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The effects of hormone therapy on carotid artery intima-media thickness and serum lipids by ApoE4 genotype

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The effects of hormone therapy on carotid artery intima-media thickness and serum lipids by ApoE4 genotype

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Content 1

THE EFFECTS OF HORMONE THERAPY ON CAROTID ARTERY INTIMA-MEDIA
THICKNESS AND SERUM LIPIDS BY APOE4 GENOTYPE

By

Huaxin Liu

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(APPLIED BIOSTATISTICS AND EPIDEMIOLOGY)

August 2016

Copyright 2016 Huaxin Liu
2

ACKNOWLEDGEMENTS
I sincerely appreciate everyone who has helped me to complete my Master’s thesis. I would
like to express my sincere gratitude to my committee chair, Dr. Wendy Mack, for her patience,
motivation, and immense knowledge. Her guidance and encouragement helped me in all the time
of completing this thesis. Her endless support broaden my knowledge and help me learn more
new things. I would also like to thank my committee members, Dr. Hooman Allayee and Dr.
Howard Hodis, for their insightful comments and invaluable feedback. A special thanks also
goes to my family: my partents Xia Zhou and Jianchuan Liu, my cousins Zhou Zhang and
Junpeng Zhou, My grandmother Congzhi Wang, for supporting me spiritually throughout my
life. Thank you, everyone!

3

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .............................................................................................................2

LIST OF TABLES ..........................................................................................................................4

LIST OF FIGURES ........................................................................................................................5

ABSTRACT ....................................................................................................................................6

CHAPTER 1: INTRODUCTION ...................................................................................................8

CHAPTER 2: MATERIALS AND METHODS ..........................................................................10
2.1. Study Population and Design ...........................................................................................10
2.2. Carotid artery intima-media thickness (CIMT) measurements ........................................11
2.3. Laboratory Measurements ................................................................................................11
2.4. Statistical Analysis ...........................................................................................................12

CHAPTER 3: RESULTS ..............................................................................................................15
3.1. Demographic and baseline characteristics .......................................................................15
3.2. The effects of HT on CIMT progression by ApoE4 genotype .........................................17
3.3. The effects of HT on total cholesterol level by ApoE4 genotype .....................................20
3.4. The effects of HT on LDL cholesterol level by ApoE4 genotype ..................................24
3.5. The effects of HT on HDL cholesterol level by ApoE4 genotype ..................................28
3.6. The effects of HT on triglyceride mean level by ApoE4 genotype ..................................31

CHAPTER 4: DISCUSSION ........................................................................................................34

REFERENCES ..............................................................................................................................38

4

LIST OF TABLES

Table 1. Demographic characteristics by ApoE4 genotype .........................................................16

Table 2. Comparison of the rate of CIMT progression (mm/year) by treatment and ApoE4
genotype ........................................................................................................................................19

Table 3. Comparison of Total cholesterol mean level (mg/dl) by treatment and ApoE4
genotype ........................................................................................................................................ 22

Table 4. Comparison of HDL cholesterol mean level (mg/dl) by treatment and ApoE4
genotype ........................................................................................................................................ 26

Table 5. Comparison of LDL cholesterol mean level (mg/dl) by treatment and ApoE4
genotype ........................................................................................................................................29

Table 6. Comparison of Triglyceride mean level (mg/dl) by treatment and ApoE4
genotype ........................................................................................................................................32

5

LIST OF FIGURES

Figure 1. Comparison of total cholesterol mean level ...................................................................23

Figure 2. Comparison of HDL cholesterol mean level ................................................................. 27

Figure 3. Comparison of LDL cholesterol mean level ................................................................. 30

Figure 4. Comparison of Triglyceride cholesterol mean level ......................................................33

