Publications by authors named "Lukasz Mateuszuk"

26 Publications

  • Page 1 of 1

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

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

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

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

ImmunoSERS microscopy for the detection of smooth muscle cells in atherosclerotic plaques.

Biosens Bioelectron 2019 May 11;133:79-85. Epub 2019 Mar 11.

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

We investigated the suitability of immuno-SERS (iSERS) microscopy for imaging of smooth muscle cells (SMCs) in atherosclerotic plaques. Localization of SMCs is achieved by using SERS-labelled antibodies direct against alpha-smooth muscle actin (SMA). The staining quality of the false-colour iSERS images obtained by confocal Raman microscopy with point mapping is compared with wide-field immunofluorescence images. Both direct (labelled primary antibody) and indirect iSERS staining (unlabelled primary and labelled secondary antibody) techniques were employed. Direct iSERS staining yields results comparable to indirect IF staining, demonstrating the suitability of iSERS in research on atherosclerosis and paving the way for future multiplexed imaging experiments.
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http://dx.doi.org/10.1016/j.bios.2019.02.068DOI Listing
May 2019

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

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

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

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

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

Comparison of Pulmonary and Systemic NO- and PGI-Dependent Endothelial Function in Diabetic Mice.

Oxid Med Cell Longev 2018 4;2018:4036709. Epub 2018 Jun 4.

Jagiellonian Centre for Experimental Pharmacology (JCET), Jagiellonian University, Bobrzyńskiego 14, 30-348 Kraków, Poland.

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (K) and contractile response to TXA analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO generation, and production of 6-keto-PGF were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI-dependent function and diminished CD141 expression.
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http://dx.doi.org/10.1155/2018/4036709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008763PMC
October 2018

Enhanced adhesion of blood platelets to intact endothelium of mesenteric vascular bed in mice with streptozotocin-induced diabetes is mediated by an up-regulated endothelial surface deposition of VWF - In vivo study.

Platelets 2018 Jul 26;29(5):476-485. Epub 2017 Jul 26.

a Department of Haemostasis and Haemostatic Disorders , Medical University of Lodz , Lodz , Poland.

Numerous in vitro experiments have confirmed that a dysfunctional endothelium is characterized by, inter alia, a higher affinity for binding of platelets and leukocytes. However, there is still no direct evidence for greater interaction between platelets and intact endothelium in in vivo animal models of diabetes. Therefore, the present study examines the pro-adhesive properties of endothelium change in vivo as an effect of streptozotocin (STZ)-induced diabetes and the role of two key platelet receptors: GPIb-IX-V and GPIIb/IIIa. Mice of C57BL strain with streptozotocin-induced diabetes were used in the study. Flow cytometry was used to assess basal activation and reactivity of platelets. Adhesion of platelets to the vascular wall was visualized with the use of intravital microscopy in mesentery. The contribution of GPIIb/IIIa and GPIb-IX-V was evaluated by the injection of Fab fragments of respective antibodies. The integrity of the endothelium and vWf expression were evaluated histochemically. Basal activation and reactivity of platelets in streptozotocin-diabetic mice were elevated. Blood platelets adhered more often to the vascular wall of diabetic mice than nondiabetic animals: 11.9 (6.4; 32.8) plt/min/mm (median [IQR]) vs 2.7 (1.3; 6.4) plt/min/mm. The injection of anti-GPIbα antibodies decreased the number of adhering platelets from 89.5 (34.0; 113.1) plt/min/mm (median [IQR]) in mice treated with isotype antibodies to 3.1 (1.7; 5.6) plt/min/mm in mice treated with blocking antibodies. The effect of GPIIb/IIIa blockage was not significant. Immunohistochemistry revealed a higher expression of vWF in the endothelium of STZ mice, but no substantial changes in endothelial morphology were detected. To conclude, the study shows that the platelets interact more frequently with the mesenteric vascular bed in mice with 1-month STZ-induced diabetes than in healthy mice. These interactions are mediated via platelet GPIb-IX-V and are driven by increased expression of vWF in endothelial cells.
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http://dx.doi.org/10.1080/09537104.2017.1332365DOI Listing
July 2018

A possible Fourier transform infrared-based plasma fingerprint of angiotensin-converting enzyme inhibitor-induced reversal of endothelial dysfunction in diabetic mice.

J Biophotonics 2018 02 31;11(2). Epub 2017 Jul 31.

