- Email: [email protected]
Ursolic acid effect on atherosclerosis: Apples and apples, or apples and oranges?
Atherosclerosis 219 (2011) 397–398
Contents lists available at SciVerse ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Invited commentary
Ursolic acid effect on atherosclerosis: Apples and apples, or apples and oranges? Lisa R. Tannock a,b,∗ a b
Division of Endocrinology and Molecular Medicine, University of Kentucky, Lexington, KY, USA Department of Veterans Affairs, Lexington, KY, USA
a r t i c l e
i n f o
Article history: Received 13 September 2011 Accepted 14 September 2011 Available online 28 September 2011 Keywords: Ursolic acid Atherosclerosis Mouse models
Ursolic acid is a phytonutrient found in several different plant species, and of potential interest to cardiovascular research due its anti-oxidative, anti-inflammatory and other potentially cardioprotective properties. Although there is a wealth of data evaluating ursolic acid’s biological effects, the literature is conflicted with some studies showing potentially beneficial effects while others show potentially negative effects. Furthermore, there is a paucity of research specifically investigating the potential effects of ursolic acid on atherosclerosis or vascular diseases. However, as illustrated by the two studies in this issue investigating the effect of ursolic acid on atherosclerosis using murine models, the controversy remains. Messner et al. [1] investigated the effect of ursolic acid supplied in the drinking water on atherosclerosis development using male apoE deficient (apoE−/−) mice fed a Western diet. Their data demonstrates a dose dependent increase in atherosclerosis in mice receiving ursolic acid. Although there were some inconsistent effects of the ursolic acid on the plasma lipid levels, they propose that the pro-atherogenic effects of ursolic acid were mediated by its effects to inhibit endothelial cell proliferation and induce cell death. Additionally they report a reduction in the serum levels of IL-5 and suggest that this may also play a causal role in ursolic acid’s induction of atherogenesis. Conversely, Ullevig et al. [2] report anti-atherogenic effects of ursolic acid. In their studies, female LDL receptor deficient (LDLR−/−) mice were first treated with streptozotocin to induce
DOIs of original articles: 10.1016/j.atherosclerosis.2011.05.025, 10.1016/j.atherosclerosis.2011.06.013 ∗ Corresponding author at: University of Kentucky, Division of Endocrinology and Molecular Medicine, Wethington Building, Room 567, 900 South Limestone Street, Lexington, KY 40536-0200, USA. Tel.: +1 859 218 1415; fax: +1 859 257 3646. E-mail address: [email protected] 0021-9150/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2011.09.029
beta cell failure and hyperglycemia, then were fed a high fat diet for 11 weeks, supplemented either with resveratrol or ursolic acid. After 11 weeks, the mice receiving ursolic acid were found to have decreased atherosclerosis measured at two sites: the en face aorta (as in Messner et al.) and the aortic root. They report no effects of ursolic acid on plasma lipid levels but did find a significant reduction in plasma glucose levels. In addition, the mice receiving ursolic acid had better survival and higher body weights than the mice on diet alone. The authors investigated the effect of ursolic acid on monocyte/macrophage function, and report decreased monocyte recruitment in mice receiving ursolic acid, which they suggest as a mechanism by which atherosclerosis is reduced. Thus, the reader is faced with two studies with conflicting reports on the effect of ursolic acid on atherosclerosis development. However, upon close scrutiny of the studies, there are so many differences between the studies that perhaps one should simply conclude that the comparisons are “apples and oranges” rather than “apples and apples”. First, the studies used very different animal models: apoE−/− versus diabetic LDLR−/− mice; the duration of study and diets differed; the atherosclerosis measurements differed; the dose of ursolic acid administered is expressed differently so not easily compared; and the mechanistic studies evaluated different cell types, albeit both pertinent to atherosclerosis. Let us address these issue by issue. First, apoE−/− and LDLR−/− mice are two of the most commonly studied murine models of atherosclerosis, yet differ strikingly in the disease development. While both are hyperlipidemic, their lipoprotein profiles differ dramatically, and the effect on atherogenesis also differs [3]. On chow diets apoE-/- mice have total cholesterol levels approximating 500 mg/dl with accumulation of VLDL remnants, whereas LDLR−/− mice have much lower total cholesterol levels (approximating 200 mg/dl) with striking accumulation of LDL. ApoE−/− mice will develop atherosclerosis spontaneously even on
