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Divergent effects of folic acid administration on inflammatory status and cholesterol levels in apoE deficient mice
608
Letters to the Editor
Divergent effects of folic acid administration on inflammatory status and cholesterol levels in apoE deficient mice Dimitris Tousoulis ⁎, Polina Kourkouti, Alexandros Briasoulis, Georgia Vogiatzi, Aggeliki Valatsou, Alkistis Pantopoulou, Evangelos Oikonomou, Despina Perrea, Christodoulos Stefanadis 1st Department of Cardiology, ‘Hippokration’ Hospital, University of Athens Medical School, Athens, Greece
a r t i c l e
i n f o
Article history: Received 5 February 2014 Accepted 15 March 2014 Available online 21 March 2014 Keywords: Atherosclerosis Folic acid Inflammation
Atherosclerosis is a chronic systematic disorder developing insidiously throughout life. Despite the identification of a large number of modifiable risk factors and the appropriate treatments, atherosclerotic cardiovascular disease remains the leading cause of death in western countries. Folate deficiency has been advocated as an atherosclerotic risk factor associated with vascular disease and carotid intima media thickness [1]. Supplementation with folic acid (FA) can also suppress atherosclerosis progression in patients at risk independently of homocysteine levels [2]. Nevertheless, FA treatment failed to decrease the risk of recurrent cardiovascular disease after acute myocardial infarction and a harmful effect was suggested [3]. Atherosclerosis progression is influenced by a balance between inflammatory and anti-inflammatory cytokines. Proinflammatory cytokines such as interleukin (IL)-6 are implicated in the progress of atherosclerotic plaques lesions [4] while, IL-10 with multifaceted anti-inflammatory properties is associated with a more favorable prognosis in patients with acute coronary syndromes [5]. As the mechanisms of FA actions are not well defined we investigate the effects of FA fortification on the inflammatory status and cholesterol levels of apolipoprotein E-deficient (apoE KO) mice fed cholesterol-rich diet, an animal model of premature atherosclerosis prone to develop atherosclerotic plaques based on a severe lipid disorder. ApoE−/− C57BL/6J mice were purchased from The Jackson Laboratory. Mice were divided in a randomized blinded manner in four groups. The first group (n = 8) was treated with regular diet. The second group (n = 16) was treated with regular diet and an aqueous solution of FA that provided each mouse with a dose of 75 mcg/kg/day. The third group (n = 8) was treated with high-fat, high-cholesterol diet (containing 21% fat, and 1.25% cholesterol) — western diet (WD). The fourth group (n = 16) was treated with WD and FA for 16 weeks. Treatment started 4 weeks after birth and lasted for 6 weeks. The study was performed in accordance with the guidelines for animal experiments of Athens University School of Medicine. At the end of the treatment period venous blood samples were centrifuged at 3000 rpm and serum/plasma was collected and stored at − 80 °C until assayed. IL-6 a potent proinflammatory cytokine and IL-10, one of the most important anti-inflammatory cytokines were measured with enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). Total cholesterol (TC) and low density ⁎ Corresponding author at: Vasilissis Sofias 114, TK 115 28, Hippokration Hospital, Athens, Greece. Tel.: + 30 213 2088099; fax: +30 213 2088676. E-mail address: [email protected] (D. Tousoulis).
