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Hypercholesterolemia in pregnant mice does not affect atherosclerosis in adult offspring
Atherosclerosis 168 (2003) 221 /228 www.elsevier.com/locate/atherosclerosis
Hypercholesterolemia in pregnant mice does not affect atherosclerosis in adult offspring Claus Madsen a,*, Frederik Dagnæs-Hansen b, Jan Møller c, Erling Falk a a
Department of Cardiology, Institute of Experimental Clinical Research, Aarhus University Hospital, DK-8200 Aarhus, Denmark b Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark c Department of Clinical Biochemistry, Aarhus University Hospital, DK-8200 Aarhus, Denmark Received 13 September 2002; received in revised form 7 February 2003; accepted 24 February 2003
Abstract In humans, maternal hypercholesterolemia during pregnancy promotes microscopical fatty streaks in the children. The mechanism is unknown. Fatty streaks are clinically silent, and many of them regress and never develop into advanced atherosclerosis. The aim of this study was to investigate whether hypercholesterolemia in pregnant mice induced more advanced atherosclerosis in their adult progeny. Hypercholesterolemic (HC) apolipoprotein E knockout (apoE / ) female mice were mated with normocholesterolemic (NC) wild-type (apoE/) males and vice versa. All parents were almost identical genetically except for apoE. Therefore, all progeny became genetically identical and heterozygous apoE / . They were born of either HC (i.e. apoE / ) or NC (i.e. apoE / ) mothers. The progeny were killed 6 months after birth and the amount of atherosclerosis in the aortic root was assessed. Females developed more atherosclerosis than males (P B/0.001) but, regardless of sex, maternal hypercholesterolemia during pregnancy had no influence on the amount of atherosclerosis in adult progeny. Males of HC mothers had lower plasma cholesterol levels than males of NC mothers. Thus, in mice, maternal hypercholesterolemia during pregnancy does not promote the development of advanced atherosclerosis in their adult progeny. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Hypercholesterolemia; Pregnancy; Mice; Programming
1. Introduction Fatty streaks, usually considered to represent early atherosclerotic lesions, are already present in the fetal life [1,2]. The amount of such early lesions appears to be increased in fetuses of the hypercholesterolemic (HC) mothers [1]. In FELIC (fate of early lesions in children) study, Napoli et al. [3] showed that these fetal lesions seemed to regress toward birth and then to progress again in the first years after birth. During childhood, the amount of fatty streaks increased with age, particularly in children born of HC mothers. That is, children born of HC mothers had an increased amount of fatty streaks, regardless of their own plasma cholesterol levels.
* Corresponding author. Tel.: /45-8949-6230; fax: /45-8949-6009. E-mail address: [email protected] (C. Madsen).
The fatty streak precedes the advanced atherosclerotic plaque [4 /6]. However, many of the fatty streaks regress and never develop into advanced atherosclerosis. While the fatty streaks are clinically silent, it is the advanced atherosclerotic plaques that are responsible for the majority of deaths in the Western world (e.g. ischemic heart disease and stroke). The risk factors of atherosclerosis in adults include hypercholesterolemia, hypertension, diabetes, and family history [7]. But although the mother’s cholesterol level may promote the development of fatty streaks during fetal life and childhood, it is unknown whether this ‘‘proatherogenic’’ effect influences the development of advanced plaques in adulthood. The homozygous apolipoprotein E-deficient / (apoE ) mouse lacks the gene coding for apoE. This homozygous mouse has high plasma cholesterol levels and develops atherosclerosis spontaneously on a normal chow diet. The heterozygous apolipoprotein E-
0021-9150/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00092-3
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deficient (apoE / ) mouse only becomes HC and develops atherosclerosis if it is fed an atherogenic diet high in cholesterol and containing cholate [8,9]. The plasma cholesterol level and the amount of atherosclerosis have been reported to be higher in females than in males [9]. We mated HC apoE / females with normocholesterolemic (NC) wild-type C57BL (apoE/) males and NC apoE/ females with HC apoE / males. Thus, all of the offspring became heterozygous apoE / and nearly identical on the rest of the genome as well. They were divided into four groups: male/female progeny of HC (apoE / ) mothers and male/female progeny of NC (apoE /) mothers.
2. Methods 2.1. Animals 2.1.1. Parents Eighty mice were obtained from M&B, Ry, Denmark, at 7 weeks of age. Half of the mice (10 males and 30 females) were wild-type C57BL/6J (apoE /), and the other half (10 males and 30 females) were apoE / mice (B6.129P2-ApoeBtm1Unc) [10] backcrossed nine generations into C57BL/6J (i.e. nearly identical genetically to the wild-type C57BL/6J except to the locus expressing apoE). The apoE / females were mated with the apoE/ males and apoE / females with apoE / males. The parents had free access to water and normal chow (Altromin 1324). 2.1.2. Pups One hundred and ten pups (apoE / ) were included in the present atherosclerosis study. They were weaned after 3 weeks and then fed normal chow (Altromin 1324) for another 3 weeks. When the offspring were 6 weeks old, the feed was changed to an atherogenic diet containing 15.8% fat, 1.25% cholesterol, and 0.5% sodium cholate (all w/w) obtained from Teklad Premier (TD 88051, Madison, WI). At all time there was free access to feed and water. The mice were weighed and killed when they were 6 months of age. The mice were housed up to five per cage in a temperature-controlled (21 8C) room with a 12-h light/ dark cycle. The Danish Experimental Animal Inspectorate approved the experimental protocol. 2.2. Plasma lipids Blood samples were obtained from mothers (pregnant and non-pregnant) and offspring. The blood was collected in heparin-coated tubes (Microvette CB 300 LH; 15 IU heparin/ml blood) by puncture of the
