Publications by authors named "Stefan Chlopicki"

207 Publications

A derivative of vitamin B applied several days after exposure reduces lethality of severely irradiated mice.

Sci Rep 2021 Apr 12;11(1):7922. Epub 2021 Apr 12.

Department of Radiobiology and Radiation Protection, Military Institute of Hygiene and Epidemiology, 4 Kozielska St., 01-163, Warsaw, Poland.

Most, if not all, of the hitherto tested substances exert more or less pronounced pro-survival effects when applied before or immediately after the exposure to high doses of ionizing radiation. In the present study we demonstrate for the first time that 1-methyl nicotinamide (MNA), a derivative of vitamin B, significantly (1.6 to 1.9 times) prolonged survival of BALB/c mice irradiated at LD (6.5 Gy), LD (7.0 Gy) or LD (7.5 Gy) of γ-rays when the MNA administration started as late as 7 days post irradiation. A slightly less efficient and only after the highest dose (7.5 Gy) of γ-rays was another vitamin B derivative, 1-methyl-3-acetylpyridine (1,3-MAP) (1.4-fold prolonged survival). These pro-survival effects did not seem to be mediated by stimulation of haematopoiesis, but might be related to anti-inflammatory and/or anti-thrombotic properties of the vitamin B derivatives. Our results show that MNA may represent a prototype of a radioremedial agent capable of mitigating the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of ionizing radiation.
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http://dx.doi.org/10.1038/s41598-021-86870-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041812PMC
April 2021

Toward Raman Subcellular Imaging of Endothelial Dysfunction.

J Med Chem 2021 Apr 6;64(8):4396-4409. Epub 2021 Apr 6.

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

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00051DOI Listing
April 2021

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

Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium.

Int J Mol Sci 2021 Feb 27;22(5). Epub 2021 Feb 27.

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

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome-lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1-30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.
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http://dx.doi.org/10.3390/ijms22052401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957661PMC
February 2021

Low Dose Curcumin Administered in Hyaluronic Acid-Based Nanocapsules Induces Hypotensive Effect in Hypertensive Rats.

Int J Nanomedicine 2021 24;16:1377-1390. Epub 2021 Feb 24.

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

Background: Vascular drug delivery becomes a promising direction in the development of novel therapeutic strategies in the treatment of cardiovascular pathologies, such as hypertension. However, targeted delivery of hydrophobic substances, with poor bioavailability, remains a challenge. Here, we described the hypotensive effects of a low dose of curcumin delivered to the vascular wall using hyaluronic acid-based nanocapsules.

Methods: The group of hypertensive TGR(m-Ren2)27 rats, was administrated respectively with the vehicle, curcumin solution or curcumin delivered using hyaluronic acid-based nanocapsules (HyC12-Cur), for 7 days each, maintaining the wash-out period between treatments. Arterial blood pressure (systolic - SBP, diastolic - DBP) and heart rate (HR) were monitored continuously using a telemetry system (Data Science International), and Mean Arterial Pressure (MAP) was calculated from SBP and DBP.

Results: In hypertensive rats, a low dose of curcumin (4.5 mg/kg) administrated in HyC12-Cur for 7 days resulted in a gradual inhibition of SBP, DBP and MAP increase without an effect on HR. At the end of HyC12-Cur - based treatment changes in SBP, DBP and MAP amounted to -2.0±0.8 mmHg, -3.9±0.7 mmHg and -3.3±0.7 mmHg, respectively. In contrast, the administration of a curcumin solution (4.5 mg/kg) did not result in a significant hypotensive effect and the animals constantly developed hypertension. Vascular delivery of capsules with curcumin was confirmed using newly developed fluorine-rich nanocapsules (HyFC10-PFOB) with a shell based on a HA derivative and similar size as HyC12-Cur. HyFC10-PFOB gave fluorine signals in rat aortas analyzed ex vivo with a F NMR technique after a single intragastric administration.