Faculty of Chemistry, Jagiellonian University, Krakow, Poland.

Angiotensin-converting enzyme inhibitors (ACE-I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)-based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased β-sheet formation. Spectral markers at 3010, 1520/1238 cm , representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE-I.
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http://dx.doi.org/10.1002/jbio.201700044DOI Listing
February 2018

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

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

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

Aims: Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis.

Methods And Results: Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular inflammation and improved endothelial function.

Conclusions: This study highlights the importance of extracellular nucleotides and adenosine metabolism in the atherosclerotic vessel in both experimental and clinical setting. The increased eADA activity marks an early stage of atherosclerosis, contributes to its progression and could represent a novel target for therapy.
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http://dx.doi.org/10.1093/cvr/cvw203DOI Listing
November 2016

CD47 and Nox1 Mediate Dynamic Fluid-Phase Macropinocytosis of Native LDL.

Antioxid Redox Signal 2017 06 31;26(16):886-901. Epub 2017 Jan 31.

1 Vascular Medicine Institute, University of Pittsburgh , Pittsburgh, Pennsylvania.

Aims: Macropinocytosis has been implicated in cardiovascular and other disorders, yet physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved.

Results: TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and NADPH oxidase 1 (Nox1) signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin-related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser-3, actin remodeling, and macropinocytotic uptake of unmodified native low-density lipoprotein (nLDL), leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE mice in vivo.

Innovation: Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to early propagation of vascular inflammatory disease.

Conclusions: The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated. Antioxid. Redox Signal. 26, 886-901.
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http://dx.doi.org/10.1089/ars.2016.6834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455613PMC
June 2017

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

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

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

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

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

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

Conclusions: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.
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http://dx.doi.org/10.1186/s12931-016-0423-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007701PMC
August 2016

Alterations in plasma biochemical composition in NO deficiency induced by L-NAME in mice analysed by Fourier Transform Infrared Spectroscopy.

J Biophotonics 2016 10 21;9(10):1098-1108. Epub 2016 Jul 21.

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Krakow, Poland.

Mouse model of nitric oxide deficiency, induced by prolonged treatment with N -nitro-L-arginine methyl ester (L-NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L-NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO-deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L-NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS-DA method provided >95% classification accuracy. Spectral features characteristic for L-NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 - 0.005) were observed between spectra collected for each time-point of the L-NAME treatment versus control subjects. We demonstrated for the first time that NO-deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.
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http://dx.doi.org/10.1002/jbio.201600141DOI Listing
October 2016

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

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

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

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.
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http://dx.doi.org/10.1016/j.jpba.2016.06.050DOI Listing
September 2016

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

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

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

1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)-deficient mice. ApoE/LDLR(-/-) mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1 α and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR(-/-) mice, an effect associated with an improvement in prostacyclin- and nitric oxide-dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.
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http://dx.doi.org/10.1124/jpet.115.228643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047228PMC
February 2016

Endothelium in spots--high-content imaging of lipid rafts clusters in db/db mice.

PLoS One 2014 28;9(8):e106065. Epub 2014 Aug 28.

Faculty of Chemistry, Jagiellonian University, Krakow, Poland; Jagiellonian Centre of Experimental Therapeutics, Krakow, Poland.

Lipid rafts (LRs) are dynamic, sterol- and sphingolipid-enriched nanodomains involved in the regulation of cellular functions and signal transduction, that upon stimuli, via (e.g. association of raft proteins and lipids), may cluster into domains of submicron or micron scale. Up to date, however, lipid raft clusters were observed only under artificially promoted conditions and their formation in vivo has not been confirmed. Using non-destructive approach involving Raman and Atomic Force Microscopy imaging we demonstrated the presence of clustered lipid rafts in endothelium of the aorta of the db/db mice that represent a reliable murine model of type 2 diabetes. The raft clusters in the aorta of diabetic mice were shown to occupy a considerably larger (about 10-fold) area of endothelial cells surface as compared to the control. Observation of pathology-promoted LRs confirms that the cellular increase of lipid content results in clustering of LRs. Clustering of LRs leads to the formation of assemblies with diameters up to 3 micrometers and increased lipid character. This massive clustering of lipid rafts in diabetes may trigger a signaling cascade leading to vascular inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106065PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148353PMC
November 2015

Quantification of plaque area and characterization of plaque biochemical composition with atherosclerosis progression in ApoE/LDLR(-/-) mice by FT-IR imaging.