6

ABSTRACT
Background – Apolipoprotein E (APOE) plays an important role in cholesterol metabolism,
which is related to the development of atherosclerosis. We evaluated whether the effects of
hormone therapy (HT) on serum lipid levels and carotid artery intima-media thickness (CIMT)
were modified by ApoE4 genotype. Using the longitudinal data from the Early vs. Late
Intervention Trial with Estradiol (ELITE) trial, we conducted subgroup analyses by ApoE4 in
relation to HT, CIMT progression, and serum lipid levels.
Methods – A total of 643 postmenopausal women, with no clinical evidence of cardiovascular
disease, were randomly assigned to receive either 17β-estradiol or placebo. Trial outcomes were
the rate of change in CIMT and lipid mean levels. Mixed effect models were used to test whether
the effect of HT on the change rate of CIMT or lipid mean levels differed by ApoE4 genotype
strata (positive or negative for at least one E4 allele). The progression rate of CIMT and lipid
mean levels in ApoE4 subgroups were estimated and statistically compared.
Results – Combining women in the early and late menopause strata, effects of HT on the CIMT
progression and on-trial mean levels serum lipid were not modified by ApoE4 genotype strata
(interaction: CIMT progression p=0.95; total cholesterol p=0.23; HDL p=0.89; LDL p=0.23;
triglyceride p=0.75). Interactions of HT × ApoE4 genotype were also not significant for the
CIMT progression or on-trial lipid mean levels when analyzed in early post-menopause or late
post-menopause strata. The mean on-trial total cholesterol level did not differ between HT and
placebo groups among all participants (P=0.33). The mean levels of high-density lipoprotein
(HDL) cholesterol levels and triglycerides were higher in HT-treated women in the total sample
(p=0.0001 and 0.02; respectively) as well as within the total E4-negative group (p=0.001 and
0.01; respectively) and E4-positive group (p=0.02 and 0.04; respectively). The mean levels of
7

low-density lipoprotein (LDL) cholesterol levels were significantly decreased by HT in the total
sample (p=0.0006) as well as within the early and late postmenopause groups (p=0.05 and 0.003;
respectively).
Conclusions – while HT showed benefits on CIMT (in early postmenopause) and on lipids, and
lipid levels also differed by ApoE4 genotype, the effects of HT on serum lipid levels (total, LDL,
HDL cholesterol and triglycerides) and CIMT progression were not modified by ApoE4
genotype.
8

CHAPTER 1: INTRODUCTION
Cardiovascular disease (CVD) is the number one cause of death among both males and
females in the United States and Europe [1]. In the United States in 2011, the overall rate of
death due to cardiovascular disease (CVD) was 229.6 per 100,000 persons. Approximately
155,000 mortalities attributable to CVD occurred among persons less than 75 years of age in the
US in 2011 [2].
Atherosclerosis is the primary underlying etiology of cardiovascular disease (CVD).
Atherosclerosis is an arterial condition characterized by arterial plaque; as atherosclerosis
progresses, plaque can narrow arteries. Clinical cardiovascular events including heart attack and
stroke are late manifestations of atherosclerosis, caused by plaque rupture.
Compared with pre-menopausal women, CVD is more prevalent in post-menopausal women,
and menopause is a cardiovascular risk factor for women [3]. Sex steroid hormones are
considered to play a crucial role in altering cardiovascular risk among menopausal women.
Observational studies report that hormone therapy (HT) reduces cardiovascular morbidity and
slows the rate of progression of subclinical atherosclerosis among postmenopausal women [3, 4-
6]. Other studies provide evidence of the beneficial effects of hormone replacement therapy
when initiated in women who are close to menopause [7].
The low-density lipoprotein (LDL) receptor is a protein which regulates the endocytosis of
LDL cholesterol from plasma into cells. The endocytosis process occurs mainly in liver and can
remove LDL cholesterol from the circulation, which can diminish the risk of CVD [8].
Apolipoprotein E (ApoE) is a polymorphic protein which has three common isoforms: alleles E2,
E3 and E4 [9]. Although apoE4 is not the most common isoform, approximately 25% of the
Caucasian population are apoE4 carriers [10]. ApoE4 enhances liver lipoprotein metabolism,
9

downregulates LDL receptors, and is associated with increased LDL cholesterol concentrations
in serum [11].
Several studies have showed that the presence of apoE4 is associated with a higher frequency
of cardiovascular disease in different populations [12-15]. ApoE4 is considered as a risk factor
for both carotid and coronary atherosclerosis. Persons with ApoE4 alleles had an increased
carotid intima-media thickness (CIMT) and a higher prevalence of coronary artery disease [16].
Cahua-Pablo et al. [17] reported that female ApoE4 carriers are more likely to have elevated
LDL cholesterol levels. However, June et al. reported that there was no relationship between the
presence of ApoE4 and the change of LDL cholesterol level during a multiple risk factor
intervention trial that evaluated the influence of apolipoprotein E phenotype on risk of coronary
events [18].
The Early versus Late Intervention Trial with Estradiol (ELITE) tested the timing hypothesis
of hormone therapy among post-menopausal women free of cardiovascular disease. The timing
hypothesis proposes that HT effects depend on the initiation of HT relative to time since
menopause [19]. The ELITE primary outcome was rate of progression of CIMT. Primary
outcome analyses supported the HT timing hypothesis in relation to CIMT progression. We used
longitudinal data from ELITE to investigate the effects of hormone therapy on CIMT and
cholesterol levels in post-menopausal women when initiating HT soon after menopause (<6 y)
versus distant from menopause (≥ 10 y). We now use the longitudinal ELITE data to conduct
subgroup analyses by apoE4 in relation to HT, CIMT progression, and cholesterol levels.