retroorbital venous plexus. From all the mice (mothers and progeny) the analyses included total cholesterol, HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), and triglycerides. Plasma lipids were measured on a Konelab 30i (Konelab Corporation, Espoo, Finland) with reagents from Konelab Corporation for total cholesterol and triglycerides, and from ABX Diagnostics (Triolab A/S, Copenhagen, Denmark) for direct measurements of HDL-C and LDL-C. 2.3. Quantification of atherosclerosis The offspring were anesthetized with 0.7 /0.9 ml Mebumal (5 mg/ml) injected intraperitoneally and exsanguinated by aspirating the maximum amount of blood from the right ventricle. Then, the mice were flushed via the left ventricle with a cardioplegic solution (cardioplex) for 1 min and perfusion fixed (phosphatebuffered 4% formaldehyde, pH 7.2) at 100 mmHg for 6 min and immersed in the fixative for 6 /12 h. Finally, the formaline was changed to phosphate-buffered saline. The heart and the ascending aorta were removed, and the heart was cut transversely into two halves of which the upper part was embedded in paraffin. The aortic root was cross-sectioned serially at 4-mm intervals modified after Paigen et al. [11]. From the proximal part of the aortic sinuses every ninth section (36 mm apart) was stained with orcein. A zero point was defined at the commissure level of the aortic leaflets. Six crosssections (the zero point, three below (if possible), and two above) were chosen and used for the assessment of the amount of atherosclerosis. These sections were transferred from microscope (Olympus BX50) to computer images using a digital camera (Olympus Camedia C-3030 Zoom) and plaque area was measured using Olympus DP-Soft. 2.4. Assessments of plaque and correction for oblique sectioning To reduce the risk of missing a real difference between groups, different methods were used to assess the amount of atherosclerosis in the aortic root. The plaque area of all three aortic sinuses together (i.e. the total area) was measured. The section containing the largest total plaque area was identified (Plaquemax) and the mean of the six sections was calculated (Plaquemean). Because absolute area measurements performed on cross-sections that are not cut perpendicular to the ascending aorta (oblique sectioning) may overestimate the size of a plaque in the aortic root, it has been suggested that the measured plaque area should be ‘‘corrected’’ by dividing it with the area of the artery accommodating the plaque [12]. Therefore, we also calculated a corrected maximum value (Plaquemax(c)) by dividing Plaquemax with the length of the internal
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elastic membrane (perimeter) raised to the second power. Of the three aortic sinuses, the one from which the left coronary artery originates (left coronary sinus (LCS)) contains the largest amount of plaque [13], and this feature was used to assess atherosclerosis in a welldefined and particularly atherosclerosis-prone area of aorta. For LCS lesions, the largest area was identified (LCS lesionmax) and the mean was calculated (LCS lesionmean) as described above for the total plaque, but to ensure consistency and avoid errors caused by tangential sectioning of the base of the sinus, only sections in which the ascending aorta contained a complete internal elastic membrane were used for the determination of LCS lesionmax. Finally, cases in which all three commissures were present within three consecutive sections were judged to represent ‘‘perfectly’’ (versus obliquely) cross-sectioned aortic roots (n /53) and were analyzed separately as described above. All decisions and measurements were performed blindly by one observer (C.M.).
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difference in the body weight between adult progeny of apoE / and apoE/ mice (Table 2). 3.2. Atherosclerosis in the adult offspring
The distributions of plasma lipid concentrations and plaque areas were skewed but the log-transformed lipid concentrations and the square roots of the plaque areas were normally distributed. Therefore, statistical analyses were performed on the transformed values. The results are given as medians (2.5 /97.5 percentiles). Unpaired and paired t -tests were used to compare (the square root of) plaque areas, (the logarithm of) lipid concentrations, and weight differences. Adjustments for multiple comparisons were not performed.
Examples of aortic cross-sections from adult progeny of both sexes are given in Fig. 1. The adult male progeny had significantly smaller plaques than the adult female progeny (P B/0.001), but plaque size varied with more than a factor 8 in all four groups of progeny (Fig. 2). All females developed mature atherosclerotic plaques, which contained both collagen- and lipid-rich components usually with cholesterol crystals. The plaque composition of the males differed a lot: some of the mice only contained a few intimal foam cells, others had a little amount of mature plaque, and finally some displayed advanced lesions containing abundant extracellular matrix and lipid. The amount of atherosclerosis was assessed in five different ways (largest plaque area (corrected and uncorrected), mean of six sections, and the largest and mean lesion area of LCS). Comparing progeny of similar sexes but born of mothers with different plasma cholesterol levels (apoE / versus apoE / mothers) revealed no significant difference in the amount of atherosclerosis, regardless of assessment method (Fig. 2 and Table 3). Finally, some sections were classified as ‘‘perfect’’ (opposite to oblique) and the five different ways of assessment were repeated on these ‘‘perfect’’ sections only. It gave the same result (data not shown). Plasma cholesterol levels (total cholesterol, HDL-C, and LDL-C) and triglycerides did not correlate with atherosclerosis (data not shown).
3. Results
4. Discussion
In the progeny group, 110 mice were included (55 of each sex). Six months later 90 (44 females and 46 males) could be used for further investigations and 20 were excluded because of death, failure to thrive (cholate is known to be hepatotoxic), aorta aneurisms, etc.
In this study, adult progeny of HC mothers did not develop more atherosclerosis than those of NC mothers. The two groups of progeny were almost identical genetically and grew up in the same environment. Thus, the only difference between the two groups was the cholesterol levels of their mothers (and fathers). In FELIC study, it was shown that children of HC mothers developed significantly more fatty streaks than those of NC mothers [3]. In an experimental model in LDL receptor-deficient mice, the authors have recently reproduced this result in male, but not in female, offspring; early atherogenesis in 3-month-old male progeny was promoted by maternal hypercholesterolemia [14]. We have evaluated the next step in atherogenesis and could not extend these observations to the development of advanced atherosclerosis later in life: maternal hypercholesterolemia did not influence the development of advanced plaque in the adult progeny.