Conclusion: These results suggest that nanocapsules based on hyaluronic acid, the ubiquitous glycosaminoglycan of the extracellular matrix and an integral part of endothelial glycocalyx, may represent a suitable approach to deliver hydrophobic, poorly bioavailable compounds, to the vascular wall.
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http://dx.doi.org/10.2147/IJN.S291945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917338PMC
March 2021

Deletion of Mcpip1 in Mcpip1Alb mice recapitulates the phenotype of human primary biliary cholangitis.

Biochim Biophys Acta Mol Basis Dis 2021 May 26;1867(5):166086. Epub 2021 Jan 26.

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.

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by progressive destruction of the intrahepatic bile ducts. The immunopathology of PBC involves excessive inflammation; therefore, negative regulators of inflammatory response, such as Monocyte Chemoattractant Protein-1-Induced Protein-1 (MCPIP1) may play important roles in the development of PBC. The aim of this work was to verify whether Mcpip1 expression protects against development of PBC. Genetic deletion of Zc3h12a was used to characterize the role of Mcpip1 in the pathogenesis of PBC in 6-52-week-old mice. We found that Mcpip1 deficiency in the liver (Mcpip1Alb) recapitulates most of the features of human PBC, in contrast to mice with Mcpip1 deficiency in myeloid cells (Mcpip1LysM mice), which present with robust myeloid cell-driven systemic inflammation. In Mcpip1Alb livers, intrahepatic bile ducts displayed proliferative changes with inflammatory infiltration, bile duct destruction, and fibrosis leading to cholestasis. In plasma, increased concentrations of IgG, IgM, and AMA autoantibodies (anti-PDC-E2) were detected. Interestingly, the phenotype of Mcpip1Alb mice was robust in 6-week-old, but milder in 12-24-week-old mice. Hepatic transcriptome analysis of 6-week-old and 24-week-old Mcpip1Alb mice showed 812 and 8 differentially expressed genes, respectively, compared with age-matched control mice, and revealed a distinct set of genes compared to those previously associated with development of PBC. In conclusion, Mcpip1Alb mice display early postnatal phenotype that recapitulates most of the features of human PBC.
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http://dx.doi.org/10.1016/j.bbadis.2021.166086DOI Listing
May 2021

Multi-omic signatures of atherogenic dyslipidaemia: pre-clinical target identification and validation in humans.

J Transl Med 2021 01 6;19(1). Epub 2021 Jan 6.

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

Background: Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH).

Methods: Liquid chromatography/mass spectrometry (LC/MS) and immunoassays were used to find out blood alterations at metabolite and protein levels in dyslipidaemic ApoE/LDLR mice and in FH patients to evaluate their human relevance.

Results: We identified 15 metabolites (inhibitors and substrates of nitric oxide synthase (NOS), low-molecular-weight antioxidants (glutamine, taurine), homocysteine, methionine, 1-methylnicotinamide, alanine and hydroxyproline) and 9 proteins (C-reactive protein, proprotein convertase subtilisin/kexin type 9, apolipoprotein C-III, soluble intercellular adhesion molecule-1, angiotensinogen, paraoxonase-1, fetuin-B, vitamin K-dependent protein S and biglycan) that differentiated FH patients from healthy controls. Most of these changes were consistently found in dyslipidaemic mice and were further amplified if mice were fed an atherogenic (Western or low-carbohydrate, high-protein) diet.

Conclusions: The alterations highlighted the involvement of an immune-inflammatory response system, oxidative stress, hyper-coagulation and impairment in the vascular function/regenerative capacity in response to dyslipidaemia that may also be directly engaged in development of atherosclerosis. Our study further identified potential biomarkers for an increased risk of atherosclerosis that may aid in clinical diagnosis or in the personalized treatment.
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http://dx.doi.org/10.1186/s12967-020-02663-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789501PMC
January 2021

Menadione-induced endothelial inflammation detected by Raman spectroscopy.