10

CHAPTER 2: MATERIALS AND METHODS
2.1. Study population and design
ELITE was a randomized, double-blind, placebo-controlled trial [20]. 643 women were
randomized to oral micronized 17 β-estradiol 1 mg/day with 4% vaginal micronized
progesterone gel for 10 days per month (treatment), or to matching placebos, within two strata
defined by time since menopause (less than 6 years, 10 or more years).
Eligible participants were healthy postmenopausal women, with a serum estradiol level lower
than 25 pg/ml, and free of clinical evidence of CVD and diabetes. All participants were
postmenopausal for less than 6 years or at least 10 years at randomization. Exclusion criteria
included: women whose menopausal status could not be defined; current use of menopausal
hormone therapy; history of cardiovascular disease, diabetes mellitus and uncontrolled
hypertension (systolic blood pressure/diastolic blood pressure > 160/110 mm Hg); fasting plasma
triglycerides (TG) higher than 500 mg/dl; serum creatinine level higher than 2.0 mg/dl; untreated
thyroid disease; life-threatening disease with prognosis of less than 5 years; liver disease; history
of deep vein thrombosis or pulmonary embolism, history of breast cancer [21].
Randomization lists were prepared by the trial statistician. Within the two strata of time since
menopause, participants were randomized to HT or placebo (1:1 ratio). Additional randomization
stratification factors were hysterectomy (yes or no) and baseline CIMT (< 0.75 or ≥0.75 mm).
Women with an intact uterus were randomized to oral micronized 17 β-estradiol 1 mg/day with
4% vaginal micronized progesterone gel for 10 days per month or placebo. Women without a
uterus were randomized to oral micronized 17β-estradiol 1 mg/day alone or to placebo. After
randomization, the frequency of participant evaluations was every month in the first 6 months,
and then every 2 months thereafter.
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2.2. Carotid artery intima-media thickness (CIMT) measurements
CIMT is the thickness of the intima and media layers of the carotid artery and was used in
ELITE as a measure of subclinical atherosclerosis progression in all randomized participants.
High-resolution B-mode ultrasound images of the right common carotid artery were performed
to complete CIMT measurements, using previously described methods [20, 22, 23]. The CIMT
measurements were performed at the baseline (two times, approximately two weeks apart) and
every 6 months during trial follow-up. The first CIMT was assessed at the screening visit and
used to determine the CIMT randomization stratification level. The second CIMT was assessed
at the baseline visit before randomization. The primary trial endpoint was the change in CIMT
measured over the trial.

2.3. Laboratory measurements
For the baseline measurement and at 6-month trial follow-ups, fasting blood samples were
obtained to measure lipids and DNA extraction for ApoE4 genotype. Total cholesterol and
triglyceride levels were measured using enzymatic assays standardized to the Centers for Disease
Control and Prevention Standardization Program. High-density lipoprotein (HDL) levels were
measured after lipoproteins containing apolipoprotein B were precipitated in whole plasma by
using heparin manganese chloride [1]. Low-density lipoprotein (LDL) levels were estimated
from measurement of total and HDL cholesterol and plasma triglyceride by using the Friedewald
equation [24].
Genomic DNA from baseline samples was extracted from the buffy coat using DNeasy
isolation kits (Qiagen, Valencia, California). The human ApoE gene is located on chromosome
19 and has three major alleles, E2, E3 and E4. ApoE genotype was defined by two SNPs,
12

rs429358 and rs7412 at codon positions 112 and 158, respectively. Determination of genotypes
for rs429358 and rs7412 was carried out using the Applied Biosystems, Inc. (ABI) TaqMan
system, as previously reported [25]. Briefly, for each SNP, a PCR reaction containing 2ng of
genomic DNA, amplification primers, and two 20-30 bp oligonucleotides encompassing the
polymorphic site performed according to the manufacturer's protocols. Genotypes were called
using allele discrimination software from ABI.