2.5. Statistical analyses
3.1. Plasma lipids and body weights Pregnant apoE / mice had a markedly more atherogenic lipid profile than pregnant apoE / mice (higher total cholesterol, LDL-C and triglycerides, and lower HDL-C; Table 1). Among offspring, maternal hypercholesterolemia did not influence the lipid values in adult female progeny. However, among adult males, progeny of HC apoE / mothers had significantly lower non-HDL-C levels compared with progeny of NC apoE / mothers (Table 2). In both sexes, there was no significant
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224 Table 1 Plasma lipids of pregnant mice Mouse
n
TC (mmol/l)
HDL-C (mmol/l)
LDL-C (mmol/l)
TG (mmol/l)
ApoE / ApoE/
28 29
10.1 (7.0 /14.4)a 1.8 (1.2 /2.8)
0.9 (0.7 /1.2)b 1.0 (0.6 /1.8)
1.9 (1.0 /3.5)a 0.1 (0.0 /0.4)
1.9 (1.3 /2.6)a 1.5 (0.8 /2.7)
TC, total cholesterol; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; and TG, triglycerides. Values are median (2.5 /97.5 percentile). Most of the cholesterol is carried in the remnant particles (can be calculated from the tabulated measured values). a P 5/0.001 compared with apoE/ . b P B/0.05 compared with apoE/ .
A few other experimental animal studies have examined the connection between maternal hypercholesterolemia during pregnancy and early atherosclerotic lesions of the offspring with conflicting results. In one study, diet-induced maternal hypercholesterolemia promoted fatty streak formation in New Zealand white rabbits [15]. In another, hypercholesterolemia in pregnant sows protected the offspring from aortic and coronary early lesions [16]. However, the swine were not inbred and only four sows were used as dams. Contrary to the present investigation, in which advanced atherosclerosis was used as endpoint, all the studies mentioned above measured early atherosclerotic lesions only. Our observations indicate that maternal hypercholesterolemia may influence the plasma lipid levels of adult offspring in mice; the non-HDL-C levels were lower in male progeny of HC (versus NC) mothers (Table 2). Although a similar change was seen in another group of chow-fed apoE/ progeny of both sexes (n /31; data not shown), it needs to be confirmed because of the inconsistent sex relation and the unexpected direction of the change. Furthermore, contrasting results have recently been reported in LDL receptor-deficient mice in which maternal hypercholesterolemia had no influence on plasma lipid levels of the offspring [14]. 4.1. Fatty streak versus advanced atherosclerosis It has previously been shown that the amount of early lesions in offspring was dependent on their mothers’
cholesterol level during pregnancy [3,14,15]. We used advanced atherosclerosis as the endpoint but did not find any difference between adult offspring of HC and NC mothers (Fig. 2). It is well established that the fatty streak is the precursor of advanced atherosclerosis [4], but the relation is not straightforward [5,6]. Fatty streaks and advanced atherosclerosis have some risk factors in common in adolescence and adulthood (e.g. cholesterol level) [17,18], but the influence of plasma cholesterol on fatty streaks in childhood is uncertain [19]. Alternative pathways than from the fatty streak to advanced atherosclerosis have also been proposed [20]. Fatty streaks are found in the aorta of all children regardless of ethnic background, but the extent of fatty streaks in childhood is not related to the amount of advanced atherosclerosis in adulthood [21]. There may be a relationship between fatty streaks and advanced lesions in the coronary arteries [21], but it also seems that the number of individuals with early lesions in the coronary artery declines from the first to the third year of age [6]. Additionally, in young people there are more fatty streaks in the thoracic aorta than in the abdominal part, and black persons and females have more early lesions than whites and males. In contrast, later in life there are less advanced atherosclerosis in the thoracic aorta compared with the abdominal part, and whites and males have more advanced lesions than blacks and females [21]. It seems that by measuring the amount of fatty streaks, at least in the aorta, it is not possible to predict the amount of advanced lesions in later life in a population.
Table 2 Plasma lipids and body weights of adult apoE/ offspring Sex
Progeny of /
n
TC (mmol/l)
HDL-C (mmol/l) a
LDL-C (mmol/l)
TG (mmol/l)
Body weight (g)
a
Female
ApoE mothers ApoE/ mothers
23 13.1 (9.1 /18.9) 21 12.6 (5.8 /27.4)
1.0 (0.1 /6.5) 2.0 (0.1 /5.3)
3.5 (2.1 /5.7) 3.3 (1.2 /9.0)
0.7 (0.4 /1.1) 0.7 (0.3 /1.6)
18.39/1.5 18.29/2.1
Male
ApoE / mothers ApoE/ mothers
22 10.9 (7.7 /15.6)b 24 12.8 (6.5 /25.1)
1.5 (0.5 /4.6) 1.1 (0.2 /6.2)
2.6 (1.6 /4.3)c 3.6 (1.3 /10.0)
0.6 (0.4 /1.1) 0.6 (0.4 /1.1)
26.39/2.3 25.49/3.7
a b c
P 5/0.01 compared with male progeny of apoE / mothers. P B/0.05 compared with male progeny of apoE / mothers. P B/0.01 compared with male progeny of apoE / mothers.
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Fig. 1. Cross-sections of the aortic root from (A) adult female offspring and (B) adult male offspring containing advanced plaque. Females contain more plaque than males. Orcein, staining elastin black.
4.2. Mice versus humans Mice with plasma cholesterol levels similar to or just a little higher than those seen in humans develop atherosclerosis, but it takes more than just a few months to develop advanced human-like lesions [22]. In general, the amount of atherosclerosis correlates with cholesterol levels when mice with a broad range of hypercholesterolemia are analyzed together. However, within the relatively narrow range of a typical experiment like
ours, such correlation is rarely established (for further discussion, see Ref. [23]). A peculiarity of the most frequently used mice models, including the present one, is a reverse relation between sex and atherosclerosis; females develop more atherosclerosis than males [24,25]. Regarding embryology and fetal nutrition, the murine and human yolk sac and placenta differ in some anatomical and functional aspects but there are many developmental similarities [26,27]. In humans, transient hyperlipidemia is common during pregnancy [28 /30],
Fig. 2. Mean plaque size (Plaquemean) in the four groups of adult apoE/ progeny. Median values for daughters of apoE / and apoE / mothers were 152,000 and 156,000 mm2, respectively, and sons of apoE / and apoE / mothers were 20,000 and 23,000 mm2, respectively. No significant difference between progeny of apoE / and apoE / mothers. This is consistent with both sexes. NS, non-significant.