Analyst 2013 Nov;138(21):6645-52

Faculty of Chemistry, Jagiellonian University, 3 Ingardena Str., 30-060 Krakow, Poland.

In this work the quantitative determination of atherosclerotic lesion area (ApoE/LDLR(-/-) mice) by FT-IR imaging is presented and validated by comparison with atherosclerotic lesion area determination by classic Oil Red O staining. Cluster analysis of FT-IR-based measurements in the 2800-3025 cm(-1) range allowed for quantitative analysis of the atherosclerosis plaque area, the results of which were highly correlated with those of Oil Red O histological staining (R(2) = 0.935). Moreover, a specific class obtained from a second cluster analysis of the aortic cross-section samples at different stages of disease progression (3, 4 and 6 months old) seemed to represent the macrophages (CD68) area within the atherosclerotic plaque.
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http://dx.doi.org/10.1039/c3an01050cDOI Listing
November 2013

Low carbohydrate, high protein diet promotes atherosclerosis in apolipoprotein E/low-density lipoprotein receptor double knockout mice (apoE/LDLR(-/-)).

Atherosclerosis 2012 Aug 17;223(2):327-31. Epub 2012 Jun 17.

Department of Human Nutrition, Faculty of Food Technology, Agricultural University of Kraków, ul. Balicka 122, 30-149 Kraków, Poland.

Although in apoE/LDLR(-/-) mice atherosclerotic plaques develop spontaneously, various atherogenic diets (e.g. Western diet) are frequently used to accelerate the disease in this model. The objective of this study was to compare the effects on atherosclerosis of Western diet and other types of high-fat, high cholesterol, hypertriglyceridemic diets with the effects of the low carbohydrate, high protein (LCHP) diet. 16-18 week old mice with pre-established atherosclerosis were assigned to experimental groups and fed for the next 10 weeks with control diet, margarine diet (margarine 7%), hypertrigliceridemic diet (fructose 62%), high-fat diet (Western diet), high cholesterol diet (egg yolk diet) or with LCHP diet. No differences in body weight were observed among experimental groups. Plasma cholesterol concentration was significantly increased in egg yolk diet- and LCHP diet-fed apoE/LDLR(-/-) mice as compared to other types of diets. Plasma concentration of triacylglycerols was significantly elevated in egg yolk diet- and LCHP diet-fed apoE/LDLR(-/-) mice. The area of atherosclerotic plaques in the aortic root was substantially increased in LCHP diet-fed mice as compared to other types of diets. Furthermore, in brachiocephalic arteries of LCHP diet-fed mice there was evidence of plaque rupture. In conclusion, the LCHP diet promoted atherosclerosis in apoE/LDLR(-/-) mice more intensively than classical Western diet and favored the development of unstable lesions.
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http://dx.doi.org/10.1016/j.atherosclerosis.2012.05.024DOI Listing
August 2012

Functional alterations in endothelial NO, PGI₂ and EDHF pathways in aorta in ApoE/LDLR-/- mice.

Prostaglandins Other Lipid Mediat 2012 Aug 24;98(3-4):107-15. Epub 2012 Mar 24.

Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College, 16 Grzegórzecka Street, 31-531 Krakow, Poland.

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI₂) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI₂-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR⁻/⁻ mice. We found that in the aorta of 2-month-old apoE/LDLR⁻/⁻ mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI₂ and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR⁻/⁻ mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI₂ and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI₂ and EDHF pathways may compensate for the loss of the biological activity of NO.
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http://dx.doi.org/10.1016/j.prostaglandins.2012.02.002DOI Listing
August 2012

Imaging of lipids in atherosclerotic lesion in aorta from ApoE/LDLR-/- mice by FT-IR spectroscopy and Hierarchical Cluster Analysis.

Analyst 2011 Dec 17;136(24):5247-55. Epub 2011 Oct 17.

Faculty of Chemistry, Jagiellonian University, Krakow, Poland.