Biochim Biophys Acta Mol Cell Res 2021 02 21;1868(2):118911. Epub 2020 Nov 21.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland; Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Str., 30-387 Krakow, Poland. Electronic address:

In this work, the effect of an early oxidative stress on human endothelial cells induced by menadione was studied using a combined methodology of label-free Raman imaging and fluorescence staining. Menadione-induced ROS-dependent endothelial inflammation in human aorta endothelial cells (HAEC) was studied with focus on changes in cytochrome, proteins, nucleic acids and lipids content and their distribution in cells. Fluorescence staining (ICAM-1, VCAM-1, vWF, LipidTox, MitoRos and DCF) was used to confirm endothelial inflammation and ROS generation. The results showed that short time, exposure to menadione did not cause their apoptosis or necrosis (Annexin V Apoptosis Detection Kit) within the 3 h timescale of measurement. On the other hand, 3 h of incubation, did result in endothelial inflammation (ICAM-1, VCAM-1, vWF) that was associated with an increased ROS formation (MitoRos and DCF) suggesting the oxidative stress-mediated inflammation. Chemometric analysis of spectral data enabled the determination of spectroscopic markers of menadione-induced oxidative stress-mediated endothelial inflammation including a decrease of the bands intensity of cytochrome (604, 750, 1128, 1315 and 1585 cm), nucleic acids bands (785 cm), proteins (1005 cm) and increased intensity of lipid bands (722, 1085, 1265, 1303, 1445 and 1660 cm), without changes in the spectroscopic signature of the cell nucleus. In conclusion, oxidative stress resulting in endothelial inflammation was featured by significant alterations in the number of biochemical changes in mitochondria and other cellular compartments detected by Raman spectroscopy. Most of these, coexisted with results from fluorescence imaging, and most importantly occurred earlier than the detection of increased ROS or markers of endothelial inflammation.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118911DOI Listing
February 2021

Bardoxolone Methyl Displays Detrimental Effects on Endothelial Bioenergetics, Suppresses Endothelial ET-1 Release, and Increases Endothelial Permeability in Human Microvascular Endothelium.

Oxid Med Cell Longev 2020 14;2020:4678252. Epub 2020 Oct 14.

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

Nrf2 is a master regulator of antioxidant cellular defence, and agents activating the Nrf2 pathway have been tested in various diseases. However, unexpected side effects of cardiovascular nature reported for bardoxolone methyl in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease (the BEACON trial) still have not been fully explained. Here, we aimed to characterize the effects of bardoxolone methyl compared with other Nrf2 activators-dimethyl fumarate and L-sulforaphane-on human microvascular endothelium. Endothelial toxicity, bioenergetics, mitochondrial membrane potential, endothelin-1 (ET-1) release, endothelial permeability, Nrf2 expression, and ROS production were assessed in human microvascular endothelial cells (HMEC-1) incubated for 3 and 24 hours with 100 nM-5 M of either bardoxolone methyl, dimethyl fumarate, or L-sulforaphane. Three-hour incubation with bardoxolone methyl (100 nM-5 M), although not toxic to endothelial cells, significantly affected endothelial bioenergetics by decreasing mitochondrial membrane potential (concentrations ≥ 3 M), decreasing spare respiratory capacity (concentrations ≥ 1 M), and increasing proton leak (concentrations ≥ 500 nM), while dimethyl fumarate and L-sulforaphane did not exert such actions. Bardoxolone methyl at concentrations ≥ 3 M also decreased cellular viability and induced necrosis and apoptosis in the endothelium upon 24-hour incubation. In turn, endothelin-1 decreased permeability in endothelial cells in picomolar range, while bardoxolone methyl decreased ET-1 release and increased endothelial permeability even after short-term (3 hours) incubation. In conclusion, despite that all three Nrf2 activators exerted some beneficial effects on the endothelium, as evidenced by a decrease in ROS production, bardoxolone methyl, the most potent Nrf2 activator among the tested compounds, displayed a distinct endothelial profile of activity comprising detrimental effects on mitochondria and cellular viability and suppression of endothelial ET-1 release possibly interfering with ET-1-dependent local regulation of endothelial permeability.
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http://dx.doi.org/10.1155/2020/4678252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584962PMC
October 2020

A new approach to study human perivascular adipose tissue of the internal mammary artery by fiber-optic Raman spectroscopy supported by spectral modelling.