2.4. Statistical analysis
Statistical analysis was performed with the Statistical Analysis System (SAS 9.3) software.
The level of statistical significance was set at a wo-sided P<0.05 for all analyses.
A linear mixed effects model with an unstructured covariance structure was used to test
whether the effect of HT on the change rate of CIMT differed by ApoE4 genotype strata. The
baseline CIMT value was the average value of screening and randomization visits. Random
effects were specified for the intercept and slope, to model individual subject deviations from the
overall mean level of the baseline CIMT and rate of change in CIMT over the trial. Independent
factors included treatment group, ApoE4 genotype (presence or absence of at least one E4
allele), follow-up time (continuous variable, measured as years since randomization), time since
menopause (<6 yrs or ≥ 10 yrs) and the two randomization stratification factors: baseline CIMT
and hysterectomy (yes or no). The main effect of follow-up time estimated the annual rate of
change in CIMT assessed over the trial. An interaction term of follow-up time × randomized
treatment tested if the CIMT rate of change differed in HT- vs. placebo-treated participants. A 3-
level interaction term of treatment × follow-up time × ApoE4 genotype (presence or absence of
at least one E4 allele) tested whether the effect of HT on rate of change of CIMT differed in
13

women with vs. without the ApoE4 genotype. In the mixed effects model, the rate of change of
CIMT in different groups, treatment × time since menopause (<6 yrs or ≥ 10 yrs) and treatment ×
ApoE4 genotype (presence or absence of at least one E4 allele), were estimated and statistically
compared by P-values and 95% CI. Using the SAS PROC MIXED procedure, the P-values for
fixed effects were used to test the association between the change rates of CIMT with
independent variables. The rates of change of CIMT were estimated by follow-up time ×
treatment group × ApoE4 genotype and follow-up time × treatment group × ApoE4 genotype ×
time since menopause; adjusted treatment group differences were estimated in each stratum
level.
Linear mixed effects models with an unstructured covariance structure were also used to test
the association of the lipid measures (total cholesterol, LDL, HDL, triglyceride) levels with HT
treatment stratified by ApoE4 genotype. The dependent variables in these models were lipid
levels from post-randomization visits (i.e., on-trial measurements). A random effect was
specified for the intercept, modeling individual subject deviations from average lipid levels
during the trial. These models included independent variables of treatment group, ApoE4
genotype (presence or absence), the visit month, time since menopause (<6 yrs or ≥ 10 yrs),
baseline lipid levels, baseline CIMT stratum, and hysterectomy (yes or no). An interaction term
of treatment × ApoE4 genotype tested whether a differential effect of HT differed by ApoE4
genotype. Baseline lipids were the average value of screening and randomization visits and were
included as model covariates. Post-randomization visit month was included as a class-level
covariate which was used to detect the different mean lipid levels between individuals at the
same time. After adjusting for baseline levels, we tested associations of lipid levels with the
stated independent variables by P-values for fixed effects. Least-square means of on-trial levels
14

of lipids were estimated by treatment group × ApoE4 genotype and time since menopause ×
treatment group × ApoE4 genotype; adjusted mean treatment group differences were estimated
in each stratum level.
15

CHAPTER 3: RESULTS
3.1. Demographic and baseline characteristics
Table 1 contrasts demographic factors and other baseline characteristics between participants
with and without an ApoE4 allele. Among the total of 643 participants, 198 women were E4
carriers and 440 women (68%) were not E4 carriers; genotype information was missing in 5
participants.
Among the E4 carriers, the mean participant age was 60.1 years; the average follow-up time
was 4.1 years. Among the women without an E4 allele, the mean participant age was 59.8 years,
and the mean follow-up time was 4.1 years. Overall, 31.0% of participants were ApoE4 carriers
but genotype distributions differed by ethnicity (p=0.008). Among non-Hispanic blacks, 43.1%
were ApoE4carriers 13.5% of Asians were carriers. Most participants were non-Hispanic white
(68.7% in the E4 carrier group, 68.9% in E4-negative group). The proportions of randomized
treatment (HT or placebo) were approximately 50% for women with E4 and without E4.
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Table 1 — Demographic characteristics by ApoE4 genotype