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capable of synthesizing its own cholesterol and does not need the supply from the mother anymore.
Table 3 Atherosclerosis in the aortic root of apoE / mice Sex and assessment
Plaque area (1000 mm2)a in progeny of ApoE / mothers
ApoE/ mothers
P -value
Females Plaquemaxb [23; 21]c Plaquemax(c) [23; 21] Plaquemean [23; 21] LCS lesionmax [22; 21] LCS lesionmean [22; 21]
179 101 152 106 85
(42 /413) (34 /203) (33 /357) (21 /26) (17 /204)
179 101 156 92 79
(24 /480) (26 /225) (13 /457) (10 /254) (8 /224)
1.00 0.98 0.90 0.44 0.69
Males Plaquemax [22; 24] Plaquemax(c) [22; 24] Plaquemean [22; 24] LCS lesionmax [21; 24] LCS lesionmean [21; 24]
35 20 20 10 8
(0 /125) (0 /70) (0 /85) (1 /56) (1 /45)
35 21 23 13 12
(0 /136) (0 /81) (0 /97) (1 /67) (0 /57)
0.97 0.87 0.70 0.62 0.28
Values for plaque area are medians (2.5 /97.5 percentile). a Units of measurements are 1000 mm2 except Plaquemax(c), which are without unit. b Plaquemax: section containing largest total plaque area (i.e. from all three sinuses). Plaquemax(c): Plaquemax corrected with the perimeter of the internal elastic membrane raised to second power. Plaquemean: mean of the total plaque area. LCS lesionmax: section with the largest lesion area in LCS. LCS lesionmean: mean of the lesion area from LCS. c [n ; n ], number of progeny of [apoE / mothers; apoE/ mothers].
but in this study the cholesterol level decreased in pregnant mice (data not shown) as previously reported for other animals [31].
4.4. Programming One well-established theory of how the fetus is influenced by the mother is the programming or imprinting theory [39,40]. In this it is stated that an insult or stimulus in a critical period or ‘‘time window’’ (e.g. in the fetal life or perinatal period) can have a lasting, lifelong effect. The way of exerting the effect is, for example, changes in cell numbers, organ structures, metabolism, and/or gene expression. One of the bestinvestigated parameters is birth weight. It has been shown in several epidemiological studies that low birth weight (caused by undernutrition in fetal life) is associated with an increased risk of developing coronary heart disease (reviewed in Refs. [39,40]), and experimental animal studies have shown that it is indeed possible to ‘‘program’’ one of the main risk factors for this disease, the plasma cholesterol level [39,40]. The results of these animal studies have, however, been ambiguous. The programming hypothesis implies that intrauterine undernutrition leads to ‘‘thrifty’’ adaptations during fetal development [41]. Therefore, if the programming hypothesis is correct, one would expect that oversupply of cholesterol (versus undernutrition) during fetal life would reduce (rather than increase) the risk of developing atherosclerosis in later life. We found no evidence for such an effect on the development of advanced lesions in apoE-deficient mice and the opposite has recently been reported for early lesion formation in LDL receptor-deficient male mice [14].
4.3. Lipoproteins crossing the placenta or yolk sac? 4.5. Limitations Various (human and animal) studies have shown that most of the fetus’ cholesterol is synthesized by the fetus itself [28,32/35]. However, at least in the references mentioned, the collection of data was done in the last half [34] or at the end of the pregnancy [28,32,33,35]. Maybe the circumstances are different in the beginning, recalling that the plasma cholesterol levels of the human fetuses and mothers are correlated in the first 6 months [1]. The placenta and yolk sac do take up maternal lipoproteins by receptor-mediated as well as receptorindependent processes [32,36], but the destiny of the lipoproteins is not completely understood [27,37]. However, release of lipids from the lipoproteins to the endodermal cells of the yolk sac, repackaging the lipids into new lipoproteins, and shipping them into the fetal circulation have been proposed [38]. Although the fetus synthesizes most of its own cholesterol, this transport may not be negligible [37]. Maybe the offspring is dependent on the cholesterol delivered from the mother in the beginning of pregnancy, but later on the fetus is
In mice, atherosclerosis develops readily in the aortic root, and therefore the assessment is often done here. However, the aortic root is not a predilection site for plaque development in humans and it is uncertain whether the results from the murine aorta can be extrapolated to, for example, the human coronary arteries. Heterozygous apoE/ mice only develop advanced atherosclerosis when fed a cholate-containing (proinflammatory) atherogenic diet and it may also have influenced the result. It should also be noticed that mice and humans differ in lipid metabolism [42] and, to a certain extent, in fetal development and nutrition.
5. Conclusion Heterozygous apoE-deficient mice developed hypercholesterolemia when fed an atherogenic diet, and advanced atherosclerosis was present at 6 months of
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age, most pronounced in females. Maternal hypercholesterolemia during pregnancy did not influence the development of advanced atherosclerosis in the adult offspring, regardless of sex.
Acknowledgements The Danish Medical Research Council, the Danish Heart Foundation, Lægeforeningens Forskningsfond, and Elin Holms Forskningspulje supported this work. We also thank Birgitte Sahl and the staff at the Animal Research Department for all their technical assistance. Thanks to Marianne Lyngbak (Department of Clinical Biochemistry, Aarhus University Hospital (Skejby)) for analysis of the lipids and to Kaspar Lund for helpful comments.
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[37] Woollett LA. Fetal lipid metabolism. Front Biosci 2001;6:D536 / 45. [38] Farese RV, Cases S, Ruland SL, et al. A novel function for apolipoprotein B: lipoprotein synthesis in the yolk sac is critical for maternal /fetal lipid transport in mice. J Lipid Res 1996;37:347 /60. [39] Barker DJ. Mothers, Babies and Health in Later Life. New York: Churchill Livingstone, 1998. [40] Waterland RA, Garza C. Potential mechanisms of metabolic imprinting that lead to chronic disease. Am J Clin Nutr 1999;69:179 /97 (see comments). [41] Petry CJ, Ozanne SE, Hales CN. Programming of intermediary metabolism. Mol Cell Endocrinol 2001;185:81 /91. [42] Breslow JL, Plump AS, Dammerman M. New mouse models of lipoprotein disorders and atherosclerosis. In: Fuster V, Ross R, Topol EJ, editors. Atherosclerosis and Coronary Disease. New York: Lippincott/Raven, 1996:363 /78.