Analyst 2021 Jan;146(1):270-276

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

Perivascular adipose tissue (PVAT) regulates vascular function and represents a novel therapeutic target in vascular diseases. In this work, a new approach based on fiber-optic Raman spectroscopy and spectral modelling was used to characterize the chemical content of the PVAT of the internal mammary artery (IMA) of patients with advanced coronary atherosclerosis (n = 10) undergoing coronary bypass surgery. Our results showed a high degree of lipid unsaturation and low carotenoid content in the PVAT of the IMA of patients with more advanced coronary artery disease. Moreover, the spectral modelling of the IMA's PVAT composition indicated that glyceryl trioleate was a major PVAT lipid and for patients with relatively low levels of β-carotene, it was accompanied by arachidonic acid and glyceryl trilinolenate. In summary, our proof-of-concept study suggests that carotenoid content and lipid unsaturation degree may reflect the PVAT functional status and a Raman-based assessment of the PVAT of the IMA could prove useful as a novel diagnostic tool to rapidly define the PVAT phenotype in a grafted artery in patients undergoing coronary bypass.
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http://dx.doi.org/10.1039/d0an01868fDOI Listing
January 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

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

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Cancers (Basel) 2020 Oct 2;12(10). Epub 2020 Oct 2.

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

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer-endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.
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http://dx.doi.org/10.3390/cancers12102850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601413PMC
October 2020

Eosinophils adhesion assay as a tool for phenotypic drug screening - The pharmacology of 1,3,5 - Triazine and 1H-indole like derivatives against the human histamine H receptor.

Eur J Pharmacol 2021 Jan 2;890:173611. Epub 2020 Oct 2.

Jagiellonian University Medical College, Faculty of Pharmacy, Department of Technology and Biotechnology of Drugs, Medyczna 9, 30-688, Kraków, Poland. Electronic address:

Histamine is a pleiotropic biogenic amine, having affinity towards four distinct histamine receptors. The existing pharmacological studies suggest the usefulness of histamine H receptor ligands in the treatment of many inflammatory and immunomodulatory diseases, including allergic rhinitis, asthma, atopic dermatitis, colitis or pruritus. Up to date, several potent histamine H receptor ligands were developed, none of which was registered as a drug yet. In this study, a series of potent indole-like and triazine derivatives were tested, in radioligand displacement and functional assays at histamine H receptor, as well as in human eosinophils adhesion assay to endothelium. For selected compounds permeability, cytotoxicity, metabolic and in vivo studies were conducted. Adhesion assay differentiated the activity of different groups of compounds with a known affinity towards the histamine H receptor. Most of the tested compounds downregulated the number of adherent cells. However, adhesion assay revealed additional properties of tested compounds that had not been detected in radioligand displacement and aequorin-based functional assays. Furthermore, for some tested compounds, these abnormal effects were confirmed during the in vivo studies. In conclusion, eosinophils adhesion assay uncovered pharmacological activity of histamine H receptor ligands that has been later confirmed in vivo, underscoring the value of well-suited cell-based phenotypic screening approach in drug discovery.
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http://dx.doi.org/10.1016/j.ejphar.2020.173611DOI Listing
January 2021

Age-related and atherosclerosis-related erythropathy in ApoE/LDLR mice.

Biochim Biophys Acta Mol Basis Dis 2020 12 17;1866(12):165972. Epub 2020 Sep 17.

