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Intermediate density lipoprotein cholesterol as the best lipoprotein predictor of atherosclerosis severity in the Watanabe Heritable Hyperlipidemic rabbit
Atherosclerosis 132 (1997) 119 – 122
Letter to the Editors
Intermediate density lipoprotein cholesterol as the best lipoprotein predictor of atherosclerosis severity in the Watanabe Heritable Hyperlipidemic rabbit Børge G. Nordestgaard a,*, Birgit Agerholm-Larsen b, Alicja Mortensen e, Birgit Fischer Hansen c, Jørgen Fischer Hansen d, Per Ibsen e, Knud Kjeldsen a b
a Department of Clinical Biochemistry, Rigshospitalet, Uni6ersity of Copenhagen, Copenhagen, Denmark Department of Clinical Biochemistry, Herle6 Hospital, Uni6ersity of Copenhagen, Herle6 Ring6ej 75, DK-2730 Herle6, Denmark c Department of Pathology, H6ido6re Hospital, Uni6ersity of Copenhagen, Copenhagen, Denmark d Department of Cardiology, H6ido6re Hospital, Uni6ersity of Copenhagen, Copenhagen, Denmark e Institute of Toxicology, National Food Agency of Denmark, Denmark
Received 1 November 1996; received in revised form 15 January 1997; accepted 24 February 1997
Keywords: Familial hypercholesterolemia; Low density lipoprotein; Postprandial lipoproteins
Dear Editors, Zilversmit proposed that atherogenesis might be a postprandial phenomena [1], implying that chylomicron remnants and even very low density lipoprotein (VLDL) remnants, the so-called intermediate density lipoprotien (IDL) particles, may share with low density lipoprotein (LDL) the ability to cause atherosclerosis. This hypothesis has gained strength over the years [2,3]. Familial hypercholesterolemia (FH), a disorder caused by defects in the LDL-receptor leading to high LDL levels and premature atherosclerosis [4], represents an important observation supporting a role for LDL as the most atherogenic lipoprotein. Both LDL and IDL levels are, however, elevated in patients with FH [4]. The relative role of these two lipoproteins or of chylomicron remnants in promoting atherosclerosis in FH, is at present unknown. In the present study we took advantage of the existence of an animal model of FH, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit that also has * Corresponding author. Present address: Department of Clinical Biochemistry, Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730, Herlev, Denmark. Tel.: +45 44 535300 ext. 3843; fax: +45 44 535332.
defective LDL-receptors [4], to examine the relative importance of chylomicron remnants, IDL and LDL in atherogenesis: fasting (24 h after last meal) and postprandial (2–4 h after the daily meal) levels of cholesterol and triglycerides (CHOD-PAP and GPO- PAP, Boehringer Mannheim) in total plasma, and in the VLDL (dB 1.006 g/ml), IDL (1.006 g/mlB dB1.019 g/ml), LDL (1.019 g/mlB dB 1.063 g/ml) and high density lipoprotein (HDL) (d\ 1.063 g/ml) fractions were compared as predictors of atherosclerosis severity in eight female and ten male, 19 month old, chow-fed, WHHL rabbits (11 homozygotes and seven heterozygotes) [5]. Atherosclerosis severity was assesed as cholesterol content in the intima-inner media of the aortic arch (from the heart to the first intercostal arteries), the thoracic aorta (to the celiac artery), and the abdominal aorta, as well as intimal area on histological cross-sections of the ascending aorta and proximal right coronary artery [5,6]. For histological evaluation of atherosclerosis severity, only results from homozygous rabbits were included in the analysis, as no significant intimal thickening was found in heterozygous animals. The Minitab programme was used for statistics.