398
L.R. Tannock / Atherosclerosis 219 (2011) 397–398
a normal chow diet, whereas LDLR−/− usually require an additional pro-atherogenic stimulus such as a high fat/high cholesterol diet. Furthermore, the deficiency of apoE is thought to be detrimental, as apoE is thought to have anti-oxidant and anti-proliferative properties [4], suggesting that the susceptibility of apoE−/− versus LDLR−/− models to atherogenic factors may differ. In addition, when challenged with diabetogenic diets apoE−/− mice are more resistant than LDLR−/− mice to elevations in glucose and insulin [5]. Thus, the responses to the diets used, and ursolic acid administered in these two studies, could be expected to differ. Second, the stage of atherosclerosis evaluated between the two studies is different, with the Ullevig study investigating relatively early, macrophage rich stages (11 weeks) and the Messner study evaluating later stages (24 weeks). There are several examples of factors that have transient effects on atherosclerosis development [6–8]. Indeed, Messner et al. acknowledge that the prolonged duration of treatment may have caused systemic toxicity that could indirectly affect the atherosclerosis development. Third, the atherosclerosis measurements differ between the two studies. Messner et al. report only one site: the en face aorta. Their data is depicted as the area of atherosclerosis in the aorta from its root to iliac bifurcation. Conversely, Ullevig et al. report both en face aortic and aortic root atherosclerosis. However, their data depicts the en face aortic atherosclerosis as percent lesion area of the aortic arch to 3 mm distal to the left subclavian artery. To convert their results to area, we can estimate that the aortic surface area of this region of the aorta is approximately 15 mm2 (personal observations) so 6% (as reported for the control mouse) is 900 m2 and 3% for the ursolic acid treated mouse is 450 m2 . Thus, the atherosclerotic area for the untreated mice in each study is comparable. However, the image shown in Fig 3A of Messner et al. suggests that a significant proportion of the atherosclerosis in the ursolic acid treated animal is distal to the upper 3 mm of the aorta, suggesting that perhaps ursolic acid is having site specific effects. In addition, the two studies report on different components of atherosclerosis plaque composition further limiting direct comparisons. Fourth, the dose and method of ursolic acid administered differ between the studies. Messner et al. report doses of 10 and 30 mM ursolic acid administered daily in drinking water, whereas Ullevig et al. report using 0.2% milled into diet, which they estimate is 300 mg/kg/d. Using a molecular weight for ursolic acid of 456.7 g/mol, and presumptive consumption of 5 ml/day drinking water and 3 g/d chow, the respective ursolic acid doses in the two studies are Messner: 0.023 and 0.068 mg/mouse/day and Ullevig 6 mg/mouse/day. The lower does used by Messner et al. likely reflects the poor water solubility of ursolic acid; however these