Jagiellonian Center for Experimental Therapeutics, Jagiellonian University, 14 Bobrzyńskiego St., 30-348 Krakow, Poland. Electronic address:

In this work we applied a multimodal approach to define the age- and atherosclerosis-related biochemical and functional alterations in red blood cells (RBCs) in ApoE/LDLR mice. Our results revealed that age-related changes in RBCs, such as decreases in RBC deformability and mean height, were more pronounced in ApoE/LDLR mice than in age-matched control mice (C57BL/6J). The decreases in phospholipid content and level of lipid unsaturation were accompanied by an increase in cholesterol esters and esterified lipids in RBC membranes in aged C57BL/6J mice. The age-related decrease in the phospholipid content was more pronounced in ApoE/LDLR mice. In contrast, the increase in the total lipid content in RBC membranes occurred only in ApoE/LDLR mice with advanced atherosclerosis. The age-related alterations also included a decrease in the ratio of turns to α-helices in the secondary structure of hemoglobin (Hb) inside intact RBCs. On the other hand, an increase in the ratio of unordered conformations to α-helices of Hb was observed only in ApoE/LDLR mice and occurred already at the age of 5-weeks. This was related to hypercholesterolemia and resulted in an increased oxygen-carrying capacity. In conclusion, progressive mechanical and functional alterations of RBCs in aged ApoE/LDLR mice were more pronounced than in age-matched C57BL/6J mice. Although, several biochemical changes in RBCs in aged ApoE/LDLR mice recapitulated age-dependent changes observed in control mice, some biochemical features of RBC membranes attributed to hypercholesterolemia were distinct and could contribute to the accelerated deterioration of RBC function in ApoE/LDLR mice.
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http://dx.doi.org/10.1016/j.bbadis.2020.165972DOI Listing
December 2020

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

Irreversible alterations in the hemoglobin structure affect oxygen binding in human packed red blood cells.

Biochim Biophys Acta Mol Cell Res 2020 11 30;1867(11):118803. Epub 2020 Jul 30.

Jagiellonian Center for Experimental Therapeutics, Jagiellonian University, 14 Bobrzynskiego St., 30-348 Krakow, Poland. Electronic address:

The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R'. On the secondary structure level, disordered protein organization involved formation of β-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R', which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118803DOI Listing
November 2020

Endothelial barrier integrity in COVID-19-dependent hyperinflammation: does the protective facet of platelet function matter?

Cardiovasc Res 2020 08;116(10):e118-e121

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

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http://dx.doi.org/10.1093/cvr/cvaa190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432357PMC
August 2020

Distinct Chemical Changes in Abdominal but Not in Thoracic Aorta upon Atherosclerosis Studied Using Fiber Optic Raman Spectroscopy.

Int J Mol Sci 2020 Jul 8;21(14). Epub 2020 Jul 8.

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

Fiber optic Raman spectroscopy and Raman microscopy were used to investigate alterations in the aorta wall and the surrounding perivascular adipose tissue (PVAT) in the murine model of atherosclerosis ( mice). Both abdominal and thoracic parts of the aorta were studied to account for the heterogenic chemical composition of aorta and its localization-dependent response in progression of atherosclerosis. The average Raman spectra obtained for both parts of aorta cross sections revealed that the chemical composition of intima-media layers along aorta remains relatively homogeneous while the lipid content in the adventitia layer markedly increases with decreasing distance to PVAT. Moreover, our results demonstrate that the increase of the lipid to protein ratio in the aorta wall correlates directly with the increased unsaturation level of lipids in PVAT and these changes occur only in the abdominal, but not in thoracic, aorta. In summary, distinct pathophysiological response in the aortic vascular wall could be uncovered by fiber optic Raman spectroscopy based on simple parameters detecting chemical contents of lipids in PVAT.
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http://dx.doi.org/10.3390/ijms21144838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402309PMC
July 2020

Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 09 11;1865(9):158758. Epub 2020 Jun 11.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland; Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Str., 30-387 Krakow, Poland.

Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6-91.3%) with an important content of arachidonic acid (8.7-19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.
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http://dx.doi.org/10.1016/j.bbalip.2020.158758DOI Listing
September 2020

Reversal of endothelial dysfunction by nicotinamide mononucleotide via extracellular conversion to nicotinamide riboside.