ApoE4-
(n=440)
ApoE4+
(n=198) P-value
a

Age, mean (SD), y 59.8 (6.9) 60.1 (6.8) 0.65
Follow up time, mean (SD), y 4.1(1.8) 4.1(1.7) 0.98
Race, n (%) 0.008
White, non-Hispanic 303(68.9) 136(68.7)
Black, non-Hispanic 33(7.5) 25(13.6)
Hispanic 59(13.4) 30(15.2)
Asian 45(10.2) 7(3.5)
Treatment, n (%) 0.69
HT 223(50.7) 97(49.0)
Placebo 217(49.3) 101(51.0)
Time since menopause, n (%) 0.34
< 6 yrs 191(43.4) 78(39.4)
≥ 10 yrs 249(56.6) 120(60.6)
Baseline CIMT stratum, n (%) 0.54
<0.75mm 225(51.1) 96(48.5)
≥0.75mm 215(48.9) 102(51.5)
Hysterectomy stratum, n (%) 0.47
No 368(83.6) 161(81.3)
Yes 72(16.4) 37(18.7)
a
Group comparisons used t-test for continuous variables or the Chi-square test for categorical variables.
17

3.2. The effects of HT on CIMT progression by ApoE4 genotype
The data from 592 participants were used to analyze the effects of HT on CIMT
progression by ApoE4 genotype (Table 2). In the total sample, combining women in the early
and late menopause strata, the effect of HT on CIMT progression was not significantly modified
by the ApoE4 genotype (p-value for the interaction term follow-up time × treatment × ApoE4
genotype = 0.95).
The estimated change rate of CIMT progression was 0.0073 mm/year among HT participants
and 0.0082mm/year among placebo participants (difference=-0.0009 mm/year; p = 0.33). In the
total sample, 408 participants were not E4 carriers and 184 participants had at least one E4 allele.
For E4 carriers, the estimated change rate of CIMT progression was 0.0078 mm/year in the HT
and 0.0087 mm/year in the placebo group (p=0.54). For women without E4, the estimated
change rate of CIMT progression was 0.0069 mm/year in the HT group and 0.0078 mm/year in
the placebo group (p=0.41). CIMT progression rates did not differ between treatment groups in
either ApoE4 genotype subgroup (p=0.54 for E4-positive and 0.41 for E4-negative participants).
Since prior studies showed support for the timing hypothesis of HT and this was the primary
hypothesis of the ELITE trial [26], the effect of HT on CIMT progression was tested in strata of
time since menopause. A total of 247 women had been menopausal for less than 6 years (early
postmenopause group). A total of 345 women had been menopausal for at least 10 years (late
postmenopause group).
In the early menopause group, the effect of HT on CIMT progression was not significantly
modified by the ApoE4 genotype (p=0.56). The estimated change rate of CIMT progression was
0.0043 mm/year in the HT group and 0.0076 mm/year in the placebo group; CIMT progression
18

significantly differed between HT and placebo groups (difference=-0.0034 mm/year; p=0.02),
with a reduced rate of CIMT progression in HT participants.
In the early menopause group, 173 women were not E4 carriers and 74 women had at least
one E4 allele. For women without the E4 allele, the estimated change rate of CIMT progression
was 0.0049mm/year in the HT group and 0.0074mm/year in the placebo group (difference=-
0.0025 mm/year; p=0.09). For E4 carriers, the estimated change rate of CIMT progression was
0.0036mm/year among HT group and 0.0078mm/year in the placebo group (difference=-0.0043
mm/year; p=0.13).
In the late menopause group, the effect of HT on CIMT progression was not significantly
modified by the ApoE4 genotype (p=0.51). The estimated change rate of CIMT progression was
0.0104 mm/year in the HT group and 0.0088 mm/year in the placebo group; CIMT progression
did not significantly differ between HT and placebo groups (difference=0.0016 mm/year;
p=0.15).
In the late menopause group, 235 women were not E4 carriers and 110 participants had at
least one E4 allele. For women without the E4 allele, the estimated change rate of CIMT
progression was 0.0089 mm/year in the HT group and 0.0081mm/year in the placebo group
(p=0.21). For E4 carriers, the estimated change rate of CIMT progression was 0.0119 mm/year in
the HT group and 0.0096 mm/year in the placebo group (p=0.50).