Hypercholesterolemia in pregnant mice does not affect atherosclerosis in adult offspring Claus Madsen a,*, Frederik Dagnæs-Hansen b, Jan Møller c, Erling Falk a a
Department of Cardiology, Institute of Experimental Clinical Research, Aarhus University Hospital, DK-8200 Aarhus, Denmark b Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark c Department of Clinical Biochemistry, Aarhus University Hospital, DK-8200 Aarhus, Denmark Received 13 September 2002; received in revised form 7 February 2003; accepted 24 February 2003
Abstract In humans, maternal hypercholesterolemia during pregnancy promotes microscopical fatty streaks in the children. The mechanism is unknown. Fatty streaks are clinically silent, and many of them regress and never develop into advanced atherosclerosis. The aim of this study was to investigate whether hypercholesterolemia in pregnant mice induced more advanced atherosclerosis in their adult progeny. Hypercholesterolemic (HC) apolipoprotein E knockout (apoE / ) female mice were mated with normocholesterolemic (NC) wild-type (apoE/) males and vice versa. All parents were almost identical genetically except for apoE. Therefore, all progeny became genetically identical and heterozygous apoE / . They were born of either HC (i.e. apoE / ) or NC (i.e. apoE / ) mothers. The progeny were killed 6 months after birth and the amount of atherosclerosis in the aortic root was assessed. Females developed more atherosclerosis than males (P B/0.001) but, regardless of sex, maternal hypercholesterolemia during pregnancy had no influence on the amount of atherosclerosis in adult progeny. Males of HC mothers had lower plasma cholesterol levels than males of NC mothers. Thus, in mice, maternal hypercholesterolemia during pregnancy does not promote the development of advanced atherosclerosis in their adult progeny. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Hypercholesterolemia; Pregnancy; Mice; Programming
1. Introduction Fatty streaks, usually considered to represent early atherosclerotic lesions, are already present in the fetal life [1,2]. The amount of such early lesions appears to be increased in fetuses of the hypercholesterolemic (HC) mothers [1]. In FELIC (fate of early lesions in children) study, Napoli et al. [3] showed that these fetal lesions seemed to regress toward birth and then to progress again in the first years after birth. During childhood, the amount of fatty streaks increased with age, particularly in children born of HC mothers. That is, children born of HC mothers had an increased amount of fatty streaks, regardless of their own plasma cholesterol levels.
* Corresponding author. Tel.: /45-8949-6230; fax: /45-8949-6009. E-mail address: [email protected] (C. Madsen).
The fatty streak precedes the advanced atherosclerotic plaque [4 /6]. However, many of the fatty streaks regress and never develop into advanced atherosclerosis. While the fatty streaks are clinically silent, it is the advanced atherosclerotic plaques that are responsible for the majority of deaths in the Western world (e.g. ischemic heart disease and stroke). The risk factors of atherosclerosis in adults include hypercholesterolemia, hypertension, diabetes, and family history [7]. But although the mother’s cholesterol level may promote the development of fatty streaks during fetal life and childhood, it is unknown whether this ‘‘proatherogenic’’ effect influences the development of advanced plaques in adulthood. The homozygous apolipoprotein E-deficient / (apoE ) mouse lacks the gene coding for apoE. This homozygous mouse has high plasma cholesterol levels and develops atherosclerosis spontaneously on a normal chow diet. The heterozygous apolipoprotein E-
0021-9150/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0021-9150(03)00092-3
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deficient (apoE / ) mouse only becomes HC and develops atherosclerosis if it is fed an atherogenic diet high in cholesterol and containing cholate [8,9]. The plasma cholesterol level and the amount of atherosclerosis have been reported to be higher in females than in males [9]. We mated HC apoE / females with normocholesterolemic (NC) wild-type C57BL (apoE/) males and NC apoE/ females with HC apoE / males. Thus, all of the offspring became heterozygous apoE / and nearly identical on the rest of the genome as well. They were divided into four groups: male/female progeny of HC (apoE / ) mothers and male/female progeny of NC (apoE /) mothers.