Biochem Pharmacol 2020 08 8;178:114019. Epub 2020 May 8.

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

Background: Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are effective substrates for NAD synthesis, which may act as vasoprotective agents. Here, we characterize the effects of NMN and NR on endothelial inflammation and dysfunction and test the involvement of CD73 in these effects.

Materials And Methods: The effect of NMN and NR on IL1β- or TNFα-induced endothelial inflammation (ICAM1 and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38, CD73), were studied in HAECs. The effect of NMN and NR on angiotensin II-induced impairment of endothelium-dependent vasodilation was analyzed in murine aortic rings. The involvement of CD73 in NMN and NR effects was tested using CD73 inhibitor-AOPCP, or CD73 mice.

Results: 24 h-incubation with NMN and NR induced anti-inflammatory effects in HAEC stimulated by IL1β or TNFα, as evidenced by a reduction in ICAM1 and vWF expression. Effects of exogenous NMN but not NR was abrogated in the presence of AOPCP, that efficiently inhibited extracellular endothelial conversion of NMN to NR, without a significant effect on the metabolism of NMN to NA. Surprisingly, intracellular NAD concentration increased in HAEC stimulated by IL1β or TNFα and this effect was associated with upregulation of NAMPT and CD73, whereas changes in CD38 expression were less pronounced. NMN and NR further increased NAD in IL1β-stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-induced response. In ex vivo aortic rings stimulated with angiotensin II for 24 h, NO-dependent vasorelaxation induced by acetylcholine was impaired. NMN and NR, both prevented Ang II-induced endothelial dysfunction in the aorta. In aortic rings taken from CD73 mice NMN effect was lost, whereas NR effect was preserved.

Conclusion: NMN and NR modulate intracellular NAD content in endothelium, inhibit endothelial inflammation and improve NO-dependent function by CD73-dependent and independent pathways, respectively. Extracellular conversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent important vasoprotective mechanisms to maintain intracellular NAD.
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http://dx.doi.org/10.1016/j.bcp.2020.114019DOI Listing
August 2020

Corrigendum to "Differential response of liver sinusoidal endothelial cells and hepatocytes to oleic and palmitic acid revealed by Raman and CARS imaging." [Biochim. Biophys. Acta Mol. Basis Dis. 1866 (2020) 165763-72].

Biochim Biophys Acta Mol Basis Dis 2020 Aug 27;1866(8):165804. Epub 2020 Apr 27.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland; Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland. Electronic address:

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http://dx.doi.org/10.1016/j.bbadis.2020.165804DOI Listing
August 2020

Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension.

Redox Biol 2020 07 18;34:101515. Epub 2020 Apr 18.

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

Arterial hypertension is the most important risk factor for the development of cardiovascular disease. Recently, aircraft noise has been shown to be associated with elevated blood pressure, endothelial dysfunction, and oxidative stress. Here, we investigated the potential exacerbated cardiovascular effects of aircraft noise in combination with experimental arterial hypertension. C57BL/6J mice were infused with 0.5 mg/kg/d of angiotensin II for 7 days, exposed to aircraft noise for 7 days at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A), or subjected to both stressors. Noise and angiotensin II increased blood pressure, endothelial dysfunction, oxidative stress and inflammation in aortic, cardiac and/or cerebral tissues in single exposure models. In mice subjected to both stressors, most of these risk factors showed potentiated adverse changes. We also found that mice exposed to both noise and ATII had increased phagocytic NADPH oxidase (NOX-2)-mediated superoxide formation, immune cell infiltration (monocytes, neutrophils and T cells) in the aortic wall, astrocyte activation in the brain, enhanced cytokine signaling, and subsequent vascular and cerebral oxidative stress. Exaggerated renal stress response was also observed. In summary, our results show an enhanced adverse cardiovascular effect between environmental noise exposure and arterial hypertension, which is mainly triggered by vascular inflammation and oxidative stress. Mechanistically, noise potentiates neuroinflammation and cerebral oxidative stress, which may be a potential link between both risk factors. The results indicate that a combination of classical (arterial hypertension) and novel (noise exposure) risk factors may be deleterious for cardiovascular health.
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http://dx.doi.org/10.1016/j.redox.2020.101515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327989PMC
July 2020

Impact of long-lasting spontaneous physical activity on bone morphogenetic protein 4 in the heart and tibia in murine model of heart failure.