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B.G. Nordestgaard et al. / Atherosclerosis 132 (1997) 119–122
In the postprandial state, VLDL cholesterol and plasma and VLDL triglycerides were significantly higher, and LDL and IDL cholesterol significantly lower, than in the fasting state (Table 1). IDL cholesterol in the postprandial state represents a mixture of IDL of hepatic origin and chylomicron remnants. Fasting and postprandial plasma, VLDL, IDL and LDL cholesterol and triglycerides were consistently positively associated, and HDL cholesterol and triglycerides negatively associated with aortic cholesterol content (Table 2). Fasting and postprandial IDL cholesterol were consistently positively associated with the intimal area, whereas fasting and postprandial LDL and VLDL cholesterol were positively associated with the intimal area in some, but not all comparisons. On stepwise multiple linear regression analysis which included postprandial and fasting VLDL, IDL, LDL and HDL cholesterol as well as postprandial and fasting total plasma triglycerides as predictors of atherosclerosis severity, fasting IDL cholesterol was the best independent predictor in four of five comparisons (Table 3). Fasting LDL cholesterol was the best independent predictor for aortic cholesterol content in the abdominal aorta. Although IDL cholesterol in the present study only constituted 12–14% of plasma cholesterol in WHHL Table 1 Cholesterol and triglycerides in plasma and lipoproteins and severity of aortic atherosclerosis of eighteen 19 month old Watanabe Heritable Hyperlipidemic rabbits Postprandial
Fasting
Cholesterol (mmol/l) Plasma VLDL IDL LDL HDL
9.259 2.76 3.78 9 1.81** 1.149 0.27* 4.069 0.97* 0.279 0.03
9.249 2.39 2.54 9 1.27 1.329 0.31 5.059 1.18 0.339 0.04
Triglycerides (mmol/l) Plasma VLDL IDL LDL HDL
6.289 2.02** 4.029 1.55** 0.429 0.13 1.599 0.41 0.259 0.04
3.649 1.13 1.74 9 0.71 0.369 0.11 1.32 9 0.35 0.229 0.02
Cholesterol content (nmol/cm2) Aortic arch Thoracic aorta Abdominal aorta Intimal area (mm2) Ascending aorta (n = 9) Proximal right coronary artery (n= 7)
33759 839 18779637 8729 287 3.939 0.92 0.689 0.10
Values are means9 S.E.M. representing 11 homozygotes and seven heterozygotes. Levels in the postprandial state significantly different from that in the fasting state at respectively the * PB0.05 and ** PB0.01 level, using paired t-test.
rabbits, IDL cholesterol was the best independent predictor of atherosclerosis severity. This finding would at first hand seem to challenge the generally held view that LDL levels is most important for atherogenesis in WHHL rabbits as well as humans with FH; however, both LDL and IDL particles interact with the LDL-receptor, and both lipoproteins are elevated in plasma of FH patients and WHHL rabbits [4,7,8]. Remnants of chylomicrons are also removed from plasma of WHHL rabbits at a reduced rate [9,10]. Our results are furthermore supported by the observation that a midband between LDL and VLDL on polyacrylamide gel electrophoresis predicts coronary atherosclerosis in both WHHL rabbits [11] and FH patients [12]; this midband could be IDL particles. IDL levels have previously been found to be strong predictors of atherosclerosis in genetically hyperlipidemic [13] and cholesterol- or casein-fed rabbits [14]. In humans without major genetic or secondary forms of hyperlipidemia, IDL and small VLDL have been shown to be independent predictors of the presence, severity, or progression of atherosclerosis [15–19]. Patients with type III hyperlipoproteinemia [20], chronic renal failure [21], or non-insulin-dependent diabetes mellitus [22] all have elevated levels of remnant lipoproteins as well as accelerated development of atherosclerosis. Finally, elevated postprandial levels of the triglycerides or remnant lipoproteins have also been found to be predictors of the presence, severity, progression or familial risk of atherosclerosis [23–29]. The present results therefore add to the now growing evidence that remnant lipoproteins may be involved directly in promoting atherosclerosis [1–3]. The mechanism by which smaller triglyceride-rich lipoproteins and particularly IDL particles could promote atherosclerosis directly, would involve transfer of these particles into the intima [30,31], where such particles appear to be retained selectively [31,32]. In contrast to LDL, smaller triglyceride-rich lipoproteins can then without prior modification be taken up directly by macrophages to produce foam cells [33], a key cell type of the atherosclerotic plaque [34]. Since increased IDL levels are associated with reduced HDL levels as well as increased levels of small dense LDL, the association between IDL and atherosclerosis could, however, be caused by reduced reverse cholesterol transport [35] or increased atherosclerosis caused by small dense LDL [36], rather than by a direct effect of IDL particles. In conclusion, the present results suggest that IDL levels may be as important as LDL levels in promoting atherosclerosis in WHHL rabbits. This finding potentially could help improve our understanding of the pathogenesis of atherosclerosis in general, and FH in particular.
