Apolipoprotein E polymorphism and atherosclerosis: association of
the
o
4 allele with defects in the internal elastic lamina
Erkki Ilveskoski
a,b, Otso Ja¨rvinen
c, Tero Sisto
c, Pekka J. Karhunen
b,
Pekka Laippala
d, Terho Lehtima¨ki
a,b,*
aDepartment of Clinical Chemistry,The Laboratory of Atherosclerosis Genetics,Tampere Uni6ersity Hospital,PO Box2000, FIN-33521Tampere,Finland
bMedical School,Uni6ersity of Tampere,and Tampere Uni6ersity Hospital,FIN-33521,Tampere,Finland cDepartment of Cardiothoracic Surgery,Tampere Uni6ersity Hospital,FIN-33521,Tampere,Finland
dTampere School of Public Health,Uni6ersity of Tampere,and Tampere Uni6ersity Hospital,FIN-33521,Tampere,Finland Received 17 June 1999; received in revised form 13 December 1999; accepted 14 January 2000
Abstract
The defects in the internal elastic lamina (IEL) have been proposed to be important for the migration of smooth muscle cells into the intima during atherosclerosis. We investigated the association of a genetic factor — apolipoprotein E (apoE) genotype — with the number of gaps in the IEL of the artery wall in 123 consecutive autopsy cases (90 male, 33 female) aged 18 – 93. At autopsy, the circumference of the IEL and the number of gaps in the IEL were measured in circular samples of the coeliac; (CA), superior mesenteric (SMA) and inferior mesenteric (IMA) arteries. In the series, the number of gaps per millimetre in the IEL of CA, SMA and IMA were associated with intimal thickening (PB0.0001,P=0.01 and P=0.005, respectively). In men, apoE genotype was significantly associated with the number of gaps in the IEL of the CA and IMA (P=0.033 and P=0.04 1, respectively). The carriers of o4/3 or o4/4 genotype had higher number of gaps in CA than the carriers of o3/3 genotype (2.3092.63 vs 1.3891.83 gaps/mm,P=0.035) and also higher number of gaps in IMA than the carriers ofo3/2 (2.1891.71 vs 0.6690.60 gaps/mm,P=0.041). The results suggest that the apoEo4 allele may be involved with IEL fragmentation in men. This may be mediated through higher serum cholesterol associated with theo4 allele. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Apolipoprotein E; Genetic polymorphism; Internal elastic lamina; Atherosclerosis; Smooth muscle cells
www.elsevier.com/locate/atherosclerosis
1. Introduction
Internal elastic lamina (IEL) is the barrier between intima and media in the arterial wall. It has been proposed by Sims et al. that defects in the IEL play a role in the pathogenesis of intimal thickening and atherosclerosis [1,2]. According to the hypothesis, the defects in the continuity of the IEL and the inability to form a reduplicated elastin membrane would allow smooth muscle cells (SMC) to proliferate into the in-tima. There is no consensus about the question on the origin of the SMC that give rise to the intimal lesion
[3]. Migration of SMC to the intima through gaps in the IEL might provide one explanation and, in fact, an association of structural changes in the IEL with inti-mal thickening and with atherosclerosis has been re-ported in humans and also in experimental animals [1,2,4 – 8].
Apolipoprotein E (apoE) polymorphism is one of the strongest and most studied genetic risk factors for atherosclerosis identified so far in general population [9]. Two point mutations in the apoE gene lead to three common alleles designated as o2, o3 and o4. These alleles result in six genotypes (o2/2,o3/2,o4/2,o3/3,o4/3 and o4/4) [10]. The apoE o4 allele has been associated with high serum total and low density lipoprotein con-centrations [11 – 13] and it is also suggested to be a risk factor for coronary artery disease [14 – 16].
* Corresponding author. Tel.: +358-3-2474066; fax: + 358-3-2475554.
E-mail address:bltele@uta.fi (T. Lehtima¨ki).