Physiol Rep 2020 04;8(8):e14412

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

Bone morphogenetic protein 4 (BMP4) plays an important role in bone remodeling and in heart failure pathogenesis. The aim of this study was to evaluate the effect of spontaneous physical activity on the expression of BMP4 in the heart and tibia of the transgenic (Tgαq*44) mice, representing a model of chronic heart failure. Tgαq*44 and wild-type FVB mice (WT) were randomly assigned either to sedentary or to trained groups undergoing 8 weeks of spontaneous wheel running. The BMP4 protein expression in heart and tibiae was evaluated using Western immunoblotting and the phosphorus and calcium in the tibiae was assessed using the X-ray microanalysis. BMP4 content in the hearts of the Tgαq*44-sedentary mice was by ~490% higher than in the WT-sedentary mice, whereas in tibiae the BMP4 content of the Tgαq*44-sedentary mice was similar to that in the WT-sedentary animals. Tgαq*44 mice revealed by ~28% poorer spontaneous physical activity than the WT mice. No effect of performed physical activity on the BMP4 content in the hearts of either in the Tgαq*44 or WT mice was observed. However, 8-week spontaneous wheel running resulted in a decrease in the BMP4 expression in tibiae (by ~43%) in the group of Tgαq*44 mice only, with no changes in their bone phosphorus and calcium contents. We have concluded that prolonged period of spontaneous physical exercise does not increase the risk of the progression of the BMP4-mediated pathological cardiac hypertrophy and does not affect bone mineral status in the chronic heart failure mice.
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http://dx.doi.org/10.14814/phy2.14412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174143PMC
April 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

Development and validation of a rapid, specific and sensitive LC-MS/MS bioanalytical method for eicosanoid quantification - assessment of arachidonic acid metabolic pathway activity in hypertensive rats.

Biochimie 2020 Apr - May;171-172:223-232. Epub 2020 Mar 13.

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

Lipid mediators such as eicosanoids maintain various physiological processes, and their alterations are involved in the development of numerous cardiovascular diseases. Therefore, the reliable assessment of their profile could be helpful in diagnosis as well as in eicosanoid biomarker-based treatment. Hence, the presented study aimed to develop and validate a new rapid, specific and sensitive LC-MS/MS method for quantification of arachidonic acid-derived eicosanoids in plasma, including lipid mediators generated via COX-, LOX- and CYP450-dependent pathways. The developed method features high sensitivity because the lower limit of quantification ranged from 0.05 to 0.50 ng mL as well as the accuracy and precision estimated within 88.88-111.25% and 1.03-11.82%, respectively. An application of a simple and fast liquid-liquid extraction procedure for sample cleaning resulted in a highly satisfactory recovery of the analytes (>88.30%). Additionally, the method was validated using artificial plasma, an approach that enabled the elimination of the matrix effect caused by an endogenous concentration of studied lipid mediators. Importantly, the presented LC-MS/MS method allowed for simultaneous quantitative and qualitative [quan/qual] analysis of the selected eicosanoids, leading to an additional improvement of the method specificity. Moreover, the validated method was successfully applied for eicosanoid profiling in rat, mouse and human plasma samples, clearly demonstrating the heterogeneity of the profile of studied lipid mediators in those species.
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http://dx.doi.org/10.1016/j.biochi.2020.03.010DOI Listing
December 2020

Differential response of liver sinusoidal endothelial cells and hepatocytes to oleic and palmitic acid revealed by Raman and CARS imaging.