lipoprotein cholesterol (LDL-C) were also measured by using routine chemical methods (colorimetric enzymatic method in a Technicon automatic analyzer RA-1000, Date-Behring Marburg GmbH, Marburg, Germany). Data analysis was performed with SPSS software, version 18.0 (SPSS Inc., Chicago, IL). At the end of the six week period in mice fed with regular diet (group 1) FA treatment (group 2) decreased IL-6 levels [8.82 (3.51– 35.37) pg/ml vs. 3.06 (1.42–3.552) pg/ml, p = 0.01]. In mice fed with WD (group 3), FA treatment (group 4) has no impact on the levels of IL-6 [4.93 (1.35–94.65) pg/ml vs. 20.25 (2.20–47.52) pg/ml, p = 0.58] (Fig. 1, panel A). There was no difference in the levels of IL-10 between mice treated with regular diet (group 1) and those treated with regular diet and FA (group 2) [4.40 (2.65–9.17) pg/ml vs. 3.60 (2.90–8.27) pg/ml, p = 0.96]. Similarly no difference was observed in IL-10 levels in mice fed with WD (group 3), and FA treatment (group 4) has no impact on the levels of IL-10 [6.53 (2.50–153.08) pg/ml vs. 9.93 (3.13–17.65) pg/ml, p = 0.67] (Fig. 1, panel B). At the end of the six week period in mice fed with regular diet (group 1) FA treatment (group 2) decreased TC levels [552 (488–623) mg/dl vs. 238 (126–247) mg/dl, p = 0.004] (Fig. 1, panel C) and LDL-C levels [469 (435–560) mg/dl vs. 181 (89–207) mg/dl, p = 0.006] (Fig. 1, panel D). In mice fed with WD (group 3), FA treatment (group 4) had no impact on TC levels [694 (398–1247) mg/dl vs. 474 (438-643) mg/dl, p = 0.24] and on LDL-C levels [650 (375–1217) mg/dl vs. 445 (384–594) mg/dl, p = 0.22] (Fig. 1, panel C). In this study, in a highly atherosclerotic model fed with additional cholesterol, we found a modest anti-inflammatory effect of FA treatment associated with a parallel improvement on lipid levels. Folate contributes to the transfer and utilization of 1-carbon moieties, reactions required for transmethylation, nucleic acid synthesis, homocysteine metabolism, and the enzymatic generation of tetrahydrobiopterin which is necessary for nitric oxide synthesis. Low folate concentrations, independently of hyperhomocysteinemia, may promote atherogenesis [1]. Folate deficiency may promote atherosclerosis not only through a decrease of antioxidative capacity and endothelial dysfunction but also through exacerbation of inflammation and adhesion of molecule concentration in the vessel wall [6]. Moreover, low IL-6 levels are found in subjects attributed a diet rich in folate [7]. Interestingly, in a mice model of early atherosclerosis we documented a favorable effect of FA fortification on IL-6 levels providing further insights into the potential mechanisms of folate action. Moreover, FA administration has no impact on anti-inflammatory IL-10 levels. IL-10 inhibits matrix degrading metalloproteinases, decreases tissue factor expression, attenuates atherogenesis and stabilizes vulnerable plaques [8]. The neutral impact of FA treatment on IL-10 levels may give some clues why despite the positive effects of folate fortification on inflammation, FA treatment failed to decrease acute coronary syndromes in humans [3]. Interestingly, we documented a positive effect of FA administration on TC and LDL-C levels in mice fed a regular diet. Importantly, the reduction in lipid levels was in parallel with the impact of FA on IL-6 levels. In conclusion, in apoE deficient mice, an early atherosclerotic model, we documented a positive impact of high dose folic acid treatment on inflammatory status and cholesterol levels. These effects are attenuated in mice with an additive atherosclerotic stimulus, a high cholesterol diet. These findings may provide further insights into the atheroprotective mechanisms of folic acid and
Letters to the Editor
609
Fig. 1. Box-plots representing the impact of folic acid administration on apoE deficient mice fed a regular diet or a western diet. Panel A: Represent the differences in interleukin-6 levels; panel B: Represent the differences in interleukin-10 levels; panel C: Represent the differences in total cholesterol levels; panel D: Represent the differences in low density lipoprotein levels.WD: western diet; IL-6: Interleukin-6; IL-10: Interleukin-10.
provide further explanations why folic acid fortification was unsuccessfully tested in humans with established cardiovascular disease. References [1] Durga J, Bots ML, Schouten EG, Kok FJ, Verhoef P. Low concentrations of folate, not hyperhomocysteinemia, are associated with carotid intima-media thickness. Atherosclerosis 2005;179:285–92. [2] Till U, Rohl P, Jentsch A, et al. Decrease of carotid intima-media thickness in patients at risk to cerebral ischemia after supplementation with folic acid, vitamins B6 and B12. Atherosclerosis 2005;181:131–5. [3] Bonaa KH, Njolstad I, Ueland PM, et al. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 2006;354:1578–88.
http://dx.doi.org/10.1016/j.ijcard.2014.03.134 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
[4] Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001;22:2243–52. [5] Heeschen C, Dimmeler S, Hamm CW, et al. Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation 2003;107:2109–14. [6] Li M, Chen J, Li YS, Feng YB, Gu X, Shi CZ. Folic acid reduces adhesion molecules VCAM-1 expression in aortic of rats with hyperhomocysteinemia. Int J Cardiol 2006;106:285–8. [7] Holt EM, Steffen LM, Moran A, et al. Fruit and vegetable consumption and its relation to markers of inflammation and oxidative stress in adolescents. J Am Diet Assoc 2009;109:414–21. [8] Smith DA, Irving SD, Sheldon J, Cole D, Kaski JC. Serum levels of the antiinflammatory cytokine interleukin-10 are decreased in patients with unstable angina. Circulation 2001;104:746–9.
