Gdansk, Gdansk, Poland c Department of Biochemistry and Clinical Physiology, Medical

Transcription

1 This article was downloaded by: [Gdanski Uniwersytet Medyczny] On: 11 July 2014, At: 08:04 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Nucleosides, Nucleotides and Nucleic Acids Publication details, including instructions for authors and subscription information: Extracellular Nucleotide Catabolism in Aortoiliac Bifurcation of Atherosclerotic ApoE/LDLr Double Knock Out Mice Barbara Kutryb-Zajac a, Paulina Zukowska a, Marta Toczek a, Magdalena Zabielska a, Marcin Lipinski b, Iwona Rybakowska c, Stefan Chlopicki d, Ewa M. Slominska a & Ryszard T. Smolenski a a Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland b Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Gdansk, Poland c Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Gdansk, Poland d Department of Experimental Pharmacology, Jagiellonian University Medical College and Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland Published online: 18 Jun To cite this article: Barbara Kutryb-Zajac, Paulina Zukowska, Marta Toczek, Magdalena Zabielska, Marcin Lipinski, Iwona Rybakowska, Stefan Chlopicki, Ewa M. Slominska & Ryszard T. Smolenski (2014) Extracellular Nucleotide Catabolism in Aortoiliac Bifurcation of Atherosclerotic ApoE/ LDLr Double Knock Out Mice, Nucleosides, Nucleotides and Nucleic Acids, 33:4-6, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or

2 howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

3 Nucleosides, Nucleotides and Nucleic Acids, 33: , 2014 Copyright C Taylor and Francis Group, LLC ISSN: print / online DOI: / EXTRACELLULAR NUCLEOTIDE CATABOLISM IN AORTOILIAC BIFURCATION OF ATHEROSCLEROTIC ApoE/LDLr DOUBLE KNOCK OUT MICE Barbara Kutryb-Zajac, 1 Paulina Zukowska, 1 Marta Toczek, 1 Magdalena Zabielska, 1 Marcin Lipinski, 2 Iwona Rybakowska, 3 Stefan Chlopicki, 4 Ewa M. Slominska, 1 and Ryszard T. Smolenski 1 1 Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland 2 Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Gdansk, Poland 3 Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Gdansk, Poland 4 Department of Experimental Pharmacology, Jagiellonian University Medical College and Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland Atherosclerosis is a consequence of diverse pathologies that could be affected by signaling mediated by nucleotides and their metabolites. Concentration of specific nucleotide derivatives in the proximity of purinergic receptors is controlled by extracellular enzymes such as ecto-nucleoside triphopsphate diphosphohydrolase (entpd), ecto-5 -nucleotidase (e5nt), and ecto-adenosine deaminase (eada). To estimate changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall, aortoiliac bifurcation of ApoE/LDLr ( / ) mice was perfused with solution containing adenosine-5 -triphosphate (ATP), adenosine-5 -monophosphate (AMP) or adenosine. Formation of the product of entpd, e5nt or eada was measured by high performance liquid chromatography (HPLC). The most significant difference between ApoE/LDLr ( / ) and wild-type mice was several times higher rate of conversion of adenosine to inosine catalyzed by eada activity. This highlights potential decrease in intravascular adenosine concentration in atherosclerosis. Keywords Nucleotides; ecto-nucleoside triphopsphate diphosphohydrolase; ecto-5 - nucleotidase; adenosine deaminase; adenosine; atherosclerosis; inflammation Received 20 September 2013; accepted 23 December This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG /09), National Science Centre of Poland (2011/01/B/NZ4/03719), and Foundation for Polish Science (TEAM/2011-8/7). Address correspondence to Ryszard T. Smolenski, Department of Biochemistry, Medical University of Gdansk, Debinki 1, Gdansk, Poland. rt.smolenski@gumed.edu.pl 323