2. Methods 2.1. Animals 2.1.1. Parents Eighty mice were obtained from M&B, Ry, Denmark, at 7 weeks of age. Half of the mice (10 males and 30 females) were wild-type C57BL/6J (apoE /), and the other half (10 males and 30 females) were apoE / mice (B6.129P2-ApoeBtm1Unc) [10] backcrossed nine generations into C57BL/6J (i.e. nearly identical genetically to the wild-type C57BL/6J except to the locus expressing apoE). The apoE / females were mated with the apoE/ males and apoE / females with apoE / males. The parents had free access to water and normal chow (Altromin 1324). 2.1.2. Pups One hundred and ten pups (apoE / ) were included in the present atherosclerosis study. They were weaned after 3 weeks and then fed normal chow (Altromin 1324) for another 3 weeks. When the offspring were 6 weeks old, the feed was changed to an atherogenic diet containing 15.8% fat, 1.25% cholesterol, and 0.5% sodium cholate (all w/w) obtained from Teklad Premier (TD 88051, Madison, WI). At all time there was free access to feed and water. The mice were weighed and killed when they were 6 months of age. The mice were housed up to five per cage in a temperature-controlled (21 8C) room with a 12-h light/ dark cycle. The Danish Experimental Animal Inspectorate approved the experimental protocol. 2.2. Plasma lipids Blood samples were obtained from mothers (pregnant and non-pregnant) and offspring. The blood was collected in heparin-coated tubes (Microvette CB 300 LH; 15 IU heparin/ml blood) by puncture of the
retroorbital venous plexus. From all the mice (mothers and progeny) the analyses included total cholesterol, HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), and triglycerides. Plasma lipids were measured on a Konelab 30i (Konelab Corporation, Espoo, Finland) with reagents from Konelab Corporation for total cholesterol and triglycerides, and from ABX Diagnostics (Triolab A/S, Copenhagen, Denmark) for direct measurements of HDL-C and LDL-C. 2.3. Quantification of atherosclerosis The offspring were anesthetized with 0.7 /0.9 ml Mebumal (5 mg/ml) injected intraperitoneally and exsanguinated by aspirating the maximum amount of blood from the right ventricle. Then, the mice were flushed via the left ventricle with a cardioplegic solution (cardioplex) for 1 min and perfusion fixed (phosphatebuffered 4% formaldehyde, pH 7.2) at 100 mmHg for 6 min and immersed in the fixative for 6 /12 h. Finally, the formaline was changed to phosphate-buffered saline. The heart and the ascending aorta were removed, and the heart was cut transversely into two halves of which the upper part was embedded in paraffin. The aortic root was cross-sectioned serially at 4-mm intervals modified after Paigen et al. [11]. From the proximal part of the aortic sinuses every ninth section (36 mm apart) was stained with orcein. A zero point was defined at the commissure level of the aortic leaflets. Six crosssections (the zero point, three below (if possible), and two above) were chosen and used for the assessment of the amount of atherosclerosis. These sections were transferred from microscope (Olympus BX50) to computer images using a digital camera (Olympus Camedia C-3030 Zoom) and plaque area was measured using Olympus DP-Soft. 2.4. Assessments of plaque and correction for oblique sectioning To reduce the risk of missing a real difference between groups, different methods were used to assess the amount of atherosclerosis in the aortic root. The plaque area of all three aortic sinuses together (i.e. the total area) was measured. The section containing the largest total plaque area was identified (Plaquemax) and the mean of the six sections was calculated (Plaquemean). Because absolute area measurements performed on cross-sections that are not cut perpendicular to the ascending aorta (oblique sectioning) may overestimate the size of a plaque in the aortic root, it has been suggested that the measured plaque area should be ‘‘corrected’’ by dividing it with the area of the artery accommodating the plaque [12]. Therefore, we also calculated a corrected maximum value (Plaquemax(c)) by dividing Plaquemax with the length of the internal
C. Madsen et al. / Atherosclerosis 168 (2003) 221 /228
elastic membrane (perimeter) raised to the second power. Of the three aortic sinuses, the one from which the left coronary artery originates (left coronary sinus (LCS)) contains the largest amount of plaque [13], and this feature was used to assess atherosclerosis in a welldefined and particularly atherosclerosis-prone area of aorta. For LCS lesions, the largest area was identified (LCS lesionmax) and the mean was calculated (LCS lesionmean) as described above for the total plaque, but to ensure consistency and avoid errors caused by tangential sectioning of the base of the sinus, only sections in which the ascending aorta contained a complete internal elastic membrane were used for the determination of LCS lesionmax. Finally, cases in which all three commissures were present within three consecutive sections were judged to represent ‘‘perfectly’’ (versus obliquely) cross-sectioned aortic roots (n /53) and were analyzed separately as described above. All decisions and measurements were performed blindly by one observer (C.M.).
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difference in the body weight between adult progeny of apoE / and apoE/ mice (Table 2). 3.2. Atherosclerosis in the adult offspring
The distributions of plasma lipid concentrations and plaque areas were skewed but the log-transformed lipid concentrations and the square roots of the plaque areas were normally distributed. Therefore, statistical analyses were performed on the transformed values. The results are given as medians (2.5 /97.5 percentiles). Unpaired and paired t -tests were used to compare (the square root of) plaque areas, (the logarithm of) lipid concentrations, and weight differences. Adjustments for multiple comparisons were not performed.
Examples of aortic cross-sections from adult progeny of both sexes are given in Fig. 1. The adult male progeny had significantly smaller plaques than the adult female progeny (P B/0.001), but plaque size varied with more than a factor 8 in all four groups of progeny (Fig. 2). All females developed mature atherosclerotic plaques, which contained both collagen- and lipid-rich components usually with cholesterol crystals. The plaque composition of the males differed a lot: some of the mice only contained a few intimal foam cells, others had a little amount of mature plaque, and finally some displayed advanced lesions containing abundant extracellular matrix and lipid. The amount of atherosclerosis was assessed in five different ways (largest plaque area (corrected and uncorrected), mean of six sections, and the largest and mean lesion area of LCS). Comparing progeny of similar sexes but born of mothers with different plasma cholesterol levels (apoE / versus apoE / mothers) revealed no significant difference in the amount of atherosclerosis, regardless of assessment method (Fig. 2 and Table 3). Finally, some sections were classified as ‘‘perfect’’ (opposite to oblique) and the five different ways of assessment were repeated on these ‘‘perfect’’ sections only. It gave the same result (data not shown). Plasma cholesterol levels (total cholesterol, HDL-C, and LDL-C) and triglycerides did not correlate with atherosclerosis (data not shown).
3. Results
4. Discussion
In the progeny group, 110 mice were included (55 of each sex). Six months later 90 (44 females and 46 males) could be used for further investigations and 20 were excluded because of death, failure to thrive (cholate is known to be hepatotoxic), aorta aneurisms, etc.
In this study, adult progeny of HC mothers did not develop more atherosclerosis than those of NC mothers. The two groups of progeny were almost identical genetically and grew up in the same environment. Thus, the only difference between the two groups was the cholesterol levels of their mothers (and fathers). In FELIC study, it was shown that children of HC mothers developed significantly more fatty streaks than those of NC mothers [3]. In an experimental model in LDL receptor-deficient mice, the authors have recently reproduced this result in male, but not in female, offspring; early atherogenesis in 3-month-old male progeny was promoted by maternal hypercholesterolemia [14]. We have evaluated the next step in atherogenesis and could not extend these observations to the development of advanced atherosclerosis later in life: maternal hypercholesterolemia did not influence the development of advanced plaque in the adult progeny.
