial injury in dyslipidaemia that is consequent to an increased reliance on oxidative metabolism in the heart. The alterations in the metabolic pattern that we identified constitute an adaptive mechanism that facilitates maintenance of metabolic equilibrium and cardiac function under normoxia. However, this adaptation could account for myocardial injury even in a mild reduction of oxygen supply.
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http://dx.doi.org/10.1016/j.metabol.2020.154400DOI Listing
January 2021

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD and ATP Depletion.

Arterioscler Thromb Vasc Biol 2020 10 13;40(10):2376-2390. Epub 2020 Aug 13.

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

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

Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD depletion.
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http://dx.doi.org/10.1161/ATVBAHA.120.314284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505148PMC
October 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

The endothelial barrier and cancer metastasis: Does the protective facet of platelet function matter?

Biochem Pharmacol 2020 06 28;176:113886. Epub 2020 Feb 28.

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

Overwhelming evidence suggests that platelets have a detrimental role in promoting cancer spread via platelet-cancer cell interactions linked to thrombotic mechanisms. On the other hand, a beneficial role of platelets in the preservation of the endothelial barrier in inflammatory conditions has been recently described, a phenomenon that could also operate in cancer-related inflammation. It is tempting to speculate that some antiplatelet strategies to combat cancer metastasis may impair the endogenous platelet-dependent mechanisms preserving endothelial barrier function. If the protective function of platelets is impaired, it may lead to increased endothelial permeability and more efficient cancer cell intravasation in the primary tumor and cancer cell extravasation at metastatic sites. In this commentary, we discuss current evidence that could support this hypothesis.
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http://dx.doi.org/10.1016/j.bcp.2020.113886DOI Listing
June 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

Comparison of Effects of Anti-thrombin Aptamers HD1 and HD22 on Aggregation of Human Platelets, Thrombin Generation, Fibrin Formation, and Thrombus Formation Under Flow Conditions.

Front Pharmacol 2019 20;10:68. Epub 2019 Feb 20.

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

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05-3 μM) and in washed platelets (WPs; 0.005-3 μM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 μM) and HD22 (0.5 μM), respectively. Neither HD1 nor HD22 (3 μM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 μM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 μM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 μM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC at 3 μM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 μM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 μM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 μM). values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure.
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http://dx.doi.org/10.3389/fphar.2019.00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391317PMC
February 2019

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

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 Cognitive Impairment Linked to Brain Endothelium Inflammation in Early Stages of Heart Failure in Mice.

J Am Heart Assoc 2018 03 26;7(7). Epub 2018 Mar 26.

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

Background: Although advanced heart failure (HF) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved.

Methods And Results: Tgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte-specific overexpression of G-αq*44 protein were studied before the end-stage HF, at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6- to 10-month-old but not in 3-month-old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood-brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E-selectin immunoreactivity, which was accompanied by increased amyloid-β accumulation in piriform cortex and increased cortical oxidative stress (8-OHdG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO-dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3- to 10-month-old Tgαq*44 mice, but it was not associated with increased platelet-dependent thrombogenicity.

Conclusions: We report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation.
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http://dx.doi.org/10.1161/JAHA.117.007694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907583PMC
March 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

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

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

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