4 324 B. Kutryb-Zajac et al. INTRODUCTION Atherosclerosis leading to cardiovascular diseases is a main cause of mortality worldwide. [1] The development of this process is characterized by a long asymptomatic phase with the critical role of local inflammation and thrombotic mechanisms in a vessel wall. [2,3] Extracellular metabolism of nucleotides plays a critical role in regulation of these mechanisms. [4] Ecto-nucleoside triphopsphate diphosphohydrolase (entpd) and ecto-5 - nucleotidase (e5nt) modulates concentrations of molecules involved in purinergic signaling: ATP, ADP, and adenosine. Besides these activities, extracellular adenosine deaminase (eada) could further modify concentration of adenosine by its conversion to inosine. [5] Enzymes responsible for extracellular nucleotide catabolism could be present in the vessel wall predominantly endothelium or on the surface of the other cells present in blood erythrocytes and in particular cells of the immune system. Besides cellular enzymes, blood plasma activities can also contribute to nucleotide metabolism. [6,7] Clarification of these complex relations is therefore interesting and important, particularly in pathological conditions such as atherosclerosis. The purpose of this study was to compare rates of conversion of ATP, AMP, and adenosine in the vessel of normal and mice with atherosclerosis (ApoE/LDLr / ). MATERIALS AND METHODS Double-knockout mice for apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLr) on the C57BL/6J x129/svj background and control wild-type mice of the same strain were maintained on standard laboratory diet. All animal care and experimental procedures complied with the EU guidelines and were approved by the local Bioethical Committee at the Medical University of Gdansk. Five male ApoE/LDLr ( / ) mice and five wild-type mice as a control (20 weeks of age, weighing g) were used. Mice from both groups of the study were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) given intraperitoneally. Abdominal aorta was then collected, cannulated, and ligated by suture just below the middle caudal artery. The perfused fragment included aortoiliac bifurcation from the middle caudal artery to ramification of the iliac arteries. Perfusion was carried out using a peristaltic pomp with Hanks Balanced Salt Solution (HBSS). The flow rate was 0.4 ml/min and the whole system was maintained at 37 C. After a 5-minute equilibration period, the substrates were added in sequence: adenosine, ATP, and AMP at 50 μm concentration. An inhibitor of adenosine deaminase-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was present at 5 μm concentration during perfusion with ATP and AMP to block conversion of adenosine to inosine. Samples of perfusion

5 Extracellular Nucleotide Catabolism 325 FIGURE 1 Concentration of entpd reactants at the inflow and outflow during perfusion of the aortoiliac bifurcation of wild-type mice (A) and ApoE/LDLr / - mice (B). Results shown as mean ± SEM, n = 5. medium both at inflow to the vessel and at outflow with each substrate were collected during each 1-minute perfusion. Concentrations of ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine, and urid acid were measured by HPLC according to the method described earlier. [8] Results are reported as the mean ± SEM. Comparison of the means was done by Student s t-test. p < 0.05 was considered significant. RESULTS Data presented on Figures 1 3 show the change in the concentrations of nucleotide metabolites during the passage of perfusion solution through the vessel. We did not observe significant differences during the infusion of ATP or AMP suggesting that activities of entpd and e5nt in ApoE/LDLr ( / ) versus wild-type mice do not differ (Figures 1 and 2). Nevertheless, some trends that suggest an increased conversion rate of ADP to AMP and AMP to adenosine in ApoE/LDLr ( / ) mice have been noted. Its full clarification needs more observation in studied age group or analysis in different age groups. Interestingly, concentration of inosine in the outflow perfusate of ApoE/LDLr ( / ) mice was six times higher than in wild type while adenosine was completely removed in ApoE/LDLr ( / ) and retained in wild-type mice (Figure 3). This suggests substantial elevation of eada activity in ApoE/LDLr ( / ) mice. DISCUSSION This work demonstrated that intravascular degradation of adenosine in atherosclerotic mice is substantially accelerated as indicated by much higher

6 326 B. Kutryb-Zajac et al. FIGURE 2 Concentration of e5nt reactants at the inflow and outflow during perfusion of the aortoiliac bifurcation of wild-type mice (A) and ApoE/LDLr / - mice (B). Results shown as mean ± SEM, n = 5. concentration of inosine in the perfusate than in wild-type mice. Extracellular adenosine acting via P1 receptors, is considered as an important antiatherosclerotic molecule through preventing thrombosis, foam cells transformation and blood pressure elevation. [9 11] The ApoE/LDLr ( / ) mice model, which we used, is commonly accepted in atherosclerosis research and has been well characterized. [12 15] Changes in the vessel wall of these mice develops gradually, but at 4 5 FIGURE 3 Concentration of eada reactants at the inflow and outflow during perfusion of the aortoiliac bifurcation of wild-type mice (A) and ApoE/LDLr / - mice (B). Results shown as mean ± SEM, p < 0.05, p < vs. wild type, n = 5.

