Publications by authors named "Patrycja Kaczara"

17 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

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

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

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

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

LSEC Fenestrae Are Preserved Despite Pro-inflammatory Phenotype of Liver Sinusoidal Endothelial Cells in Mice on High Fat Diet.

Front Physiol 2019 12;10. Epub 2019 Feb 12.

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

Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2-8 week) and late (15-20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE and PGF) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.
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http://dx.doi.org/10.3389/fphys.2019.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379824PMC
February 2019

Kidney injury by cyclosporine A is aggravated in heme oxygenase-1 deficient mice and involves regulation of microRNAs.

Acta Biochim Pol 2018 Nov;65(4):613-620

1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Kardio-Med Silesia, Zabrze, Poland.

Cyclosporine A (CsA), a widely used immunosuppressive drug, exerts nephrotoxic activities, as demonstrated by increased tubulointerstitial fibrosis, inflammation and podocyte damage. Recently, a number of microRNAs expressed in the kidney have been reported to be elevated during renal damage. Our aim was to investigate the effect of CsA on selected microRNAs in the mouse kidney after CsA treatment. Moreover, as heme oxygenase-1 (HO-1, encoded by the Hmox1 gene) was shown to play a protective role during kidney disorders, we assessed whether HO-1 deficiency in vivo influences the CsA-regulated microRNAs' expression. We have observed that the pro-fibrotic miR-21 and pro-apoptotic miR-34a expression was upregulated in kidneys of HO-1 deficient mice and it was further enhanced by CsA. Concomitantly, the level of anti-fibrotic microRNAs, belonging to miR-29 and miR-200 families, was down-regulated after CsA treatment. Generally, Hmox1 knock-out (Hmox1-/-) animals were more susceptible to CsA treatment, as the mortality rate was 4 out of 9 Hmox1-/- mice, and increased fibrosis (Tgfb2, Pai1), inflammation (Il6) and apoptosis (Cdkn1a-p21) were noticed in the HO-1 deficient kidneys. In summary, our data demonstrate that CsA induces significant changes in the expression of renal microRNAs and emphasize HO-1 deficiency as an important factor contributing to the CsA-mediated renal toxicity.
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http://dx.doi.org/10.18388/abp.2018_2658DOI Listing
November 2018

Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

Pharmacol Res 2018 10 6;136:160-171. Epub 2018 Sep 6.

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

Interactions between cancer cells and the endothelium play a crucial role during metastasis. Here we examined the effects of a carbon monoxide-releasing molecule (CORM-401) and a nitric oxide donor (PAPA NONOate) given alone or in combination on breast cancer cell adhesion and transmigration across the lung microvascular endothelium. We further explored whether the effects of CO and NO on cancer-endothelial cells interactions are linked with changes in cellular bioenergetics in breast cancer or endothelial cells. We found that CORM-401 and PAPA NONOate alone or in combination markedly decreased transmigration of breast cancer cells across human lung microvascular endothelial cells (hLMVEC), while cancer cell adhesion to the endothelium was diminished only by a combination of the two compounds. In hLMVECs, CORM-401 decreased glycolysis and stimulated mitochondrial respiration, while in breast cancer cells CORM-401 decreased both glycolysis and mitochondrial respiration. In contrast, PAPA NONOate decreased mitochondrial respiration and slightly stimulated glycolysis in both cell lines. When both donors were given together, mitochondrial respiration and glycolysis were both profoundly inhibited, and cancer-endothelial cells interactions were additively suppressed. Intercellular adhesion molecule-1 (ICAM-1), involved in breast cancer cell adhesion to hLMVECs, was downregulated by CORM-401 and PAPA NONOate, when applied alone, while a combination of both compounds did not cause any enhancement of ICAM-1 downregulation. In conclusion, our findings demonstrate that CO and NO differently affect cellular bioenergetics of cancer and endothelial cells and suggest that this phenomenon may contribute to additive anti-adhesive and anti-transmigratory effects of CO and NO. Pharmacological attenuation of metabolism represents a novel, effective way to prevent cancer cell interactions with the endothelium, that is an energy-demanding process.
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http://dx.doi.org/10.1016/j.phrs.2018.09.005DOI Listing
October 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

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

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

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

Carbon monoxide (CO) modulates mitochondrial respiration, but the mechanisms involved are not completely understood. The aim of the present study was to investigate the acute effects of CO on bioenergetics and metabolism in intact EA.hy926 endothelial cells using live cell imaging techniques. Our findings indicate that CORM-401, a compound that liberates CO, reduces ATP production from glycolysis, and induces a mild mitochondrial depolarization. In addition, CO from CORM-401 increases mitochondrial calcium and activates complexes I and II. The subsequent increase in mitochondrial respiration leads to ATP production through oxidative phosphorylation. Thus, our results show that nonactivated endothelial cells rely primarily on glycolysis, but in the presence of CO, mitochondrial Ca increases and activates respiration that shifts the metabolism of endothelial cells from glycolysis- to oxidative phosphorylation-dependent ATP production.
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http://dx.doi.org/10.1002/1873-3468.12434DOI Listing
October 2016

Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules.

Free Radic Biol Med 2016 08 20;97:320-329. Epub 2016 Jun 20.

Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. Electronic address:

To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV-vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects - modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.06.012DOI Listing
August 2016

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

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Biochim Biophys Acta 2015 Oct 14;1847(10):1297-309. Epub 2015 Jul 14.

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

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.
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http://dx.doi.org/10.1016/j.bbabio.2015.07.004DOI Listing
October 2015

Comparative endothelial profiling of doxorubicin and daunorubicin in cultured endothelial cells.