Spectroscopy-based approaches can provide an insight into the biochemical composition of a tissue sample. In the present work Fourier transform infrared (FT-IR) spectroscopy was used to develop a reliable methodology to study the content of free fatty acids, triglycerides, cholesteryl esters as well as cholesterol in aorta from mice with atherosclerosis (ApoE/LDLR(-/-) mice). In particular, distribution and concentration of palmitic, oleic and linoleic acid derivatives were analyzed. Spectral analysis of pure compounds allowed for clear discrimination between free fatty acids and other similar moieties based on the carbonyl band position (1699-1710 cm(-1) range). In order to distinguish cholesteryl esters from triglycerides a ratio of carbonyl band to signal at 1010 cm(-1) was used. Imaging of lipids in atherosclerotic aortic lesions in ApoE/LDLR(-/-) mice was followed by Hierarchical Cluster Analysis (HCA). The aorta from C57Bl/6J control mice (fed with chow diet) was used for comparison. The measurements were completed with an FT-IR spectrometer equipped with a 128 × 128 FPA detector. In cross-section of aorta from ApoE/LDLR(-/-) mice a region of atherosclerotic plaque was clearly identified by HCA, which was later divided into 2 sub-regions, one characterized by the higher content of cholesterol, while the other by higher contents of cholesteryl esters. HCA of tissues deposited on normal microscopic glass, hence limited to the 2200-3800 cm(-1) spectral range, also identified a region of atherosclerotic plaque. Importantly, this region correlates with the area stained by standard histological staining for atherosclerotic plaque (Oil Red O). In conclusion, the use of FT-IR and HCA may provide a novel tool for qualitative and quantitative analysis of contents and distribution of lipids in atherosclerotic plaque.
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http://dx.doi.org/10.1039/c1an15311kDOI Listing
December 2011

Mildronate, a regulator of energy metabolism, reduces atherosclerosis in apoE/LDLR-/- mice.

Pharmacology 2009 27;83(5):287-93. Epub 2009 Mar 27.

University of Latvia, Riga, Latvia.

Background/aims: Mildronate, an inhibitor of L-carnitine biosynthesis and transport, is used in clinics as a modulator of cellular energy metabolism and is a cardioprotective drug. L-Carnitine is a pivotal molecule in fatty acid oxidation pathways and its regulation in vasculature might be a promising approach for antiatherosclerotic treatment. This study was performed to evaluate the effects of mildronate treatment on the progression of atherosclerosis and the content of L-carnitine in the vascular wall.

Methods: ApoE/LDLR(-/-) mice received mildronate at doses of 30 and 100 mg/kg for 4 months. Lipid profile was measured in plasma and atherosclerotic lesions were analyzed in whole aorta and aortic sinus. L-Carnitine concentration was assessed in rat aortic tissues after 2 weeks of treatment with mildronate at a dose of 100 mg/kg.

Results: The chronic treatment with mildronate at a dose of 100 mg/kg significantly reduced the size of atherosclerotic plaques in the aortic roots and in the whole aorta, and slightly decreased the free cholesterol level. In addition, mildronate treatment decreased L-carnitine concentration in rat aortic tissues.

Conclusions: Long-term mildronate treatment decreases L-carnitine content in aortic tissues and attenuates the development of atherosclerosis in apoE/LDLR(-/-) mice.
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http://dx.doi.org/10.1159/000210015DOI Listing
July 2009

Activation of nicotinamide N-methyltrasferase and increased formation of 1-methylnicotinamide (MNA) in atherosclerosis.

Pharmacol Rep 2009 Jan-Feb;61(1):76-85

Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, Medical College, Kraków, Poland.

Nicotinamide N-methyltrasferase (NMMT) catalyzes the conversion of nicotinamide (NA) to 1-methylnicotinamide (MNA). Recent studies have reported that exogenous MNA exerts anti-thrombotic and anti-inflammatory activity, suggesting that endogenous NMMT-derived MNA may play a biological role in the cardiovascular system. In the present study, we assayed changes in hepatic NNMT activity and MNA plasma levels along the progression of atherosclerosis in apoE/LDLR(-/-) mice, as compared to age-matched wild-type mice. Atherosclerosis progression in apoE/LDLR(-/-) mice was quantified in aortic root, while hepatic NNMT activity and MNA plasma concentrations were concomitantly measured in 2-, 3-, 4-, and 6-month-old mice. In apoE/LDLR(-/-) mice, atherosclerotic plaques developed in the aortic roots beginning at the age of 3 months and gradually increased in size, macrophage content, and inflammation intensity over time, as detected by Oil-Red O staining, CD68 immunostaining, and in situ zymography (MMP2/MMP9 activity). Hepatic NNMT activity was upregulated approximately two-fold in apoE/LDLR(-/-) mice by the age of 2 months, as compared to wild-type mice (1.03 +/- 0.14 vs. 0.64 +/- 0.23 pmol/min/mg, respectively). MNA plasma concentrations were also elevated approximately two-fold (0.30 +/- 0.13 vs. 0.17 +/- 0.04 micromol/l, respectively). As atherosclerosis progressed, hepatic NMMTactivity and MNA plasma concentrations increased five-fold in 6-month-old apoE/LDLR(-/-) mice at the stage of advanced atherosclerotic plaques (NMMT activity: 2.29 +/- 0.34 pmol/min/mg, MNA concentration: 1.083 +/- 0.33 micromol/l). In summary, the present study demonstrated that the progression of vascular inflammation and atherosclerosis was associated with the upregulation of hepatic NNMT activity and subsequent increase in endogenous MNA plasma levels. Given the anti-thrombotic and anti-inflammatory properties of exogenous MNA, robust activation of an endogenous NA-MNA pathway in atherosclerosis may play an important compensatory role.
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http://dx.doi.org/10.1016/s1734-1140(09)70009-xDOI Listing
June 2009