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Table 2 Univariate correlation coefficients of severity of atherosclerosis as a function of cholesterol and triglycerides in plasma and lipoproteins in 19 month old Watanabe Heritable Hyperlipidemic rabbits Independent vari- Dependent variables ables Cholesterol content Aortic arch (n= Thoracic aorta 18) (n =18)
Intimal area Abdominal aorta (n=18)
Ascending aorta (n =9)
Proximal right coronary artery (n= 7)
Postprandial cholesterol Plasma 0.78 VLDL 0.82 IDL 0.91 LDL 0.88 HDL −0.62
0.77 0.87 0.90 0.90 −0.57
0.72 0.81 0.89 0.91 −0.54
NS NS 0.78 0.68 NS
0.82 NS 0.76 0.76 NS
Postprandial triglycerides Plasma 0.72 VLDL 0.51 IDL 0.81 LDL 0.82 HDL −0.66
0.74 0.51 0.85 0.83 −0.72
0.66 NS 0.82 0.77 −0.72
NS NS NS 0.67 NS
NS NS NS NS NS
Fasting cholesterol Plasma 0.89 VLDL 0.85 IDL 0.92 LDL 0.90 HDL −0.56
0.88 0.88 0.92 0.91 −0.56
0.85 0.82 0.90 0.94 −0.53
0.71 0.75 0.84 NS NS
0.87 NS 0.89 NS NS
Fasting triglycerides Plasma 0.77 VLDL 0.65 IDL 0.79 LDL 0.82 HDL −0.51
0.82 0.65 0.87 0.86 −0.64
0.74 0.56 0.79 0.79 −0.66
0.71 NS NS 0.73 NS
NS NS NS NS NS
To obtain data with a near-normal distribution, plasma, VLDL and IDL triglycerides, VLDL cholesterol, aortic cholesterol content and intimal area were all transformed logarithmically (log10) before the statistical analysis. NS, not statistically significant (P\0.05).
Acknowledgements Hanne Damm, Kurt S. Jensen and Lone Christensen provided skilful technical assistance and Erna Quist Table 3 Ranking of lipoprotein lipids as independent predictors of severity of atherosclerosis in 19 month old Watanabe Heritable Hyperlipidemic rabbits First rank Cholesterol content Aortic arch Thoracic aorta Abdominal aorta Intimal area Ascending aorta Proximal right coronary artery
R2
Fasting IDL cholesterol 0.84 Fasting IDL cholesterol 0.85 Fasting LDL cholesterol 0.88 Fasting IDL cholesterol 0.71 Fasting IDL cholesterol 0.80
In none of the five analyses did a second independent predictor statistical significance (PB0.05).
typed the manuscript. Børge G. Nordestgaard was supported by the Danish Heart Foundation and ‘Overlæge Johan Boserup and Lise Boserups Legat’ during these studies. References [1] Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473 – 485. [2] Slyper AH. A fresh look at the atherogenic remnant hypothesis. Lancet 1992;340:289 – 291. [3] Zilversmit DB. Atherogenic nature of triglycerides, postprandial lipidemia, and triglyceride-rich remnant lipoproteins. Clin Chem 1995;41:153 – 158. [4] Goldstein JL, Hobbs HH, Brown MS. Familial Hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1985:1981 – 2030. [5] Hansen BF, Mortensen A, Hansen JF, Ibsen P, Frandsen H, Nordestgaard BG. Atherosclerosis in Watanabe Heritable Hyperlipidaemic rabbits. Evaluation of macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables. APMIS 1994;102:177 – 190.