Biochim Biophys Acta Mol Basis Dis 2020 06 10;1866(6):165763. Epub 2020 Mar 10.

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland; Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland. Electronic address:

Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid. We aimed to observe the changes in the composition of LDs associated with metabolism and degradation of lipids. We were able to characterize the diversity in the formation of LDs in endothelium as compared to hepatocytes, as well as the differences in the formation of LDs and degradation manner with respect to the used fatty acid. Thus, for the first time the spectral characteristics of LDs formed in endothelial cells after incubation with oleic and palmitic acid is presented, including the time-dependent changes in their chemical composition.
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http://dx.doi.org/10.1016/j.bbadis.2020.165763DOI Listing
June 2020

Revisiting pharmacology of oxidative stress and endothelial dysfunction in cardiovascular disease: Evidence for redox-based therapies.

Free Radic Biol Med 2020 09 1;157:15-37. Epub 2020 Mar 1.

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

According to the latest Global Burden of Disease Study data, non-communicable diseases in general and cardiovascular disease (CVD) in particular are the leading cause of premature death and reduced quality of life. Demographic shifts, unhealthy lifestyles and a higher burden of adverse environmental factors provide an explanation for these findings. The expected growing prevalence of CVD requires enhanced research efforts for identification and characterisation of novel therapeutic targets and strategies. Cardiovascular risk factors including classical (e.g. hypertension, diabetes, hypercholesterolaemia) and non-classical (e.g. environmental stress) factors induce the development of endothelial dysfunction, which is closely associated with oxidant stress and vascular inflammation and results in CVD, particularly in older adults. Most classically successful therapies for CVD display vasoprotective, antioxidant and anti-inflammatory effects, but were originally designed with other therapeutic aims. So far, only a few 'redox drugs' are in clinical use and many antioxidant strategies have not met expectations. With the present review, we summarise the actual knowledge on CVD pathomechanisms, with special emphasis on endothelial dysfunction, adverse redox signalling and oxidative stress, highlighting the preclinical and clinical evidence. In addition, we provide a brief overview of established CVD therapies and their relation to endothelial dysfunction and oxidative stress. Finally, we discuss novel strategies for redox-based CVD therapies trying to explain why, despite a clear link between endothelial dysfunction and adverse redox signalling and oxidative stress, redox- and oxidative stress-based therapies have not yet provided a breakthrough in the treatment of endothelial dysfunction and CVD.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.02.026DOI Listing
September 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

Heterogeneity of chemical composition of lipid droplets in endothelial inflammation and apoptosis.

Biochim Biophys Acta Mol Cell Res 2020 06 18;1867(6):118681. Epub 2020 Feb 18.

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

Lipid droplets (LDs) play regulatory role in various cells but their significance in endothelial pathophysiology is still not well understood. Here, we studied LDs in in situ endothelial cells (ECs) in isolated blood vessels stimulated with pro-inflammatory or pro-apoptotic stimuli using Raman and fluorescence imaging. Endothelial inflammation induced by murine TNF-α (mTNF-α) was featured by overexpression of ICAM-1, vWF, increased production of PGI, and was associated with the formation of low number of LDs. However in the presence of atglistatin, the inhibitor of triacyclglycerols hydrolysis, the number of LDs significantly increased. In contrast, in endothelium stimulated by human TNF-α (hTNF-α) or FasL, apart from endothelial inflammation, displayed also apoptosis as evidenced by high annexin expression and significant LDs formation. Raman imaging confirmed that LDs were localized in endothelium and revealed significant heterogeneity in biochemical composition of endothelial LDs that dependent on endothelial stimuli. Repertoire of LDs included LDs rich in highly unsaturated lipids, assigned to the inflammation, as well as LDs featured by more saturated lipids linked to apoptosis, where Raman signals indicating content of cholesterol and phospholipids were higher for endothelial apoptosis in comparison to endothelial inflammation. The heterogeneity in chemical composition of LDs suggested more complex pathophysiological role of endothelial LDs then previously appreciated.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118681DOI Listing
June 2020