strikingly different doses could certainly contribute to the opposing results observed. Finally, the cell types evaluated are different. While both endothelial cells and macrophages are clearly pertinent to atherosclerosis development, it is not at all surprising that these cell types may respond differently to ursolic acid. Both studies include carefully conducted in vitro experiments using their cell types of interest, and each study reports concordance between their in vitro and in vivo findings, suggesting their conclusions are valid. However, the mechanisms evaluated clearly differ between the two studies, thus do not refute each other. Thus, although it appears that Atherosclerosis has published, back to back, two conflicting studies on the effects of ursolic acid on atherosclerosis, this is not the case. Numerous differences between the studies can easily account for the different outcomes reported, thus the results are not necessarily in conflict. Unfortunately, this leaves the reader confused, and the potential impact of ursolic acid on cardiovascular disease unclear. Perhaps this will inspire a comparative study where the effects of ursolic acid on endothelial cells, macrophages, and other cell types pertinent to atherosclerosis are compared directly. At a minimum these apparently discrepant results call attention to the potential confounders of atherosclerosis studies – models selected, diets used, study duration, and dose of agent of interest. References [1] Messner B, Zeller I, Ploner C, et al. Ursolic acid causes DNA-damage, P53mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo. Atherosclerosis 2011;219:402–8. [2] Ullevig SL, Zhao Q, Zamora D, Asmis R. Ursolic acid protects diabetic mice against monocyte dysfunction and accelerated atherosclerosis. Atherosclerosis 2011;219:409–17. [3] Veniant MM, Withycombe S, Young SG. Lipoprotein size and atherosclerosis susceptibility in Apoe(−/−) and Ldlr(−/−) mice. Arterioscler Thromb Vasc Biol 2001;21:1567–70. [4] Davignon J, Cohn JS, Mabile L, Bernier L. Apolipoprotein E and atherosclerosis: insight from animal and human studies. Clin Chim Acta 1999;286: 115–43. [5] Schreyer SA, Lystig TC, Vick CM, LeBoeuf RC. Mice deficient in apolipoprotein E but not LDL receptors are resistant to accelerated atherosclerosis associated with obesity. Atherosclerosis 2003;171:49–55. [6] Tannock LR, Kirk EA, King VL, LeBoeuf RC, Wight TN, Chait A. Glucosamine supplementation accelerates early but not late atherosclerosis in LDL receptor deficient mice. J Nutr 2006;136:2856–61. [7] Song L, Leung C, Schindler C. Lymphocytes are important in early atherosclerosis. J Clin Invest 2001;108:251–9. [8] Harats D, Shaish A, George J, et al. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 2000;20:2100–5.
Contents lists available at SciVerse ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Invited commentary
Ursolic acid effect on atherosclerosis: Apples and apples, or apples and oranges? Lisa R. Tannock a,b,∗ a b
Division of Endocrinology and Molecular Medicine, University of Kentucky, Lexington, KY, USA Department of Veterans Affairs, Lexington, KY, USA
a r t i c l e
i n f o
Article history: Received 13 September 2011 Accepted 14 September 2011 Available online 28 September 2011 Keywords: Ursolic acid Atherosclerosis Mouse models
Ursolic acid is a phytonutrient found in several different plant species, and of potential interest to cardiovascular research due its anti-oxidative, anti-inflammatory and other potentially cardioprotective properties. Although there is a wealth of data evaluating ursolic acid’s biological effects, the literature is conflicted with some studies showing potentially beneficial effects while others show potentially negative effects. Furthermore, there is a paucity of research specifically investigating the potential effects of ursolic acid on atherosclerosis or vascular diseases. However, as illustrated by the two studies in this issue investigating the effect of ursolic acid on atherosclerosis using murine models, the controversy remains. Messner et al. [1] investigated the effect of ursolic acid supplied in the drinking water on atherosclerosis development using male apoE deficient (apoE−/−) mice fed a Western diet. Their data demonstrates a dose dependent increase in atherosclerosis in mice receiving ursolic acid. Although there were some inconsistent effects of the ursolic acid on the plasma lipid levels, they propose that the pro-atherogenic effects of ursolic acid were mediated by its effects to inhibit endothelial cell proliferation and induce cell death. Additionally they report a reduction in the serum levels of IL-5 and suggest that this may also play a causal role in ursolic acid’s induction of atherogenesis. Conversely, Ullevig et al. [2] report anti-atherogenic effects of ursolic acid. In their studies, female LDL receptor deficient (LDLR−/−) mice were first treated with streptozotocin to induce
