Effect of L-arginine on circulating endothelial progenitor cells in hypercholesterolemic rabbits

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in sufficient agreement with invasive thermodilution measurements in this study with patients suffering from severe chronic heart failure. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [15]. References [1] Torre-Amione G, Young JB, Colucci W, et al. Hemodynamic and clinical effects of tezosentan, an intravenous dual endothelin receptor antagonist, in patients hospitalized because of acute decompensated heart failure. J Am Coll Cardiol 2003;42:140–7. [2] VMAC investigators. Intravenous nesiritide versus nitroglycerin for treatment of decompensated congestive heart failure: a randomized contolled trial. JAMA 2002;287:1531–40. [3] Connors Jr AF, Speroff T, Dawson NV, et al. The effectiveness of right heart catheterization in the initial care of critically ill patients. JAMA 1996;276:889–97. [4] Binancy C, Califf RM, Hasselblad V, et al. Evaluation study of congestive heart failure and pulmonary artery catheterization effectiveness: the ESCAPE trial. JAMA 2005;294:1625–33. [5] Shah MR, Hasselblad V, Stevenson L, et al. Impact of the pulmonary artery catheter in critically ill patients. JAMA 2005;294:1664–70. [6] Moshkovitz Y, Kaluski E, Milo O, et al. Recent developments in cardiac output determination by bioimpedance: comparison with invasive cardiac output and potential cardiovascular applications. Curr Opin Cardiol 2004;19:229–37.

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[7] Yung GL, Fedullo PF, Kinninger K, Johnson W, Channick RN. Comparison of impedance cardiography to direct Fick and thermodilution cardiac output determination in pulmonary arterial hypertension. CHF 2004;10(2 suppl 2):7–10. [8] Vijayaraghavan K, Crum S, Cherukuri S, Barnett-Avery L. Association of impedance cardiography parameters with changes in functional and quality-of-life measures in patients with chronic heart failure. CHF 2004;10(2 suppl 2):22–7. [9] Drazner MH, Thompson B, Rosenberg PB, et al. Comparison of impedance cardiography with invasive hemodynamic measurements in patients with heart failure secondary to ischemic or nonischemic cardiomyopathy. Am J Cardiol 2002;89:993–5. [10] Albert NM, Hail MD, Li J, Young JB. Equivalence of the bioimpedance and thermodilution methods in measuring cardiac output in hospitalized patients with advanced, decompensated chronic heart failure. Am J Crit Care 2004;13(6):469–79. [11] Spinale FG, Reines HD, Crawford Jr FA. Comparison of bioimpedance and themodilution methods for determining cardiac output: experimental and clinical studies. Ann Thorac Surg 1988;45:421–5. [12] Fuller HD. The validity of cardiac output measurements by thoracic impedance: a meta-analysis. Clin Invest Med 1992;15:103–12. [13] Packer M, Abraham WT, Mehra MR, et al. Utility of impedance cardiography for the identification of short-term risk of clinical decompensation in stable patients with chronic heart failure. J Am Coll Cardiol 2006;47:2245–52. [14] Bland JM, Altman DG. Agreement between methods of measurement with multiple observations per individual. J Biopharm Stat 2007;17(4): 571–82. [15] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.

0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2008.11.201

Effect of L-arginine on circulating endothelial progenitor cells in hypercholesterolemic rabbits Shaghayegh Haghjooy Javanmard a,⁎, Yousof Gheisari a , Masoud Soleimani b , Mehdi Nematbakhsh a , Alireza Monajemi a a

Applied Physiology Research Center, Isfahan University of Medical Sciences, Hezar jerib Avenue, Isfahan, Iran b Hematology Department, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran Received 10 July 2008; accepted 30 November 2008 Available online 24 January 2009

Keywords: Atherosclerosis; Endothelial progenitor cells; L-arginine; Nitric Oxide

Atherosclerosis risk factors cause injury to endothelial cells as well as apoptosis and lead to a progressive loss of endothelial integrity [1]. In endothelial injury, adjacent

⁎ Corresponding author. Tel.: +98 311 7922295; fax: +98 311 6682006. E-mail address: [email protected] (S.H. Javanmard).

endothelial cells proliferate, and re-endothelialize the denuded luminal surface[1]. Additionally, adult peripheral blood contains Endothelial Progenitors Cells (EPCs) which have a prominent role in the re-endothelization phenomena at sites of endothelial injury[2] and [3]. They can also affect surrounding cells through the secretion of angiogenic growth factors [4].