Letters to the Editor
Divergent effects of folic acid administration on inflammatory status and cholesterol levels in apoE deficient mice Dimitris Tousoulis ⁎, Polina Kourkouti, Alexandros Briasoulis, Georgia Vogiatzi, Aggeliki Valatsou, Alkistis Pantopoulou, Evangelos Oikonomou, Despina Perrea, Christodoulos Stefanadis 1st Department of Cardiology, ‘Hippokration’ Hospital, University of Athens Medical School, Athens, Greece
a r t i c l e
i n f o
Article history: Received 5 February 2014 Accepted 15 March 2014 Available online 21 March 2014 Keywords: Atherosclerosis Folic acid Inflammation
Atherosclerosis is a chronic systematic disorder developing insidiously throughout life. Despite the identification of a large number of modifiable risk factors and the appropriate treatments, atherosclerotic cardiovascular disease remains the leading cause of death in western countries. Folate deficiency has been advocated as an atherosclerotic risk factor associated with vascular disease and carotid intima media thickness [1]. Supplementation with folic acid (FA) can also suppress atherosclerosis progression in patients at risk independently of homocysteine levels [2]. Nevertheless, FA treatment failed to decrease the risk of recurrent cardiovascular disease after acute myocardial infarction and a harmful effect was suggested [3]. Atherosclerosis progression is influenced by a balance between inflammatory and anti-inflammatory cytokines. Proinflammatory cytokines such as interleukin (IL)-6 are implicated in the progress of atherosclerotic plaques lesions [4] while, IL-10 with multifaceted anti-inflammatory properties is associated with a more favorable prognosis in patients with acute coronary syndromes [5]. As the mechanisms of FA actions are not well defined we investigate the effects of FA fortification on the inflammatory status and cholesterol levels of apolipoprotein E-deficient (apoE KO) mice fed cholesterol-rich diet, an animal model of premature atherosclerosis prone to develop atherosclerotic plaques based on a severe lipid disorder. ApoE−/− C57BL/6J mice were purchased from The Jackson Laboratory. Mice were divided in a randomized blinded manner in four groups. The first group (n = 8) was treated with regular diet. The second group (n = 16) was treated with regular diet and an aqueous solution of FA that provided each mouse with a dose of 75 mcg/kg/day. The third group (n = 8) was treated with high-fat, high-cholesterol diet (containing 21% fat, and 1.25% cholesterol) — western diet (WD). The fourth group (n = 16) was treated with WD and FA for 16 weeks. Treatment started 4 weeks after birth and lasted for 6 weeks. The study was performed in accordance with the guidelines for animal experiments of Athens University School of Medicine. At the end of the treatment period venous blood samples were centrifuged at 3000 rpm and serum/plasma was collected and stored at − 80 °C until assayed. IL-6 a potent proinflammatory cytokine and IL-10, one of the most important anti-inflammatory cytokines were measured with enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA). Total cholesterol (TC) and low density ⁎ Corresponding author at: Vasilissis Sofias 114, TK 115 28, Hippokration Hospital, Athens, Greece. Tel.: + 30 213 2088099; fax: +30 213 2088676. E-mail address: [email protected] (D. Tousoulis).