There is evidence from the studies on animal models that hypercholesterolemia induces changes in the IEL structure [17,18]. Therefore, it can be suggested that apoE polymorphism plays a role in the IEL fragmenta-tion. In our study of 123 consecutive autopsy cases, we tested for the first time the hypothesis that apoE poly-morphism is associated with defects in the IEL of the arterial wall.
2. Materials and methods
2.1. Subjects
An autopsy series of 123 consecutive medical (n=42) and forensic (n=81) autopsies was collected in the Tampere University Hospital in 1993 to evaluate atherosclerosis in abdominal arteries. Subjects were both men (n=90, 73%) and women (n=33; 27%) aged 18 – 93 years (mean 62.0 years, SD917.7). Medical histories were retrospectively obtained from hospital records and the history of hypertension, diabetes melli-tus, lipid lowering medication and smoking was recorded. The study was approved by the Ethics Com-mittee of the Tampere University Hospital.
2.2. Autopsies and measurements of the histologic changes in mesenteric arteries
The body-mass index (BMI) was calculated by divid-ing the weight (kg) of the cadaver by height (m) squared. The measurements of the atherosclerosis are described in detail in Ja¨rvinen et al. [19]. Briefly, the pathologist who performed the autopsy graded atherosclerosis of the coronary and cerebral arteries, and for the analysis, the findings were classified as: 0, no lesions or only fatty change and 1, more advanced atherosclerotic lesions. Abdominal viscera was removed en bloc and coeliac artery (CA), and superior and inferior mesenteric arteries (SMA, IMA) were investi-gated. From the CA, SMA and IMA, segments with a length of 0.5 cm were cut at the level of 1.0 cm distal to their aortic ostia, and these circular samples were em-bedded in paraffin and stained with Masson’s trichrome for light-microscopic examination. The circumference of the IEL and the thickness of the intima were mea-sured in millimetres (MOP 3 image-analysis system, Reichert-Jung, Eching, Germany). The intimal thick-ness was defined as the measure from the lumen to the IEL at the area of greatest intimal thickness [20]. The intimal thickness was divided into two categories ac-cording to the median (Md) value of the series (in CA and IMA Md=0.1 mm, in SMA Md=0.2 mm, respec-tively): 0=no intimal thickening, 1=intimal thicken-ing. Further, the number of the gaps in the IEL (see Fig. 1) was counted, divided by the circumference of the IEL and expressed as gaps per mm.
2.3. DNA isolation and apoE genotyping
DNA was isolated from the paraffin embedded sam-ples of the mesenteric artery wall by using the method of QIamp Tissue Kit (QIAGEN Inc., USA). ApoE genotypes were determined by PCR and following re-striction enzyme digestion [21,22]. The PCR conditions were denaturation at 95°C, annealing at 62°C and extension at 72°C for 40 cycles followed by final exten-sion at 72°C. The fragments were visualised on 5% MetaPhor (FMC) agarose gels.
2.4. Statistical methods
Data are expressed as mean9SD, unless otherwise specified. Comparisons were done between three geno-type groups:o3/2,o3/3, ando4/3 or o4/4. For statistical analysis, one subject with o2/2 and all three subjects witho4/2 were excluded because of the too low number of subjects in these groups. Comparisons between con-tinuous variables between genotypes were done using one-way analysis of variance (ANOVA), and thex2-test was used to compare the genotype distributions. We used two-way analysis of covariance (ANCOVA) to compare the number of gaps of the IEL in different genotype groups. Data was analysed in square-root form, but the results are displayed as crude. ApoE genotype group and intimal thickness were used as factors in the ANCOVA. Age and BMI were taken as covariates to eliminate their confounding effect. Fi-nally, the differences between three genotype groups were tested using Scheffe´ post-hoc test. Computation was carried out with Statistica for Windows 5.1 (Stat-Soft Inc., Tulsa, Oklahoma) on a PC.