19

Table 2 — Comparison of the rate of CIMT progression (mm/year) by treatment and ApoE4 genotype
HT (SE) Placebo (SE) Difference of rate (SE) P-value
a
P-value for interaction
b

Total sample (n=592) 0.0073 (0.0007) 0.0082 (0.0006) -0.0009 (0.0009) 0.33 0.95
ApoE4 - (n=408) 0.0069 (0.0007) 0.0078 (0.0007) -0.0083 (0.0010) 0.41
ApoE4 + (n=184) 0.0078 (0.0011) 0.0087 (0.0011) -0.0010 (0.0015) 0.54
Menopause < 6 years(n=247) 0.0043 (0.0011) 0.0076 (0.0011) -0.0034 (0.0015) 0.02 0.56
ApoE4 - (n=173) 0.0049 (0.0011) 0.0074 (0.0012) -0.0025 (0.0016) 0.09
ApoE4 + (n=74) 0.0036 (0.0018) 0.0078 (0.0017) -0.0043 (0.0025) 0.13
Menopause >= 10 years(n=345) 0.0104 (0.0008) 0.0088 (0.0008) 0.0016 (0.0011) 0.15 0.51
ApoE4 - (n=235) 0.0089 (0.0009) 0.0081 (0.0009) 0.0010 (0.0013) 0.21
ApoE4 + (n=110) 0.0119 (0.0013) 0.0096 (0.0013) 0.0023 (0.0018) 0.50
a
P-values are for the difference of estimated change rates of CIMT progression.
b
P-values are for the interaction term time since randomization × treatment × ApoE4.

20

3.3. The effects of HT on total cholesterol level by ApoE4 genotype
Among all participants, 561 subjects were available for analysis of HT effects on total
cholesterol by ApoE4 genotype (Table 3). For the total sample, the effects of HT on total
cholesterol level was not significantly modified by ApoE4 genotype (p=0.23). The estimated
mean on-trial level of total cholesterol among women randomized to HT was 214.4 (SD=1.5)
mg/dl, and among participants randomized to placebo was 216.2 (SD=1.5) mg/dl. The mean on-
trial total cholesterol did not differ between HT and placebo groups (p=0.33).
Among the women who were ApoE4 negative (n=388), the mean on-trial level of total
cholesterol was marginally significantly lower in the HT group compared to the placebo group
(difference=-3.9 mg/dl, P=0.053). Among E4 carriers (n=173), treatment groups did not differ on
on-trial total cholesterol (difference=0.4 mg/dl; p=0.89).
Within time since menopause strata, 241 women had been menopausal for less than 6 years
(early menopause group) and 320 women had been menopausal for at least 10 years (late
menopause group).
In the early menopause group, mean on-trial levels of total cholesterol were 217.7 mg/dl
among HT group and 219.6 mg/dl among placebo group (difference=-1.9 mg/dl; p=0.49). For 71
women with E4 allele, the mean level was 221.6 mg/dl among HT group and 220.7 mg/dl among
placebo group (p=0.84). For 170 women who were E4 negative, the mean levels were 213.8
mm/year among HT group and 218.5 mg/dl among placebo group (p=0.12).
In late menopause group, mean on-trial levels of total cholesterol were 213.5 mg/dl among
HT group and 215.3 mg/dl among placebo group (p=0.46). There were 218 women without E4
allele and 102 E4 carriers in late menopause group. For E4 carriers, mean levels of total
cholesterol were not significantly different between treatment groups (p=0.93). For women who
21

were ApoE4 negative, the mean levels were also not significantly different between HT and
placebo groups (p=0.24).
22

Table 3 — Comparison of total cholesterol mean level (mg/dl) by treatment and ApoE4 genotype
HT (SE) Placebo (SE) Difference of means (SE) P-value
a
P-value for interaction
b

Total sample (n=561) 214.4 (1.5) 216.2 (1.5) -1.8 (1.8) 0.33 0.23
ApoE4 - (n=388) 211.5 (1.6) 215.4 (1.6) -3.9 (2.0) 0.053
ApoE4 + (n=173) 217.4 (2.3) 217.0 (2.3) 0.4 (3.0) 0.89
Menopause < 6 years (n=241) 217.7 (3.3) 219.6 (3.5) -1.9 (2.7) 0.49 0.30
ApoE4 - (n=170) 213.8 (3.3) 218.5 (3.6) -4.7 (3.0) 0.12
ApoE4 + (n=71) 221.6 (4.3) 220.7 (4.5) 0.9 (4.6) 0.84
Menopause >= 10 years (n=320) 213.5 (1.8) 215.3 (1.7) -1.8 (2.4) 0.46 0.56
ApoE4 - (n=218) 211.6 (2.0) 214.7 (2.0) -3.1 (2.7) 0.24
ApoE4 + (n=102) 215.5 (2.9) 215.9 (2.7) -0.4 (3.9) 0.93
P-values were generated using mixed models with an unstructured covariance structure.
a
P-values are for the difference of LS means.
b
P-values are for interaction term treatment × ApoE4.