lipoprotein cholesterol (LDL-C) were also measured by using routine chemical methods (colorimetric enzymatic method in a Technicon automatic analyzer RA-1000, Date-Behring Marburg GmbH, Marburg, Germany). Data analysis was performed with SPSS software, version 18.0 (SPSS Inc., Chicago, IL). At the end of the six week period in mice fed with regular diet (group 1) FA treatment (group 2) decreased IL-6 levels [8.82 (3.51– 35.37) pg/ml vs. 3.06 (1.42–3.552) pg/ml, p = 0.01]. In mice fed with WD (group 3), FA treatment (group 4) has no impact on the levels of IL-6 [4.93 (1.35–94.65) pg/ml vs. 20.25 (2.20–47.52) pg/ml, p = 0.58] (Fig. 1, panel A). There was no difference in the levels of IL-10 between mice treated with regular diet (group 1) and those treated with regular diet and FA (group 2) [4.40 (2.65–9.17) pg/ml vs. 3.60 (2.90–8.27) pg/ml, p = 0.96]. Similarly no difference was observed in IL-10 levels in mice fed with WD (group 3), and FA treatment (group 4) has no impact on the levels of IL-10 [6.53 (2.50–153.08) pg/ml vs. 9.93 (3.13–17.65) pg/ml, p = 0.67] (Fig. 1, panel B). At the end of the six week period in mice fed with regular diet (group 1) FA treatment (group 2) decreased TC levels [552 (488–623) mg/dl vs. 238 (126–247) mg/dl, p = 0.004] (Fig. 1, panel C) and LDL-C levels [469 (435–560) mg/dl vs. 181 (89–207) mg/dl, p = 0.006] (Fig. 1, panel D). In mice fed with WD (group 3), FA treatment (group 4) had no impact on TC levels [694 (398–1247) mg/dl vs. 474 (438-643) mg/dl, p = 0.24] and on LDL-C levels [650 (375–1217) mg/dl vs. 445 (384–594) mg/dl, p = 0.22] (Fig. 1, panel C). In this study, in a highly atherosclerotic model fed with additional cholesterol, we found a modest anti-inflammatory effect of FA treatment associated with a parallel improvement on lipid levels. Folate contributes to the transfer and utilization of 1-carbon moieties, reactions required for transmethylation, nucleic acid synthesis, homocysteine metabolism, and the enzymatic generation of tetrahydrobiopterin which is necessary for nitric oxide synthesis. Low folate concentrations, independently of hyperhomocysteinemia, may promote atherogenesis [1]. Folate deficiency may promote atherosclerosis not only through a decrease of antioxidative capacity and endothelial dysfunction but also through exacerbation of inflammation and adhesion of molecule concentration in the vessel wall [6]. Moreover, low IL-6 levels are found in subjects attributed a diet rich in folate [7]. Interestingly, in a mice model of early atherosclerosis we documented a favorable effect of FA fortification on IL-6 levels providing further insights into the potential mechanisms of folate action. Moreover, FA administration has no impact on anti-inflammatory IL-10 levels. IL-10 inhibits matrix degrading metalloproteinases, decreases tissue factor expression, attenuates atherogenesis and stabilizes vulnerable plaques [8]. The neutral impact of FA treatment on IL-10 levels may give some clues why despite the positive effects of folate fortification on inflammation, FA treatment failed to decrease acute coronary syndromes in humans [3]. Interestingly, we documented a positive effect of FA administration on TC and LDL-C levels in mice fed a regular diet. Importantly, the reduction in lipid levels was in parallel with the impact of FA on IL-6 levels. In conclusion, in apoE deficient mice, an early atherosclerotic model, we documented a positive impact of high dose folic acid treatment on inflammatory status and cholesterol levels. These effects are attenuated in mice with an additive atherosclerotic stimulus, a high cholesterol diet. These findings may provide further insights into the atheroprotective mechanisms of folic acid and
Letters to the Editor
609
Fig. 1. Box-plots representing the impact of folic acid administration on apoE deficient mice fed a regular diet or a western diet. Panel A: Represent the differences in interleukin-6 levels; panel B: Represent the differences in interleukin-10 levels; panel C: Represent the differences in total cholesterol levels; panel D: Represent the differences in low density lipoprotein levels.WD: western diet; IL-6: Interleukin-6; IL-10: Interleukin-10.
provide further explanations why folic acid fortification was unsuccessfully tested in humans with established cardiovascular disease. References [1] Durga J, Bots ML, Schouten EG, Kok FJ, Verhoef P. Low concentrations of folate, not hyperhomocysteinemia, are associated with carotid intima-media thickness. Atherosclerosis 2005;179:285–92. [2] Till U, Rohl P, Jentsch A, et al. Decrease of carotid intima-media thickness in patients at risk to cerebral ischemia after supplementation with folic acid, vitamins B6 and B12. Atherosclerosis 2005;181:131–5. [3] Bonaa KH, Njolstad I, Ueland PM, et al. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 2006;354:1578–88.
http://dx.doi.org/10.1016/j.ijcard.2014.03.134 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
[4] Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6-174G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001;22:2243–52. [5] Heeschen C, Dimmeler S, Hamm CW, et al. Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes. Circulation 2003;107:2109–14. [6] Li M, Chen J, Li YS, Feng YB, Gu X, Shi CZ. Folic acid reduces adhesion molecules VCAM-1 expression in aortic of rats with hyperhomocysteinemia. Int J Cardiol 2006;106:285–8. [7] Holt EM, Steffen LM, Moran A, et al. Fruit and vegetable consumption and its relation to markers of inflammation and oxidative stress in adolescents. J Am Diet Assoc 2009;109:414–21. [8] Smith DA, Irving SD, Sheldon J, Cole D, Kaski JC. Serum levels of the antiinflammatory cytokine interleukin-10 are decreased in patients with unstable angina. Circulation 2001;104:746–9.