Table 1
Characteristics of the subjects by apoE genotype.
o3/3 (n=70) o4/3 oro4/4 (n=37) P
History of hypertension (yes/no) 11
–/11
History of myocardial infarction (yes/no) –
6/6
Coronary artery atherosclerosis (yes/no) 34/36 22/15 0.56c – 7/5
Cerebral artery atherosclerosis (yes/no) 36/34 16/2 10.59c –
Intimal thickening(yes/no)
31/38 16/19 0.85c 8
aNumber of subjects missing in the analysis.
bOne-way ANOVA was used to compare the variable between genotype. c
x2-test was used to compare the variable between genotype.
3. Results
3.1. ApoE genotype and clinical characteristics
ApoE allele frequencies were o2=0.070, o3=0.756, and o4=0.175. In the whole sample, none of the sub-ject characteristics varied significantly by apoE geno-type (see Table 1). ApoE was not associated with dichotomised score of coronary or cerebral atheroscle-rosis or with intimal thickening of the mesenteric arter-ies in this sample.
3.2. ApoE genotype and the defects of the IEL in mesenteric arteries
The ANCOVA revealed a significant association of the intima thickness with the IEL defects in the CA. The group with intimal thickening was associated with higher number of IEL defects than the group with no intimal thickening (2.5092.47 versus 0.7390.79 gaps/
mm, PB0.0001). ApoE genotype had a significant effect on the number of gaps in the IEL of the CA (P=0.027), the carriers of the o4/3 or o4/4 genotype expressing higher number of defects than carriers of o3/3 genotype (2.0892.46 vs 1.2891.70 gaps/mm, P=0.032 in Scheffe´ post-hoc test) (see Table 2; Fig. 2A). In SMA and IMA, the intimal thickness was also significantly associated with the IEL defects (P=0.01 and P=0.005, respectively). However, in these vessels no association of the apoE genotype with the number of IEL defects was observed (P=0.93 for SMA and P=0.66 for IMA).
We performed a subgroup analysis of the association between apoE genotype and IEL defects for only male subjects to eliminate the confounding effect of sex (see
Table 2 and Fig. 2B – D). For women, no subgroup analysis could be performed because of too low number of subjects. In men, intimal thickness was also signifi-cantly associated with IEL gaps in all three arteries. Further, we found that in men, apoE genotype was significantly associated with IEL defects both in the CA (P=0.033) and IMA (P=0.041). The men witho4/3 or o4/4 genotype had higher number of IEL gaps in their CA than the men with o3/3 (2.3092.63 vs 1.3891.83 gaps/mm,P=0.035 in Scheffe´ post-hoc test) (Fig. 2B). In IMA, the IEL of the men with the o4 allele was characterised by higher number of gaps than the IEL of the men with the o3/2 genotype (2.1891.71 vs 0.669
0.60 gaps/mm,P=0.041 in Scheffe´ post-hoc test) (Fig. 2D). ApoE genotype was not associated with the IEL defects in the SMA (Fig, 2C). No significant apoE genotype-by-intimal thickness interaction on the
num-Table 2
The number of IEL gaps per mm, in mesenteric arteries by apoE genotypea
* The P-value for the main effect of the apoE genotype in AN-COVA.
†PB0.05 foro4/3 oro4/4 versuso3/3 in Scheffe´ post-hoc test. ‡PB0.05 for
Fig. 2. The number of defects in the IEL of the mesenteric arteries by apoE genotype group in all subjects (A) and in the subgroup of men (B,C,D). The bars indicate means and the whiskers indicate SEM. The analysis of covariance (ANCOVA) was used (the main effect is shown in the figure), and the results were adjusted for age and BMI. To test the difference between three genotypes Scheffe´ post-hoc tests were performed. (A) *P=0.032 foro4/3 oro4/4 group versuso3/3. (B) †P=0.035 foro3/3 oro4/4 group versuso3/3. (C) NS, not significant. (D) ‡P=0.041 for o4/3 oro4/4 group versuso3/2.
ber of the IEL gaps was observed in any of the AN-COVA models.