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[6] Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497– 507. [7] Yamada N, Shames DM, Havel RJ. Effect of low density lipoprotein receptor deficiency on the metabolism of apolipoprotein B-100 in blood plasma. Kinetic studies in normal and Watanabe heritable hyperlipidemic rabbits. J Clin Invest 1987;80:507 – 515. [8] Rosenfeld ME, Tsukada T, Gown AM, Ross R. Fatty streak initation in Watanabe Heritable Hyperlipidemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 1987;7:9 – 23. [9] Beaumont JL, Assadollahi F. Retinyl palmitate labeled intestinally derived lipoproteins accumulate in the circulation of WHHL rabbits. Atherosclerosis 1990;85:103–111. [10] Bowler A, Redgrave TG, Mamo JCL. Chylomicron-remnant clearance in homozygote and heterozygote Watanabe Heritable Hyperlipidaemic rabbits is defective. Biochem J 1991;276:381 – 386. [11] Shiomi M, Ito T, Shiraishi M, Watanabe Y. Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: Risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis. Atherosclerosis 1992;96:43 – 52. [12] Tashiro J, Nishide T, Shinomiya M, Shirai K, Saito Y, Yoshida S, Yamashita M, Ohskima H, Murayama H. The ‘midband’ lipoprotein is a coronary risk factor in Japanese patients with familial hypercholesterolaemia. Scand J Clin Lab Invest 1993;53:335 – 338. [13] Nordestgaard BG, Lewis B. Intermediate density lipoprotein levels are strong predictors of the extent of aortic atherosclerosis in the St. Thomas’s Hospital rabbit strain. Atherosclerosis 1991;87:39 – 46. [14] Daley SJ, Herderick EE, Cornhill JF, Rogers KA. Cholesterolfed and casein-fed rabbit models of atherosclerosis. Part 1: Differing lesion area and volume despite equal plasma cholesterol levels. Arterioscler Thromb 1994;14:95–104. [15] Tatami R, Mabuchi H, Ueda K, Ueda R, Toshihiro H, Kametani T, Ito S, Koizumi J, Ohta M, Miyamoto S, Nakayana A, Kanaya H, Oiwake H, Genda A, Takeda R. Intermediatedensity lipoprotein and cholesterol-rich very low density lipoprotein in angiographically determined coronary artery disease. Circulation 1981;64:1174–1184. [16] Reardon MF, Nestel PJ, Craig IH, Harper RW. Lipoprotein predictors of the severity of coronary artery disease in men and women. Circulation 1985;71:881–888. [17] Steiner G, Schwartz L, Shumak S, Poapst M. The association of increased levels of intermediate-density lipoproteins with smoking and with coronary artery disease. Circulation 1987;75:124 – 130. [18] Krauss RM, Lindgren FT, Williams PT, Kelsey SF, Brensike J, Vranizan K, Detre KM, Levy RI. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men. Lancet 1987; II:62– 66. [19] Phillips NR, Waters D, Havel RJ. Plasma lipoproteins and progression of coronary artery disease evaluated by angiography and clinical events. Circulation 1993;88:2762–2770. [20] Mahley RW, Rall SC. Type III hyperlipoproteinemia (Dysbetalipoproteinemia): The role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds), The Metabolic and Molecular Bases of Inherited Disease, 7th edn. New York: McGraw-Hill, 1995:1953 – 1980.