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It has been shown that the cardiovascular disease risk factors such as age, hypercholesterolemia and diabetes reduce the number and functional activity of EPCs [3,5–7]. Therefore, interventions that increase the number of EPCs and/or improve the function of EPC might be promising strategies for the prevention and treatment of early atherosclerosis. Previous studies have used erythropoietin [8], VEGF [9], stromal-derived growth factor [9], Granulocytecolony stimulating factor [10], statins [11], estrogen [12], and physical activity [13] to increase the number of EPCs and restored the injured endothelium. It has been shown that these interventions activate the Nitric Oxide (NO) dependent mechanisms [14] and [15]. It has recently been shown that Larginine (the precursor of NO synthesis) supplementation potentiates the effects of moderate physical exercise by increasing significantly EPCs and VEGF serum levels in C57BL/6J mice [16]. The purpose of the present study was to investigate the effect of L-arginine on circulating EPC, nitrite, and vWF levels in a rabbit model of hypercholesterolemia. This study was reviewed and approved by the Ethics Committee of Isfahan University of Medical Sciences. 26 white male rabbits weighing 2.3 ± 0.2 kg were obtained from the Razi Institute of Iran. After 24 h fasting, venous blood was sampled for lipids and biomarkers measurement. Then the animals were randomly assigned in 3 groups. The rabbits were fed 1% cholesterol diet (Hypercholesterolemic (HC) group, n = 10) or 1% cholesterol diet with oral L-arginine (3% in drinking water) (HC + L-arg group, n = 10) or standard diet (control group, n = 6) for 4 weeks. By the end of 4 weeks, the blood samples were taken by cardiac puncture and the abdominal aortas were isolated from sodium pentobarbital anesthetized animals. The animals euthanized by an overdose of sodium pentobarbital. Total cholesterol and LDL-cholesterol levels were measured by standard enzymatic kit (Pars Azmoon Co, Iran). The plasma levels of nitrite (stable NO metabolite) were measured using a colorimetric assay kit (R&D Systems, Minneapolis, USA) that involves the Griess reaction. Plasma VEGF and vWF concentration was measured by ELISA kits (R&D Systems, Minneapolis, MN) and (Cedarlane Co, Canada) respectively. The formalin fixed paraffin-embedded specimens were prepared from the entire aorta, sectioned at 5 µm, stained with haematoxylin and eosin, and examined by light microscopy to measure fatty streaks by two pathologists in a doubleblinded manner. Fatty streaks formation was determined by intima thickness, and media thickness measurement in 20 sections. The data were averaged and were used to obtain the IMT ratio (intima thickness/ media thickness). Flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMC) as described [17]. PBMC were isolated by density gradient centrifugation with Ficoll from 10 mL of heparinized blood. Immediately after isolation 4 × 106 PBMC were stained employing monoclonal antibodies: fluorescein isothiocyanate (FITC)–conjugate–CD34

Table 1 The levels of total cholesrtol, LDL, nitrite and vWF in three groups of the study. HC (n = 10) Total cholesterol(mg/dl) Before experiment After 4 weeks p(before and after) LDL-cholesterol(mg/dl) Before experiment After 4 weeks p(before and after) Nitrite(µmol/l) Before experiment After 4 weeks p(before and after) VWF(IU/dl) Before experiment After 4 weeks p(before and after)

HC + L-arg (n = 10)