23

208
209
210
211
212
213
214
215
216
217
218
ApoE4 - (n=388) ApoE4 + (n=173)
211.49
217.37
215.42
216.95
Figure 1 ：Comparison of total cholesterol mean level
(mg/dl) (n=561)
HT
Placebo
E4 - : p=0.053
E4+ : p= 0.89
24

3.4. The effects of HT on HDL cholesterol level by ApoE4 genotype
561 subjects were available to test the effects of HT on HDL cholesterol level by ApoE4
genotype (Table 4). The effect of HT on mean on-trial HDL cholesterol level was not
significantly modified by ApoE4 genotype (p=0.89).
The overall mean level in HT group was 72.8 mg/dl, and in placebo group was 70.4 mg/dl.
The estimated mean level of HDL cholesterol was significantly higher in HT group than in
placebo group (difference=2.5 mg/dl; P=0.0001).
The mean levels of HDL cholesterol increased with HT. Among 388 E4 carriers, mean levels
of HDL cholesterol were 73.7 mg/dl in HT group and 71.1 mg/dl in placebo group. The
difference between treatment groups was significant (difference=2.6 mg/dl; p=0.02). Among 173
women without E4 allele mean levels of HDL cholesterol were 72.0 mg/dl in HT group and 69.6
mg/dl in placebo group. The difference of mean levels was significant (difference =2.4 mg/dl;
p=0.001).
In the early menopause group, the mean levels of HDL cholesterol were 72.1 mg/dl among
HT group and 70.3 mg/dl among placebo group (p=0.61). There were 71 E4 carriers and 170
participants without E4 allele in early menopause group. Among women without E4 allele, the
mean levels of HDL cholesterol were 71.0 mg/dl among HT group and 68.5 mg/dl among
placebo group (p=0.02). Among E4 carriers, the mean levels of HDL cholesterol were 73.2
mg/dl among HT group and 72.2 mg/dl among placebo group (p=0.50).
In the late menopause group, the overall mean levels were 73.8 mg/dl among HT group and
70.7 mg/dl among placebo group. The difference of mean levels was significant (difference=3.1
mg/dl; p=0.0004). For 102 E4 carriers, the estimated mean level of HDL cholesterol was
25

significantly higher in HT group than in placebo group (difference=4.0 mg/dl; P=0.01). For 218
women without E4 allele, the mean level of HDL cholesterol was significantly higher in HT
group than in placebo group (difference=2.1 mg/dl; p=0.03).

26

Table 4 — Comparison of HDL cholesterol mean level (mg/dl) by treatment and ApoE4 genotype
HT(SE) Placebo (SE ) Difference of means (SE) P-value
a
P-value for interaction
b

Total sample (n=561) 72.8 (0.5) 70.4 (0.5) 2.5 (0.7) 0.0001 0.89
ApoE4 - (n=173) 72.0 (0.6) 69.6 (0.6) 2.4 (0.7) 0.001
ApoE4 + (n=388) 73.7 (0.8) 71.1 (0.8) 2.6 (1.1) 0.02
Menopause < 6 years (n=241) 72.1 (1.2) 70.3 (1.2) 1.8 (1.0) 0.61 0.45
ApoE4 - (n=170) 71.0 (1.2) 68.5 (1.3) 2.5 (1.1) 0.02
ApoE4 + (n=71) 73.2 (1.5) 72.2 (1.6) 1.1 (1.6) 0.50
Menopause >= 10 years (n=320) 73.8 (0.7) 70.7 (0.6) 3.1 (0.9) 0.0004 0.28
ApoE4 - (n=218) 73.1 (0.7) 70.9 (0.7) 2.1 (1.0) 0.03
ApoE4 + (n=102) 74.4 (1.1) 70.4 (1.0) 4.0 (1.5) 0.01
All values are expressed as P-value, significant values are showed in bold.
P-values were generated using mixed models with an unstructured covariance structure.
a
P-values are for the difference of LS means.
b
P-values are for interaction term treatment × ApoE4.