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[21] Nestel PJ, Fidge NH, Tan MH. Increased lipoprotein-remnant formation in chronic renal failure. New Engl J Med 1982;307:329 – 333. [22] Kasama T, Yoshino G, Iwatani I, et al. Increased cholesterol concentration in intermediate density lipoprotein fraction of normolipidemic non-insulin-dependent diabetics. Atherosclerosis 1987;63:263 – 266. [23] Simons LA, Dwyer T, Simons J, Bernstein L, Mock P, Poonia NS, Balasubramaniam S, Baron D, Branson J, Morgan J, Roy P. Chylomicrons and chylomicron remnants in coronary artery disease: A case-control study. Atherosclerosis 1987;65:181–189. [24] Simpson HS, Williamson CM, Olivecrona T, Pringle S, Maclean J, Lorimer AR, Bonnefous F, Bogaievsky Y, Packard CJ, Shepherd J. Postprandial lipemia, fenofibrate and coronary artery disease. Atherosclerosis 1990;85:193 – 202. [25] Groot PHE, van Stiphout WAHJ, Krauss XH, Jansen H, van Tol A, van Ramshorst E, Chin-On S, Hofman A, Cresswell SR, Havekes L. Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arterioscler Thromb 1991;11:653 – 662. [26] Ryu JE, Howard G, Craven TE, Bond MG, Hagaman AP, Crouse JR. Postprandial triglyceridemia and carotid atherosclerosis in middle-aged subjects. Stroke 1992;23:823 – 828. [27] Patsch JR, Miesenbo¨ck G, Hopferwieser T, Mu¨hlberger V, Knapp E, Dunn JK, Gotto AM Jr, Patsch W. Relation of triglyceride metabolism and coronary artery disease. Studies in the postprandial state. Arterioscler Thromb 1992;12:1336–1345. [28] Uiterwaal CSPM, Grobbee DE, Witteman JCM, van Stiphout WAHJ, Krauss XH, Havekes LM, de Bruijn AM, van Tol A, Hofman A. Postprandial triglyceride response in young adult men and familial risk for coronary atherosclerosis. Ann Intern Med 1994;121:576 – 583. [29] Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A. Postprandial lipoproteins and progression of coronary atherosclerosis. Atherosclerosis 1994;106:83 – 97. [30] Shaikh M, Wootton R, Nordestgaard BG, Baskerville P, Lumley JS, La Ville AE, Quiney J, Lewis B. Quantitative studies of transfer in vivo of low density, Sf 12-60, and Sf 60 –400 lipoproteins between plasma and arterial intima in humans. Arterioscler Thromb 1991;11:569 – 577. [31] Nordestgaard BG, Wootton R, Lewis B. Selective retention of VLDL, IDL, and LDL in the arterial intima of genetically hyperlipidemic rabbits in vivo. Molecular size as a determinant of fractional loss from the intima-inner media. Arterioscler Thromb Vasc Biol 1995;15:534 – 542. [32] Rapp JH, Lespine A, Hamilton RL, Colyvas N, Chaumeton AH, Tweedie-Hardman J, Kotite L, Kunitake ST, Havel RJ, Kane JP. Triglyceride-rich lipoproteins isolated by selectedaffinity anti-apolipoprotein B immunosorption from human atherosclerotic plaque. Arterioscler Thromb 1994;14:1767–1774. [33] Bradley WA, Gianturco SH. Triglyceride-rich lipoproteins and atherosclerosis: Pathophysiological considerations. J Intern Med 1994;236, suppl 736:33 – 39. [34] Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the committee on vascular lesions of the council on arteriosclerosis, American Heart Association. Arterioscler Thromb 1994;14:840 – 856. [35] Barter PJ, Rye KA. High density lipoproteins and coronary heart disease. Atherosclerosis 1996;121:1 – 12. [36] Austin Plasma triglyceride and coronary heart disease. Arterioscler Thromb 1991;11:2 – 14.