2.5. Statistical analyses
3.1. Plasma lipids and body weights Pregnant apoE / mice had a markedly more atherogenic lipid profile than pregnant apoE / mice (higher total cholesterol, LDL-C and triglycerides, and lower HDL-C; Table 1). Among offspring, maternal hypercholesterolemia did not influence the lipid values in adult female progeny. However, among adult males, progeny of HC apoE / mothers had significantly lower non-HDL-C levels compared with progeny of NC apoE / mothers (Table 2). In both sexes, there was no significant
C. Madsen et al. / Atherosclerosis 168 (2003) 221 /228
224 Table 1 Plasma lipids of pregnant mice Mouse
n
TC (mmol/l)
HDL-C (mmol/l)
LDL-C (mmol/l)
TG (mmol/l)
ApoE / ApoE/
28 29
10.1 (7.0 /14.4)a 1.8 (1.2 /2.8)
0.9 (0.7 /1.2)b 1.0 (0.6 /1.8)
1.9 (1.0 /3.5)a 0.1 (0.0 /0.4)
1.9 (1.3 /2.6)a 1.5 (0.8 /2.7)
TC, total cholesterol; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; and TG, triglycerides. Values are median (2.5 /97.5 percentile). Most of the cholesterol is carried in the remnant particles (can be calculated from the tabulated measured values). a P 5/0.001 compared with apoE/ . b P B/0.05 compared with apoE/ .
A few other experimental animal studies have examined the connection between maternal hypercholesterolemia during pregnancy and early atherosclerotic lesions of the offspring with conflicting results. In one study, diet-induced maternal hypercholesterolemia promoted fatty streak formation in New Zealand white rabbits [15]. In another, hypercholesterolemia in pregnant sows protected the offspring from aortic and coronary early lesions [16]. However, the swine were not inbred and only four sows were used as dams. Contrary to the present investigation, in which advanced atherosclerosis was used as endpoint, all the studies mentioned above measured early atherosclerotic lesions only. Our observations indicate that maternal hypercholesterolemia may influence the plasma lipid levels of adult offspring in mice; the non-HDL-C levels were lower in male progeny of HC (versus NC) mothers (Table 2). Although a similar change was seen in another group of chow-fed apoE/ progeny of both sexes (n /31; data not shown), it needs to be confirmed because of the inconsistent sex relation and the unexpected direction of the change. Furthermore, contrasting results have recently been reported in LDL receptor-deficient mice in which maternal hypercholesterolemia had no influence on plasma lipid levels of the offspring [14]. 4.1. Fatty streak versus advanced atherosclerosis It has previously been shown that the amount of early lesions in offspring was dependent on their mothers’
cholesterol level during pregnancy [3,14,15]. We used advanced atherosclerosis as the endpoint but did not find any difference between adult offspring of HC and NC mothers (Fig. 2). It is well established that the fatty streak is the precursor of advanced atherosclerosis [4], but the relation is not straightforward [5,6]. Fatty streaks and advanced atherosclerosis have some risk factors in common in adolescence and adulthood (e.g. cholesterol level) [17,18], but the influence of plasma cholesterol on fatty streaks in childhood is uncertain [19]. Alternative pathways than from the fatty streak to advanced atherosclerosis have also been proposed [20]. Fatty streaks are found in the aorta of all children regardless of ethnic background, but the extent of fatty streaks in childhood is not related to the amount of advanced atherosclerosis in adulthood [21]. There may be a relationship between fatty streaks and advanced lesions in the coronary arteries [21], but it also seems that the number of individuals with early lesions in the coronary artery declines from the first to the third year of age [6]. Additionally, in young people there are more fatty streaks in the thoracic aorta than in the abdominal part, and black persons and females have more early lesions than whites and males. In contrast, later in life there are less advanced atherosclerosis in the thoracic aorta compared with the abdominal part, and whites and males have more advanced lesions than blacks and females [21]. It seems that by measuring the amount of fatty streaks, at least in the aorta, it is not possible to predict the amount of advanced lesions in later life in a population.
Table 2 Plasma lipids and body weights of adult apoE/ offspring Sex
Progeny of /
n
TC (mmol/l)
HDL-C (mmol/l) a
LDL-C (mmol/l)
TG (mmol/l)
Body weight (g)
a
Female
ApoE mothers ApoE/ mothers
23 13.1 (9.1 /18.9) 21 12.6 (5.8 /27.4)
1.0 (0.1 /6.5) 2.0 (0.1 /5.3)
3.5 (2.1 /5.7) 3.3 (1.2 /9.0)
0.7 (0.4 /1.1) 0.7 (0.3 /1.6)
18.39/1.5 18.29/2.1
Male
ApoE / mothers ApoE/ mothers
22 10.9 (7.7 /15.6)b 24 12.8 (6.5 /25.1)
1.5 (0.5 /4.6) 1.1 (0.2 /6.2)
2.6 (1.6 /4.3)c 3.6 (1.3 /10.0)
0.6 (0.4 /1.1) 0.6 (0.4 /1.1)
26.39/2.3 25.49/3.7
a b c
P 5/0.01 compared with male progeny of apoE / mothers. P B/0.05 compared with male progeny of apoE / mothers. P B/0.01 compared with male progeny of apoE / mothers.
C. Madsen et al. / Atherosclerosis 168 (2003) 221 /228
225
Fig. 1. Cross-sections of the aortic root from (A) adult female offspring and (B) adult male offspring containing advanced plaque. Females contain more plaque than males. Orcein, staining elastin black.