Control (n = 6)

109.40 ± 12.04 127.8 ± 12.3 118.6 ± 12.1 2130.9 ± 171.8⁎ 2109.03 ± 171.8⁎ 138.2 ± 9.8 b0.05 b0.05 N0.05 89.2 ± 8.2 109.6 ± 4.5 1418.9 ± 164.6⁎ 1079.6 ± 118.2⁎ b0.05 b0.05

86.6 ± 7.6 88.7 ± 5.1 N0.05

9.45 ± 1.1 11.63 ± 1.4 † N0.05

10.77 ± 1.2 15.43 ± 0.8 ⁎ b0.05

9.77 ± 1.3 8.2 ± 1.4 N0.05

0.42 ± .09 0.99 ± .1 ⁎ b0.05

0.55 ± .06 0.49 ± 0.04 ⁎⁎ N0.05

0.23 ± 0.05 0.32 ± 0.04 N0.05

Values are mean ± S.E. HC: hypercholesterolemic animals, HC + L-arg: Hypercholesterolemic diet with oral L-arginine treated animals. LDL: low density lipoprotein cholesterol, vWF: von Willebrand Factor. IU: International Unit. ⁎ p b 0.05 significantly different from control group. ⁎⁎ p b 0.05 significantly different from HC group. †

p b 0.05 significantly different from HC+L-arg group.

antibody (eBioscience), and phycoerythrin (PE)–conjugated–VEGF receptor 2 (KDR) antibody, (eBioscience). Corresponding isotype-matched FITC or PE-conjugated antibody was used as negative control. The cells were quantified using a PAS III Partec cytometer (Germany), and Flomax Software and expressed as number of cells/106 total events. The difference within groups was evaluated by a Paired Student's t-test while the difference between groups was evaluated by a one-way ANOVA followed by Bonferroni's t-test within groups. Statistical significance was accepted at p b 0.05. The cholesterol-rich diet induced a significant increase of total cholesterol, LDL-cholesterol in both HC and HC + Larg groups (p b 0.05) (Table 1), while lipid levels in the control group remained unchanged through the study. After 4 weeks, animals of the HC + L-arg and the HC group had similar levels of total cholesterol, and LDL-cholesterol (p N 0.05); while all of them were significantly higher than control group (p b 0.05) (Table 1). L-arginine supplementation resulted in, significantly increased level of nitrite (p b 0.05); while there were no significant changes in nitrite levels of HC and control groups through the study (p N 0.05). The nitrite levels were significantly higher in HC + L-arg group than the HC and control groups (p b 0.05) (Table 1). After 4 weeks on hypercholesterolemic diet the plasma levels of vWF significantly increased in HC group (p b 0.05) while the animals of the HC + L-arg and control groups had similar levels of vWF before and after study (p N 0.05). The plasma levels of vWF were significantly

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References

Fig. 1. Determination of EPC number in rabbits. The rabbits were on hypercholesterolemic diet with or without L-arginine supplementation. A control group of 6 rabbits received a normal diet during the study. FACS computed counting was used to determine the EPC number based on the coexpression of hematopoietic stem cell marker CD 34 and VEGF-receptor 2 (VEGFR2). The result expressed as number of cells/106 peripheral blood mononuclear cells (PBMC). HC: Hypercholesterolemic animals, HC + L-arg: Hypercholesterolemic animals with oral L-arginine supplementation P b 0.05.