Letter to the Editors
Intermediate density lipoprotein cholesterol as the best lipoprotein predictor of atherosclerosis severity in the Watanabe Heritable Hyperlipidemic rabbit Børge G. Nordestgaard a,*, Birgit Agerholm-Larsen b, Alicja Mortensen e, Birgit Fischer Hansen c, Jørgen Fischer Hansen d, Per Ibsen e, Knud Kjeldsen a b
a Department of Clinical Biochemistry, Rigshospitalet, Uni6ersity of Copenhagen, Copenhagen, Denmark Department of Clinical Biochemistry, Herle6 Hospital, Uni6ersity of Copenhagen, Herle6 Ring6ej 75, DK-2730 Herle6, Denmark c Department of Pathology, H6ido6re Hospital, Uni6ersity of Copenhagen, Copenhagen, Denmark d Department of Cardiology, H6ido6re Hospital, Uni6ersity of Copenhagen, Copenhagen, Denmark e Institute of Toxicology, National Food Agency of Denmark, Denmark
Received 1 November 1996; received in revised form 15 January 1997; accepted 24 February 1997
Keywords: Familial hypercholesterolemia; Low density lipoprotein; Postprandial lipoproteins
Dear Editors, Zilversmit proposed that atherogenesis might be a postprandial phenomena [1], implying that chylomicron remnants and even very low density lipoprotein (VLDL) remnants, the so-called intermediate density lipoprotien (IDL) particles, may share with low density lipoprotein (LDL) the ability to cause atherosclerosis. This hypothesis has gained strength over the years [2,3]. Familial hypercholesterolemia (FH), a disorder caused by defects in the LDL-receptor leading to high LDL levels and premature atherosclerosis [4], represents an important observation supporting a role for LDL as the most atherogenic lipoprotein. Both LDL and IDL levels are, however, elevated in patients with FH [4]. The relative role of these two lipoproteins or of chylomicron remnants in promoting atherosclerosis in FH, is at present unknown. In the present study we took advantage of the existence of an animal model of FH, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit that also has * Corresponding author. Present address: Department of Clinical Biochemistry, Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730, Herlev, Denmark. Tel.: +45 44 535300 ext. 3843; fax: +45 44 535332.
defective LDL-receptors [4], to examine the relative importance of chylomicron remnants, IDL and LDL in atherogenesis: fasting (24 h after last meal) and postprandial (2–4 h after the daily meal) levels of cholesterol and triglycerides (CHOD-PAP and GPO- PAP, Boehringer Mannheim) in total plasma, and in the VLDL (dB 1.006 g/ml), IDL (1.006 g/mlB dB1.019 g/ml), LDL (1.019 g/mlB dB 1.063 g/ml) and high density lipoprotein (HDL) (d\ 1.063 g/ml) fractions were compared as predictors of atherosclerosis severity in eight female and ten male, 19 month old, chow-fed, WHHL rabbits (11 homozygotes and seven heterozygotes) [5]. Atherosclerosis severity was assesed as cholesterol content in the intima-inner media of the aortic arch (from the heart to the first intercostal arteries), the thoracic aorta (to the celiac artery), and the abdominal aorta, as well as intimal area on histological cross-sections of the ascending aorta and proximal right coronary artery [5,6]. For histological evaluation of atherosclerosis severity, only results from homozygous rabbits were included in the analysis, as no significant intimal thickening was found in heterozygous animals. The Minitab programme was used for statistics.