DOIs of original articles: 10.1016/j.atherosclerosis.2011.05.025, 10.1016/j.atherosclerosis.2011.06.013 ∗ Corresponding author at: University of Kentucky, Division of Endocrinology and Molecular Medicine, Wethington Building, Room 567, 900 South Limestone Street, Lexington, KY 40536-0200, USA. Tel.: +1 859 218 1415; fax: +1 859 257 3646. E-mail address: [email protected] 0021-9150/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.atherosclerosis.2011.09.029
beta cell failure and hyperglycemia, then were fed a high fat diet for 11 weeks, supplemented either with resveratrol or ursolic acid. After 11 weeks, the mice receiving ursolic acid were found to have decreased atherosclerosis measured at two sites: the en face aorta (as in Messner et al.) and the aortic root. They report no effects of ursolic acid on plasma lipid levels but did find a significant reduction in plasma glucose levels. In addition, the mice receiving ursolic acid had better survival and higher body weights than the mice on diet alone. The authors investigated the effect of ursolic acid on monocyte/macrophage function, and report decreased monocyte recruitment in mice receiving ursolic acid, which they suggest as a mechanism by which atherosclerosis is reduced. Thus, the reader is faced with two studies with conflicting reports on the effect of ursolic acid on atherosclerosis development. However, upon close scrutiny of the studies, there are so many differences between the studies that perhaps one should simply conclude that the comparisons are “apples and oranges” rather than “apples and apples”. First, the studies used very different animal models: apoE−/− versus diabetic LDLR−/− mice; the duration of study and diets differed; the atherosclerosis measurements differed; the dose of ursolic acid administered is expressed differently so not easily compared; and the mechanistic studies evaluated different cell types, albeit both pertinent to atherosclerosis. Let us address these issue by issue. First, apoE−/− and LDLR−/− mice are two of the most commonly studied murine models of atherosclerosis, yet differ strikingly in the disease development. While both are hyperlipidemic, their lipoprotein profiles differ dramatically, and the effect on atherogenesis also differs [3]. On chow diets apoE-/- mice have total cholesterol levels approximating 500 mg/dl with accumulation of VLDL remnants, whereas LDLR−/− mice have much lower total cholesterol levels (approximating 200 mg/dl) with striking accumulation of LDL. ApoE−/− mice will develop atherosclerosis spontaneously even on
398
L.R. Tannock / Atherosclerosis 219 (2011) 397–398
a normal chow diet, whereas LDLR−/− usually require an additional pro-atherogenic stimulus such as a high fat/high cholesterol diet. Furthermore, the deficiency of apoE is thought to be detrimental, as apoE is thought to have anti-oxidant and anti-proliferative properties [4], suggesting that the susceptibility of apoE−/− versus LDLR−/− models to atherogenic factors may differ. In addition, when challenged with diabetogenic diets apoE−/− mice are more resistant than LDLR−/− mice to elevations in glucose and insulin [5]. Thus, the responses to the diets used, and ursolic acid administered in these two studies, could be expected to differ. Second, the stage of atherosclerosis evaluated between the two studies is different, with the Ullevig study investigating relatively early, macrophage rich stages (11 weeks) and the Messner study evaluating later stages (24 weeks). There are several examples of factors that have transient effects on atherosclerosis development [6–8]. Indeed, Messner et al. acknowledge that the prolonged duration of treatment may have caused systemic toxicity that could indirectly affect the atherosclerosis development. Third, the atherosclerosis measurements differ between the two studies. Messner et al. report only one site: the en face aorta. Their data is depicted as the area of atherosclerosis in the aorta from its root to iliac bifurcation. Conversely, Ullevig et al. report both en face aortic and aortic root atherosclerosis. However, their data depicts the en face aortic