higher in HC group compare to HC + L-arg and control groups (p b 0.05) (Table 1). At the end of study there were no fatty streak lesions in control and L-arginine treated groups aortas while IMT ratio was 0.33 ± 0.1 in HC group (p b 0.05). L-arginine supplementation was associated with a significant increase in EPC number, while the animals of the HC and the control group had similar levels of circulating EPCs (Fig. 1). The findings of our study corroborated the preventive role of L-arginine; as L-arginine supplementation led to no fatty streaks formation, and no significant vWF increment—a reliable marker of endothelial damage [18]. The preventive role of L-arginine has been linked to its positive effect on NO bioavailability [19]. Anti-atherogenic property of L-arginine may be evaluated from the standpoint of its effects on regenerative capacity; as the results of our study showed EPC numbers were significantly higher in L-arginine treated group. This finding is in agreement with previous studies in which, systemic infusion of vascular progenitor cells in animal models of hyperlipidemia enhances regeneration processes of the diseased endothelium and thereby prevents atherosclerosis progression [20,21]. L-arginine may be an “ideal” substance because it limits endothelial injury as well as increases EPCs. L-arginine could have significant clinical implications as a safe and feasible intervention to prevent atherosclerosis. This study was supported by Isfahan University of Medical sciences, Isfahan, Iran and Stem Cell Technology CO. Tehran, Iran. Special thanks to Dr Mahin Nikoogoftar and Dr Minoo Saeidi for their technical supports. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [22].

[1] Dimmeler S, Zeiher AM. Vascular repair by circulating endothelial progenitor cells: the missing link in atherosclerosis? J Mol Med 2004;82(10):671–7. [2] Asahara T, Murohara T, Sullivan A, Silver M, van der ZR, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275(5302):964–7. [3] Werner N, Nickenig G. Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy? Arterioscler Thromb Vasc Biol 2006;26(2):257–66. [4] Rehman J, Li J, Orschell CM, March KL. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 2003;107(8):1164–9. [5] Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348(7):593–600. [6] Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, Wang T, Gregg D, Ramaswami P, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003;108(4):457–63. [7] Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89(1):E1–7. [8] Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003;102(4):1340–6. [9] Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, et al. Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1. Ann N Y Acad Sci 2001;938: 36–45. [10] Tan Y, Shao H, Eton D, Yang Z, Alonso-Diaz L, Zhang H, et al. Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor. Cardiovasc Res 2007;73(4): 823–32. [11] Dimmeler S, Aicher A, Vasa M, Mildner-Rihm C, Adler K, Tiemann M, et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest 2001;108(3): 391–7. [12] Iwakura A, Luedemann C, Shastry S, Hanley A, Kearney M, Aikawa R, et al. Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury. Circulation 2003;108(25): 3115–21. [13] Gertz K, Priller J, Kronenberg G, Fink KB, Winter B, Schrock H, et al. Physical activity improves long-term stroke outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization and cerebral blood flow. Circ Res 2006;99(10):1132–40. [14] Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, TechnauIhling K, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003;9(11): 1370–6. [15] Sasaki K, Heeschen C, Aicher A, Ziebart T, Honold J, Urbich C, et al. Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy. Proc Natl Acad Sci U S A 2006;103(39): 14537–41. [16] Fiorito C, Balestrieri ML, Crimi E, et al. Effect of L-arginine on circulating endothelial progenitor cells and VEGF after moderate physical training in mice. Int J Cardiol Jun 6 2008;126(3):421–3. [17] Van Craenenbroeck EM, Conraads VM, Van Bockstaele DR, et al. Quantification of circulating endothelial progenitor cells: a methodological comparison of six flow cytometric approaches. J Immunol Methods Mar 20 2008;332(1–2):31–40. [18] Constans J, Conri C. Circulating markers of endothelial function in cardiovascular disease. Clin Chim Acta 2006;368(1–2):33–47.

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[19] Siasos G, Tousoulis D, Antoniades C, Stefanadi E, Stefanadis C. L-Arginine, the substrate for NO synthesis: an alternative treatment for premature atherosclerosis? Int J Cardiol 2007;116(3):300–8. [20] Wassmann S, Werner N, Czech T, Nickenig G. Improvement of endothelial function by systemic transfusion of vascular progenitor cells. Circ Res 2006;99(8):e74–83. 0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2008.11.203

[21] Werner N, Junk S, Laufs U, Link A, Walenta K, Bohm M, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res 2003;93(2):e17–24. [22] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131(2): 149–50.