Platelets augment respiratory burst in neutrophils activated by selected species of gram-positive or gram-negative bacteria.

Folia Histochem Cytobiol 2008 ;46(3):383-8

Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.

Neutrophils and platelets circulate in blood system and play important physiological roles as part of immunological system. Neutrophils are the first line of host defense against various intruders, and platelets are satellite cells cooperating with other components of defense system. Recent studies report about the cooperation among these types of cells. We analyzed the effect of platelets on oxygen burst in neutrophils triggered by Staphylococcus aureus and Escherichia coli bacteria in vitro. The effect of platelets on oxygen burst in neutrophils was measured by luminol enhanced chemiluminescence. Opsonized and non-opsonized bacteria were used as activators. Activation of neutrophils with live non-opsonized and opsonized bacteria in the presence of platelets increased the oxygen burst as compared to the same system without platelets. The gram-positive bacteria (Staphylococcus aureus) were causing higher activation than gram-negative bacteria (Escherichia coli). This work demonstrate that platelets potentate the response of neutrophils augmenting their respiratory burst in vitro when triggered by bacteria.
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http://dx.doi.org/10.2478/v10042-008-0052-1DOI Listing
January 2009

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR -/- mice.

Folia Histochem Cytobiol 2008 ;46(2):143-6

Department of Histology, Jagiellonian University Medical College, Kraków, Poland.

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment.
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http://dx.doi.org/10.2478/v10042-008-0021-8DOI Listing
August 2008

Ticlopidine attenuates progression of atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice.

Eur J Pharmacol 2007 Feb 15;556(1-3):129-35. Epub 2006 Nov 15.

Jagiellonian University Medical College, Krakow, Poland.

Platelets are involved in the development of atherothrombosis. However, the anti-atherosclerotic effects of thienopiridines have not been, as yet, proven. We analyzed the effects of ticlopidine on atherogenesis in apolipoprotein E/low density lipoprotein receptor double knockout (apoE/LDLR(-/-)) mice. 2-month-old apoE/LDLR(-/-) mice fed a Western diet (21% fat, 0.15% cholesterol) were treated with ticlopidine (90 mg/kg/day) for a period of 4 months. In 6-month-old apoE/LDLR(-/-) mice treated with ticlopidine and in their non-treated counterparts we analyzed: cholesterol and triglyceride levels, the size of atherosclerotic plaques in aortic roots (oil red-O staining, cross-section method), and in the whole aorta (Sudan IV staining, en face method), the number of macrophages in atherosclerotic plaque (CD68 staining), as well as the endothelial function in the isolated thoracic aorta. Concentrations of total cholesterol and triglycerides in plasma were not altered by treatment with ticlopidine. However, the size of atherosclerotic plaques measured in aortic roots by the cross-section method and the number of macrophages estimated by anti-CD68 staining were significantly reduced by ticlopidine treatment. In contrast, the effect of ticlopidine on the area covered by plaques in the whole aorta (en face analysis) was not statistically significant. Importantly, acetylcholine-induced vasodilation in isolated aorta was improved in ticlopidine-treated apoE/LDLR(-/-) mice as compared to their non-treated counterparts. In conclusion, ticlopidine attenuates the progression of atherosclerosis and improves the endothelial function in apoE/LDLR(-/-) mice.
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http://dx.doi.org/10.1016/j.ejphar.2006.11.028DOI Listing
February 2007