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B.G. Nordestgaard et al. / Atherosclerosis 132 (1997) 119–122
In the postprandial state, VLDL cholesterol and plasma and VLDL triglycerides were significantly higher, and LDL and IDL cholesterol significantly lower, than in the fasting state (Table 1). IDL cholesterol in the postprandial state represents a mixture of IDL of hepatic origin and chylomicron remnants. Fasting and postprandial plasma, VLDL, IDL and LDL cholesterol and triglycerides were consistently positively associated, and HDL cholesterol and triglycerides negatively associated with aortic cholesterol content (Table 2). Fasting and postprandial IDL cholesterol were consistently positively associated with the intimal area, whereas fasting and postprandial LDL and VLDL cholesterol were positively associated with the intimal area in some, but not all comparisons. On stepwise multiple linear regression analysis which included postprandial and fasting VLDL, IDL, LDL and HDL cholesterol as well as postprandial and fasting total plasma triglycerides as predictors of atherosclerosis severity, fasting IDL cholesterol was the best independent predictor in four of five comparisons (Table 3). Fasting LDL cholesterol was the best independent predictor for aortic cholesterol content in the abdominal aorta. Although IDL cholesterol in the present study only constituted 12–14% of plasma cholesterol in WHHL Table 1 Cholesterol and triglycerides in plasma and lipoproteins and severity of aortic atherosclerosis of eighteen 19 month old Watanabe Heritable Hyperlipidemic rabbits Postprandial
Fasting
Cholesterol (mmol/l) Plasma VLDL IDL LDL HDL
9.259 2.76 3.78 9 1.81** 1.149 0.27* 4.069 0.97* 0.279 0.03
9.249 2.39 2.54 9 1.27 1.329 0.31 5.059 1.18 0.339 0.04
Triglycerides (mmol/l) Plasma VLDL IDL LDL HDL
6.289 2.02** 4.029 1.55** 0.429 0.13 1.599 0.41 0.259 0.04
3.649 1.13 1.74 9 0.71 0.369 0.11 1.32 9 0.35 0.229 0.02
Cholesterol content (nmol/cm2) Aortic arch Thoracic aorta Abdominal aorta Intimal area (mm2) Ascending aorta (n = 9) Proximal right coronary artery (n= 7)
33759 839 18779637 8729 287 3.939 0.92 0.689 0.10
Values are means9 S.E.M. representing 11 homozygotes and seven heterozygotes. Levels in the postprandial state significantly different from that in the fasting state at respectively the * PB0.05 and ** PB0.01 level, using paired t-test.
rabbits, IDL cholesterol was the best independent predictor of atherosclerosis severity. This finding would at first hand seem to challenge the generally held view that LDL levels is most important for atherogenesis in WHHL rabbits as well as humans with FH; however, both LDL and IDL particles interact with the LDL-receptor, and both lipoproteins are elevated in plasma of FH patients and WHHL rabbits [4,7,8]. Remnants of chylomicrons are also removed from plasma of WHHL rabbits at a reduced rate [9,10]. Our results are furthermore supported by the observation that a midband between LDL and VLDL on polyacrylamide gel electrophoresis predicts coronary atherosclerosis in both WHHL rabbits [11] and FH patients [12]; this midband could be IDL particles. IDL levels have previously been found to be strong predictors of atherosclerosis in genetically hyperlipidemic [13] and cholesterol- or casein-fed rabbits [14]. In humans without major genetic or secondary forms of hyperlipidemia, IDL and small VLDL have been shown to be independent predictors of the presence, severity, or progression of atherosclerosis [15–19]. Patients with type III hyperlipoproteinemia [20], chronic renal failure [21], or non-insulin-dependent diabetes mellitus [22] all have elevated levels of remnant lipoproteins as well as accelerated development of atherosclerosis. Finally, elevated postprandial levels of the triglycerides or remnant lipoproteins have also been found to be predictors of the presence, severity, progression or familial risk of atherosclerosis [23–29]. The present results therefore add to the now growing evidence that remnant lipoproteins may be involved directly in promoting atherosclerosis [1–3]. The mechanism by which smaller triglyceride-rich lipoproteins and particularly IDL particles could promote atherosclerosis directly, would involve transfer of these particles into the intima [30,31], where such particles appear to be retained selectively [31,32]. In contrast to LDL, smaller triglyceride-rich lipoproteins can then without prior modification be taken up directly by macrophages to produce foam cells [33], a key cell type of the atherosclerotic plaque [34]. Since increased IDL levels are associated with reduced HDL levels as well as increased levels of small dense LDL, the association between IDL and atherosclerosis could, however, be caused by reduced reverse cholesterol transport [35] or increased atherosclerosis caused by small dense LDL [36], rather than by a direct effect of IDL particles. In conclusion, the present results suggest that IDL levels may be as important as LDL levels in promoting atherosclerosis in WHHL rabbits. This finding potentially could help improve our understanding of the pathogenesis of atherosclerosis in general, and FH in particular.