7 Extracellular Nucleotide Catabolism 327 months there is a particular breakthrough in the progression of inflammation, inflammatory cells infiltration in the area of atherosclerotic plaque, and the activity of matrix metalloproteinases. [16,17] The point at which the abdominal aorta bifurcates into the common iliac arteries, known as the aortoiliac bifurcation, is particularly vulnerable for atherosclerosis development through the interaction between hemodynamic forces, blood pressure and the local flow patterns. [18 20] Disrupted barrier function of the endothelium modulated by wall shear stress are involved in the infiltration and accumulation of atherogenic lipids within the arterial wall. [21] Our results indicated an increased activity of an adenosine-degrading enzyme in the atherosclerotic vessel wall. The enzyme responsible for this effect: ecto-adenosine deaminase is highly expressed on the immune cells surface and has a pivotal role in the regulation and enhancement of the inflammatory response. [22,23] An elevated activity of eada in the ApoE/LDLr ( / ) mice vessel wall may result from the infiltration by lymphocytes and/or macrophages, in which early atherosclerotic plaque is abundant. Consequently, intensification of inflammation and, hence, progression of atherosclerosis can occur due to reduced level of extracellular adenosine. The measurement of adenosine concentration in human blood is difficult due to its short half-life (<1 s) and data on its concentration in patients with atherosclerosis is limited. Besides quick metabolism of adenosine, local differences at cellular level can further complicate the interpretation of direct measurements of adenosine in blood. [24] It can be, however, stated with certainty that high expression of eada at the cell surface will lead to reduction of adenosine receptor activation. Clinical studies in patients with atherosclerosis demonstrated elevated activity of serum adenosine deaminase in patients with unstable angina. [25] This observation, which is consistent with our data, may indicate that an increase in adenosine degrading enzyme activity in the atherosclerotic vessel may be of clinical importance, but its full elucidation requires further studies. REFERENCES 1. Minino, A.M.; Murphy, S.L.; Xu, J.; Kochanek, K.D. Deaths: final data for Natl. Vital. Stat. Rep. 2011, 59, Pastrana, J.L.; Sha, X.; Virtue, A.; Mai, J.; Cueto, R.; Lee, I.A.; Wang, H.; Yang, X.F. Regulatory T cells and atherosclerosis. J. Clin. Exp. Cardiol. 2012, S12, Herbin, O.; Ait-Oufella, H.; Yu, W.; Fredrikson, G.N.; Aubier, B.; Perez, N.; Barateau, V.; Nilsson, J.; Tedgui, A.; Mallat, Z. Regulatory T-cell response to apolipoprotein B100-derived peptides reduces the development and progression of atherosclerosis in mice. Arterioscler Thromb. Vasc. Biol. 2012, 32, Hasko, G.; Cronstein, B.N. Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 2004, 25,