4. Discussion
Our results suggest for the first time that a genetic risk factor for atherosclerosis — apoE genotype — is associated with the defects in the IEL of the artery wall. The carriers of o4/3 or o4/4 genotype had highest number of gaps in their IEL, and this association was more pronounced in men than in both men and women. The importance of this finding must be care-fully reviewed.
At present, the meaning of the injury of the IEL during the atherosclerotic process is unclear. Pieces of evidence have been reported supporting the hypothesis that the fragmentation of the IEL plays a role in the intimal thickening [1,2,6,7], and intimal thickness was significantly associated with the IEL gaps also in the present study. Other kind of injury of the IEL has also
Dis-continuities of the IEL have been detected already at very early age, when no atherosclerosis has yet devel-oped [5,31]. The IEL defects might thus allow SMC to migrate into the intima [2] and thereby lead to intimal thickening, and later, atherosclerosis. Together, the fragmentation of the IEL probably has a role both in the initial phase atherosclerosis and in the progression of the lesions.
We found that the o4/3 and o4/4 genotypes were associated with the higher number of gaps in the IEL of the CA and IMA than theo3/3 oro3/2. What might be the possible mechanism by which apoE o4 allele is related to the IEL defects? This allele is associated with high serum total and LDL cholesterol concentrations [11 – 13]. In pigs, experimental hypercholesterolernia has been reported to be associated with discontinuity of the IEL in the coronary arteries [17,18]. This provides one possible mechanism of action. the high level of serum cholesterol in subjects with o4 allele might lead to defects in the IEL. There are no prior studies on this topic, and nor has the effect of hypercholesterolemia on the IEL been studied in human subjects which makes this discussion highly speculative. In addition, the causal relationship is unknown. The association of apoE genotype with IEL defects might as well be just a marker of increased atherosclerosis related to the o4 allele. However, there is evidence on the interaction of lipid fractions with elastin, which makes the described mechanism possible. It has been suggested that arterial elastin interact with serum LDL or very low-density lipoproteins in a way which leads to a transfer of lipids to the elastin [32]. In addition, fatty acids have been found to accumulate in elastin in atherosclerosis [33], which is an important factor in the fragmentation of elastin, and maybe also of IEL. Further, decreased elasticity of the carotid artery has been found to be associated with increased oxidized LDL levels [34].
None of the risk factors for atherosclerosis consid-ered in this study explains why the carriers of the o4 allele had the highest number of gaps in their IEL (Table 1). We analysed the male subjects as a separate group to control the possible confounding effect of sex, and in men, the association was found also in the IMA. No analysis for women in separate could be performed, so we cannot draw any conclusions about possible gender-bound association. We found no association of apoE genotype with the number of gaps in the IEL of SMA, which may be due differences in the hemody-namic conditions of the arteries. In the present study, the association of the apoE polymorphism with the defects of the IEL was studied in the mesenteric arter-ies, in which atherosclerosis is a relatively common finding in older populations [19,35,36]. It would be valuable to investigate the association reported here also in the coronary arteries. Because the sample size in our autopsy series is relatively small, the novel results
presented here must be verified in more extensive material.
In conclusion, apoE polymorphism might be in-volved in the formation of structural changes in the IEL, possibly mediated through effects on cholesterol metabolism. Our results offer one possible mechanism how apoE genotype affects the risk for atherosclerosis. Further investigation is needed to assess the role of IEL defects and apoE polymorphism in the complex process of atherosclerosis.
Acknowledgements
The study was supported by grants from the Medical Research Fund of the Tampere University Hospital, Finnish Foundation for Cardiovascular Research, the Yrjo¨ Jahnson Foundation, the Elli and Elvi Oksanen Fund of the Pirkanmaa Fund under the auspices of the Finnish Cultural Foundation, and the Finnish Medical Foundation. The authors thank Merja Lehtinen for skillful technical assistance. We appreciate Professor Markku Pelto-Huikko’s help with the photograph and Professor Seppo Nikkari’s careful review of the manuscript.
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