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Table 2 Univariate correlation coefficients of severity of atherosclerosis as a function of cholesterol and triglycerides in plasma and lipoproteins in 19 month old Watanabe Heritable Hyperlipidemic rabbits Independent vari- Dependent variables ables Cholesterol content Aortic arch (n= Thoracic aorta 18) (n =18)
Intimal area Abdominal aorta (n=18)
Ascending aorta (n =9)
Proximal right coronary artery (n= 7)
Postprandial cholesterol Plasma 0.78 VLDL 0.82 IDL 0.91 LDL 0.88 HDL −0.62
0.77 0.87 0.90 0.90 −0.57
0.72 0.81 0.89 0.91 −0.54
NS NS 0.78 0.68 NS
0.82 NS 0.76 0.76 NS
Postprandial triglycerides Plasma 0.72 VLDL 0.51 IDL 0.81 LDL 0.82 HDL −0.66
0.74 0.51 0.85 0.83 −0.72
0.66 NS 0.82 0.77 −0.72
NS NS NS 0.67 NS
NS NS NS NS NS
Fasting cholesterol Plasma 0.89 VLDL 0.85 IDL 0.92 LDL 0.90 HDL −0.56
0.88 0.88 0.92 0.91 −0.56
0.85 0.82 0.90 0.94 −0.53
0.71 0.75 0.84 NS NS
0.87 NS 0.89 NS NS
Fasting triglycerides Plasma 0.77 VLDL 0.65 IDL 0.79 LDL 0.82 HDL −0.51
0.82 0.65 0.87 0.86 −0.64
0.74 0.56 0.79 0.79 −0.66
0.71 NS NS 0.73 NS
NS NS NS NS NS
To obtain data with a near-normal distribution, plasma, VLDL and IDL triglycerides, VLDL cholesterol, aortic cholesterol content and intimal area were all transformed logarithmically (log10) before the statistical analysis. NS, not statistically significant (P\0.05).
Acknowledgements Hanne Damm, Kurt S. Jensen and Lone Christensen provided skilful technical assistance and Erna Quist Table 3 Ranking of lipoprotein lipids as independent predictors of severity of atherosclerosis in 19 month old Watanabe Heritable Hyperlipidemic rabbits First rank Cholesterol content Aortic arch Thoracic aorta Abdominal aorta Intimal area Ascending aorta Proximal right coronary artery
R2
Fasting IDL cholesterol 0.84 Fasting IDL cholesterol 0.85 Fasting LDL cholesterol 0.88 Fasting IDL cholesterol 0.71 Fasting IDL cholesterol 0.80
In none of the five analyses did a second independent predictor statistical significance (PB0.05).
typed the manuscript. Børge G. Nordestgaard was supported by the Danish Heart Foundation and ‘Overlæge Johan Boserup and Lise Boserups Legat’ during these studies. References [1] Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473 – 485. [2] Slyper AH. A fresh look at the atherogenic remnant hypothesis. Lancet 1992;340:289 – 291. [3] Zilversmit DB. Atherogenic nature of triglycerides, postprandial lipidemia, and triglyceride-rich remnant lipoproteins. Clin Chem 1995;41:153 – 158. [4] Goldstein JL, Hobbs HH, Brown MS. Familial Hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, 1985:1981 – 2030. [5] Hansen BF, Mortensen A, Hansen JF, Ibsen P, Frandsen H, Nordestgaard BG. Atherosclerosis in Watanabe Heritable Hyperlipidaemic rabbits. Evaluation of macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables. APMIS 1994;102:177 – 190.
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