8 328 B. Kutryb-Zajac et al. 5. Dong, R.P.; Kameoka, J.; Hegen, M.; Tanaka, T.; Xu, Y.; Schlossman, S. F.; Morimoto, C. Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response. J. Immunol. 1996, 156, Robson, S.C.; Sevigny, J.; Zimmermann, H. The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2006, 2, Salmi, M.; Jalkanen, S. Cell-surface enzymes in control of leukocyte trafficking. Nat. Rev. Immunol. 2005, 5, Smolenski, R.T.; Lachno, D.R.; Ledingham, S. J.M.; Yacoub, M.H. Determination of 16 nucleotides, nucleosides and bases using high-performance liquid-chromatography and its application to the study of purine metabolism in hearts for transplantation. J. Chromatogr. Biomed. Appl. 1990, 527, Koupenova, M.; Johnston-Cox, H.; Ravid, K. Regulation of atherosclerosis and associated risk factors by adenosine and adenosine receptors. Curr. Atheroscler. Rep. 2012, 14, Hasko, G.; Pacher, P. Regulation of macrophage function by adenosine. Arterioscler Thromb. Vasc. Biol. 2012, 32, Reiss, A.B.; Cronstein, B.N. Regulation of foam cells by adenosine. Arterioscler Thromb. Vasc. Biol. 2012, 32, Kostogrys, R.B.; Franczyk-Zarow, M.; Maslak, E.; Gajda, M.; Mateuszuk, L.; Jackson, C.L.; Chlopicki, S. Low carbohydrate, high protein diet promotes atherosclerosis in apolipoprotein E/low-density lipoprotein receptor double knockout mice (apoe/ldlr-/-). Atherosclerosis 2012, 223, Gajda, M.; Banas, K.; Banas, A.; Jawien, J.; Mateuszuk, L.; Chlopicki, S.; Kwiatek, W.M.; Cichockil, T.; Falkenberg, G. Distribution of selected elements in atherosclerotic plaques of apoe/ldlr-double knockout mice assessed by synchrotron radiation-induced micro-xrf spectrometry. X-Ray Spectrom. 2008, 37, Csanyi, G.; Gajda, M.; Franczyk-Zarow, M.; Kostogrys, R.; Gwozdz, P.; Mateuszuk, L.; Sternak, M.; Wojcik, L.; Zalewska, T.; Walski, M.; Chlopicki, S. Functional alterations in endothelial NO, PGI(2) and EDHF pathways in aorta in ApoE/LDLR-/- mice. Prostaglandins Other Lipid Mediat. 2012, 98, Jawien, J.; Gajda, M.; Rudling, M.; Mateuszuk, L.; Olszanecki, R.; Guzik, T. J.; Cichocki, T.; Chlopicki, S.; Korbut, R. Inhibition of five lipoxygenase activating protein (FLAP) by MK-886 decreases atherosclerosis in apoe/ldlr-double knockout mice. Eur. J. Clin. Invest. 2006, 36, Mateuszuk, L.; Khomich, T.I.; Slominska, E.; Gajda, M.; Wojcik, L.; Lomnicka, M.; Gwozdz, P.; Chlopicki, S. Activation of nicotinamide N-methyltrasferase and increased formation of 1- methylnicotinamide (MNA) in atherosclerosis. Pharmacol. Rep. 2009, 61, Gajda, M.; Jawien, J.; Mateuszuk, L.; Lis, G.J.; Radziszewski, A.; Chopicki, S.; Litwin, J.A. Triple immunofluorescence labeling of atherosclerotic plaque components in apoe/ldlr-/- mice. Folia Histochemica Et Cytobiologica 2008, 46, Bassiouny, H.S.; Zarins, C.K.; Kadowaki, M.H.; Glagov, S. Hemodynamic stress and experimental aortoiliac atherosclerosis. J. Vasc. Surg. 1994, 19, Tse, J.; Martin-McNaulty, B.; Halks-Miller, M.; Kauser, K.; DelVecchio, V.; Vergona, R.; Sullivan, M.E.; Rubanyi, G.M. Accelerated atherosclerosis and premature calcified cartilaginous metaplasia in the aorta of diabetic male Apo E knockout mice can be prevented by chronic treatment with 17 beta-estradiol. Atherosclerosis 1999, 144, Nakashima, Y.; Plump, A.S.; Raines, E.W.; Breslow, J.L.; Ross, R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994, 14, Ding, Z.; Fan, Y.; Deng, X.; Sun, A.; Kang, H. 3,3 -Dioctadecylindocarbocyanine-low-density lipoprotein uptake and flow patterns. Exp. Biol. Med. (Maywood). 2010, 235, Lluis, C.; Franco, R.; Cordero, O. Ecto-ADA in the development of the immune system. Immunol. Today. England 1998, Souza Vdo, C.; Schlemmer, K.B.; Noal, C.B.; Jaques, J.A.; Zimmermann, C.E.; Leal, C.A.; Fleck, J.; Casali. E.A.; Morsch, V.M.; Schetinger, M.R.; Leal, D.B. E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas disease. Parasitol. Int. 2012, 61, Yegutkin, G.G.; Mikhailov, A.; Samburski, S.S.; Jalkanen, S. The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor. Mol. Biol. Cell 2006, 17, Rani, S.H.; Rao, D.V.; Prakash, S.M.; Jyothy, A. Serum adenosine deaminase activity and C-reactive protein levels in unstable angina. India: Indian J. Hum. Genet. 2003,