4.2. Mice versus humans Mice with plasma cholesterol levels similar to or just a little higher than those seen in humans develop atherosclerosis, but it takes more than just a few months to develop advanced human-like lesions [22]. In general, the amount of atherosclerosis correlates with cholesterol levels when mice with a broad range of hypercholesterolemia are analyzed together. However, within the relatively narrow range of a typical experiment like
ours, such correlation is rarely established (for further discussion, see Ref. [23]). A peculiarity of the most frequently used mice models, including the present one, is a reverse relation between sex and atherosclerosis; females develop more atherosclerosis than males [24,25]. Regarding embryology and fetal nutrition, the murine and human yolk sac and placenta differ in some anatomical and functional aspects but there are many developmental similarities [26,27]. In humans, transient hyperlipidemia is common during pregnancy [28 /30],
Fig. 2. Mean plaque size (Plaquemean) in the four groups of adult apoE/ progeny. Median values for daughters of apoE / and apoE / mothers were 152,000 and 156,000 mm2, respectively, and sons of apoE / and apoE / mothers were 20,000 and 23,000 mm2, respectively. No significant difference between progeny of apoE / and apoE / mothers. This is consistent with both sexes. NS, non-significant.
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226
capable of synthesizing its own cholesterol and does not need the supply from the mother anymore.
Table 3 Atherosclerosis in the aortic root of apoE / mice Sex and assessment
Plaque area (1000 mm2)a in progeny of ApoE / mothers
ApoE/ mothers
P -value
Females Plaquemaxb [23; 21]c Plaquemax(c) [23; 21] Plaquemean [23; 21] LCS lesionmax [22; 21] LCS lesionmean [22; 21]
179 101 152 106 85
(42 /413) (34 /203) (33 /357) (21 /26) (17 /204)
179 101 156 92 79
(24 /480) (26 /225) (13 /457) (10 /254) (8 /224)
1.00 0.98 0.90 0.44 0.69
Males Plaquemax [22; 24] Plaquemax(c) [22; 24] Plaquemean [22; 24] LCS lesionmax [21; 24] LCS lesionmean [21; 24]
35 20 20 10 8
(0 /125) (0 /70) (0 /85) (1 /56) (1 /45)
35 21 23 13 12
(0 /136) (0 /81) (0 /97) (1 /67) (0 /57)
0.97 0.87 0.70 0.62 0.28
Values for plaque area are medians (2.5 /97.5 percentile). a Units of measurements are 1000 mm2 except Plaquemax(c), which are without unit. b Plaquemax: section containing largest total plaque area (i.e. from all three sinuses). Plaquemax(c): Plaquemax corrected with the perimeter of the internal elastic membrane raised to second power. Plaquemean: mean of the total plaque area. LCS lesionmax: section with the largest lesion area in LCS. LCS lesionmean: mean of the lesion area from LCS. c [n ; n ], number of progeny of [apoE / mothers; apoE/ mothers].
but in this study the cholesterol level decreased in pregnant mice (data not shown) as previously reported for other animals [31].
4.4. Programming One well-established theory of how the fetus is influenced by the mother is the programming or imprinting theory [39,40]. In this it is stated that an insult or stimulus in a critical period or ‘‘time window’’ (e.g. in the fetal life or perinatal period) can have a lasting, lifelong effect. The way of exerting the effect is, for example, changes in cell numbers, organ structures, metabolism, and/or gene expression. One of the bestinvestigated parameters is birth weight. It has been shown in several epidemiological studies that low birth weight (caused by undernutrition in fetal life) is associated with an increased risk of developing coronary heart disease (reviewed in Refs. [39,40]), and experimental animal studies have shown that it is indeed possible to ‘‘program’’ one of the main risk factors for this disease, the plasma cholesterol level [39,40]. The results of these animal studies have, however, been ambiguous. The programming hypothesis implies that intrauterine undernutrition leads to ‘‘thrifty’’ adaptations during fetal development [41]. Therefore, if the programming hypothesis is correct, one would expect that oversupply of cholesterol (versus undernutrition) during fetal life would reduce (rather than increase) the risk of developing atherosclerosis in later life. We found no evidence for such an effect on the development of advanced lesions in apoE-deficient mice and the opposite has recently been reported for early lesion formation in LDL receptor-deficient male mice [14].
4.3. Lipoproteins crossing the placenta or yolk sac? 4.5. Limitations Various (human and animal) studies have shown that most of the fetus’ cholesterol is synthesized by the fetus itself [28,32/35]. However, at least in the references mentioned, the collection of data was done in the last half [34] or at the end of the pregnancy [28,32,33,35]. Maybe the circumstances are different in the beginning, recalling that the plasma cholesterol levels of the human fetuses and mothers are correlated in the first 6 months [1]. The placenta and yolk sac do take up maternal lipoproteins by receptor-mediated as well as receptorindependent processes [32,36], but the destiny of the lipoproteins is not completely understood [27,37]. However, release of lipids from the lipoproteins to the endodermal cells of the yolk sac, repackaging the lipids into new lipoproteins, and shipping them into the fetal circulation have been proposed [38]. Although the fetus synthesizes most of its own cholesterol, this transport may not be negligible [37]. Maybe the offspring is dependent on the cholesterol delivered from the mother in the beginning of pregnancy, but later on the fetus is
In mice, atherosclerosis develops readily in the aortic root, and therefore the assessment is often done here. However, the aortic root is not a predilection site for plaque development in humans and it is uncertain whether the results from the murine aorta can be extrapolated to, for example, the human coronary arteries. Heterozygous apoE/ mice only develop advanced atherosclerosis when fed a cholate-containing (proinflammatory) atherogenic diet and it may also have influenced the result. It should also be noticed that mice and humans differ in lipid metabolism [42] and, to a certain extent, in fetal development and nutrition.
5. Conclusion Heterozygous apoE-deficient mice developed hypercholesterolemia when fed an atherogenic diet, and advanced atherosclerosis was present at 6 months of
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age, most pronounced in females. Maternal hypercholesterolemia during pregnancy did not influence the development of advanced atherosclerosis in the adult offspring, regardless of sex.
Acknowledgements The Danish Medical Research Council, the Danish Heart Foundation, Lægeforeningens Forskningsfond, and Elin Holms Forskningspulje supported this work. We also thank Birgitte Sahl and the staff at the Animal Research Department for all their technical assistance. Thanks to Marianne Lyngbak (Department of Clinical Biochemistry, Aarhus University Hospital (Skejby)) for analysis of the lipids and to Kaspar Lund for helpful comments.
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