Toxicol In Vitro 2015 Apr 18;29(3):512-21. Epub 2014 Dec 18.

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:

Although anthracycline antibiotics have been successfully used for nearly half a century in the treatment of various malignancies, their use is limited by their cardiac and vascular toxicities, and the mechanisms of these toxicities are still not entirely clear. Herein, we comprehensively characterized cytotoxic effects of two structurally related anthracyclines, doxorubicin and daunorubicin. In nanomolar concentrations, both drugs induced DNA damage and increased nuclear area that were associated with their accumulation in the nucleus (doxorubicin ⩾50 nM and daunorubicin ⩾25 nM) as evidence by Raman microspectroscopy at 3820-4245 cm(-1). At low micromolar concentrations, doxorubicin (⩾5 μM) and daunorubicin (⩾1 μM) increased the generation of reactive oxygen species, decreased intracellular reduced glutathione, induced an alteration in endothelial elasticity and caused a reorganization of the F-actin cytoskeleton. In isolated mouse aortic rings, doxorubicin (⩾50 μM) was less potent than daunorubicin (⩾5 μM) in impairing the endothelium-dependent response. In summary, using a comprehensive endothelial profiling approach, we demonstrated clear-cut differences in the potencies to induce endotheliotoxic responses for two structurally similar chemotherapeutics, at a nuclear, cytosolic and membrane levels. Furthermore, our results suggest that the differences in the endothelial toxicities of doxorubicin and daunorubicin are linked to differences in their nuclear accumulation and the DNA damage-triggered response of the endothelium.
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http://dx.doi.org/10.1016/j.tiv.2014.12.009DOI Listing
April 2015

Melanosome-iron interactions within retinal pigment epithelium-derived cells.

Pigment Cell Melanoma Res 2012 Nov 1;25(6):804-14. Epub 2012 Oct 1.

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

Melanosomes were recently shown to protect ARPE-19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound-to-melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H(2) O(2) , but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.
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http://dx.doi.org/10.1111/pcmr.12008DOI Listing
November 2012

Dynamic analyses reveal cytoprotection by RPE melanosomes against non-photic stress.

Mol Vis 2011 9;17:2864-77. Epub 2011 Nov 9.

Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI 53226-4812, USA.

Purpose: Isolated melanosomes are known to have antioxidant properties but whether the granules perform an antioxidant function within cells is unclear. The aim of this study was to determine whether retinal pigment epithelium (RPE) melanosomes are competent to protect cultured cells against non-photic oxidative stress induced by treatment with H(2)O(2).

Methods: Porcine melanosomes, either untreated or irradiated with visible light to simulate age-related melanin photobleaching, were introduced by phagocytosis into ARPE-19 cells. Cells were treated with H(2)O(2) using two delivery methods: as a pulse, or by continuous generation following addition of glucose oxidase to the medium. Cell survival in melanosome-containing cells was compared to survival in cells containing phagocytosed control latex beads using two real-time cell death assays.

Results: Following H(2)O(2) delivery by either method, greater resistance to critical concentrations of H(2)O(2) was seen for cells containing melanosomes than for cells containing beads. Melanosome-mediated protection manifested as a delay in the time of onset of cell death and a slower rate of cell death over time. Photobleaching diminished the stress resistance conferred by the pigment granules. Individual cells in co-cultures were differentially sensitive to oxidative stress depending upon their particle content. Additional features of the time course of the cell death response were revealed by the dynamic analyses conducted over hours post oxidant treatment.

Conclusions: The results show, for the first time, that melanosomes perform a cytoprotective function within cultured cells by acting as an antioxidant. The outcomes imply that melanosomes perform functions within RPE cells aside from those related to light irradiation, and also suggest that susceptibility to ubiquitous pro-oxidizing agents like H(2)O(2) is partly determined by discrete features of individual RPE cells such as their granule content.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224837PMC
April 2012

Dynamics of H2O2 availability to ARPE-19 cultures in models of oxidative stress.

Free Radic Biol Med 2010 Apr 25;48(8):1064-70. Epub 2010 Jan 25.

Department of Biophysics, Jagiellonian University, 30-387 Kraków, Poland.

Oxidative injury to cells such as the retinal pigment epithelium (RPE) is often modeled using H(2)O(2)-treated cultures, but H(2)O(2) concentrations are not sustained in culture medium. Here medium levels of H(2)O(2) and cytotoxicity were analyzed in ARPE-19 cultures after H(2)O(2) delivery as a single pulse or with continuous generation using glucose oxidase (GOx). When added as a pulse, H(2)O(2) is rapidly depleted (within 2 h); cytotoxicity at 24 h, determined by the MTT assay for mitochondrial function, is unaffected by medium replacement at 2 h. Continuous generation of H(2)O(2) produces complex outcomes. At low GOx concentrations, H(2)O(2) levels are sustained by conditions under which generation matches depletion, but when GOx concentrations produce cytotoxic levels of H(2)O(2), oxidant depletion accelerates. Acceleration results partly from the release of contents from oxidant-damaged cells as indicated by testing depletion after controlled membrane disruption with detergents. Cytotoxicity analyses show that cells can tolerate short exposure to high H(2)O(2) doses delivered as a pulse but are susceptible to lower chronic doses. The results provide broadly applicable guidance for using GOx to produce sustained H(2)O(2) levels in cultured cells. This approach will be specifically useful for modeling chronic stress relevant to RPE aging and have a wider value for studying cellular effects of sublethal oxidant injury and for evaluating antioxidants that may protect significantly against mild but not lethal stress.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839027PMC
April 2010