atherosclerosis as percent lesion area of the aortic arch to 3 mm distal to the left subclavian artery. To convert their results to area, we can estimate that the aortic surface area of this region of the aorta is approximately 15 mm2 (personal observations) so 6% (as reported for the control mouse) is 900 m2 and 3% for the ursolic acid treated mouse is 450 m2 . Thus, the atherosclerotic area for the untreated mice in each study is comparable. However, the image shown in Fig 3A of Messner et al. suggests that a significant proportion of the atherosclerosis in the ursolic acid treated animal is distal to the upper 3 mm of the aorta, suggesting that perhaps ursolic acid is having site specific effects. In addition, the two studies report on different components of atherosclerosis plaque composition further limiting direct comparisons. Fourth, the dose and method of ursolic acid administered differ between the studies. Messner et al. report doses of 10 and 30 mM ursolic acid administered daily in drinking water, whereas Ullevig et al. report using 0.2% milled into diet, which they estimate is 300 mg/kg/d. Using a molecular weight for ursolic acid of 456.7 g/mol, and presumptive consumption of 5 ml/day drinking water and 3 g/d chow, the respective ursolic acid doses in the two studies are Messner: 0.023 and 0.068 mg/mouse/day and Ullevig 6 mg/mouse/day. The lower does used by Messner et al. likely reflects the poor water solubility of ursolic acid; however these
strikingly different doses could certainly contribute to the opposing results observed. Finally, the cell types evaluated are different. While both endothelial cells and macrophages are clearly pertinent to atherosclerosis development, it is not at all surprising that these cell types may respond differently to ursolic acid. Both studies include carefully conducted in vitro experiments using their cell types of interest, and each study reports concordance between their in vitro and in vivo findings, suggesting their conclusions are valid. However, the mechanisms evaluated clearly differ between the two studies, thus do not refute each other. Thus, although it appears that Atherosclerosis has published, back to back, two conflicting studies on the effects of ursolic acid on atherosclerosis, this is not the case. Numerous differences between the studies can easily account for the different outcomes reported, thus the results are not necessarily in conflict. Unfortunately, this leaves the reader confused, and the potential impact of ursolic acid on cardiovascular disease unclear. Perhaps this will inspire a comparative study where the effects of ursolic acid on endothelial cells, macrophages, and other cell types pertinent to atherosclerosis are compared directly. At a minimum these apparently discrepant results call attention to the potential confounders of atherosclerosis studies – models selected, diets used, study duration, and dose of agent of interest. References [1] Messner B, Zeller I, Ploner C, et al. Ursolic acid causes DNA-damage, P53mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo. Atherosclerosis 2011;219:402–8. [2] Ullevig SL, Zhao Q, Zamora D, Asmis R. Ursolic acid protects diabetic mice against monocyte dysfunction and accelerated atherosclerosis. Atherosclerosis 2011;219:409–17. [3] Veniant MM, Withycombe S, Young SG. Lipoprotein size and atherosclerosis susceptibility in Apoe(−/−) and Ldlr(−/−) mice. Arterioscler Thromb Vasc Biol 2001;21:1567–70. [4] Davignon J, Cohn JS, Mabile L, Bernier L. Apolipoprotein E and atherosclerosis: insight from animal and human studies. Clin Chim Acta 1999;286: 115–43. [5] Schreyer SA, Lystig TC, Vick CM, LeBoeuf RC. Mice deficient in apolipoprotein E but not LDL receptors are resistant to accelerated atherosclerosis associated with obesity. Atherosclerosis 2003;171:49–55. [6] Tannock LR, Kirk EA, King VL, LeBoeuf RC, Wight TN, Chait A. Glucosamine supplementation accelerates early but not late atherosclerosis in LDL receptor deficient mice. J Nutr 2006;136:2856–61. [7] Song L, Leung C, Schindler C. Lymphocytes are important in early atherosclerosis. J Clin Invest 2001;108:251–9. [8] Harats D, Shaish A, George J, et al. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 2000;20:2100–5.