27

67
68
69
70
71
72
73
74
ApoE4 - (n=388) ApoE4 + (n=173)
71.99
73.68
69.59
71.11
Figure 2: Comparison of HDL cholesterol mean level (mg/dl)
(n=561)
HT
Placebo
E4 - : p=0.0001
E4+ : p=0.02
28

3.5. The effects of HT on LDL cholesterol level by ApoE4 genotype
The sample size for this analysis is 561 (Table 5). The effect of HT on LDL cholesterol level
was not modified by ApoE4 genotype (p=0.23). The mean level of LDL cholesterol decreased
with HT.
The overall mean levels of LDL cholesterol were 119.2 mg/dl among HT group and 124.9
mg/dl among placebo group. The estimated mean level of LDL cholesterol was significantly
lower in HT group than in placebo group (difference=-5.7 mg/dl; p=0.0006). There are 388 E4
carriers and 173 non-E4 participants in total. For women who were ApoE4 negative, the mean
levels of LDL cholesterol were 116.8 mg/dl among HT group and 124.5 mg/dl among placebo
group (p<.0001). For E4 carriers, the mean levels of LDL cholesterol were 121.7 mg/dl among
HT group and 125.4 mg/dl among placebo group (p=0.18).
In the early menopause group, mean levels of LDL cholesterol were 122.4 mg/dl among HT
group and 127.4 mg/dl among placebo group (p=0.05). The difference of LDL cholesterol
between treatment groups was -5.0 mg/dl (p=0.05). In early menopause group, there were 71 E4
carriers and 170 participants without E4. For E4 carriers, mean levels of LDL cholesterol were
126.0 mg/dl among HT group and 126.9 mg/dl among placebo group (p=0.83). For women who
were ApoE4 negative, mean levels were 118.78 mg/dl among HT group and 127.9 mg/dl among
placebo group (p=0.001).
In the late menopause group, total mean levels of LDL cholesterol were 117.3 mg/dl among
HT group and 123.7 mg/dl in the placebo group (p=0.003). For 218 women who were ApoE4
negative, the mean level of LDL cholesterol in the HT group was significantly lower than in the
placebo group (difference=-6.1 mg/dl; p=0.01).
29

Table 5 — Comparison of LDL cholesterol mean level (mg/dl) by treatment and ApoE4 genotype
HT (SE) Placebo (SE) Difference of means (SE) P-value
a
P-value for interaction
b

Total sample (n=561) 119.22(1.36) 124.94(1.38) -5.71(1.67) 0.0006 0.23
ApoE4- (n=173) 116.76(1.46) 124.48(1.51) -7.72(1.85) <.0001
ApoE4+ (n=388) 121.69(2.09) 125.39(2.08) -3.71 (2.77) 0.18
Menopause < 6 years (n=241) 122.37(3.08) 127.39(3.28) -5.02(2.53) 0.05 0.10
ApoE4 - (n=170) 118.78(3.06) 127.92(3.32) -9.15(2.78) 0.001
ApoE4 + (n=71) 125.96(3.98) 126.85(4.14) -0.88 (4.23) 0.83
Menopause >= 10 years (n=320) 117.30 (1.64) 123.66(1.56) -6.36(2.17) 0.003 0.90
ApoE4 - (n=218) 116.05(1.82) 122.15(1.79) -6.10(2.43) 0.01
ApoE4 + (n=102) 118.54(2.64) 125.16(2.49) -6.62(3.57) 0.06
All values are expressed as P-value, significant values are showed in bold.
P-values were generated using mixed models with an unstructured covariance structure.
a
P-values are for the difference of LS means.
b
P-values are for interaction term treatment*ApoE4 genotype.

30

112
114
116
118
120
122
124
126
ApoE4 - (n=388) ApoE4 + (n=173)
116.76
121.69
124.48
125.39
Figure 3: Comparison of LDL cholesterol mean level
(mg/dl) (n=561)
HT
Placebo
E4 - : p<0.0001
E4+ : p=0.18
31

3.6. The effects of HT on triglyceride mean level by ApoE4 genotype
561 subjects were available to test the effects of HT on triglyceride (Table 6). The two-way
interaction term for HT by ApoE4 genotype was not statistically significant (p=0.75).
The mean levels of triglycerides increased with HT. The overall estimated triglyceride mean
level was 112.9 mg/dl in the HT group, and in the placebo group was 104.7 mg/dl. The mean
level of triglyceride in the HT group was significantly higher than in the placebo group
(difference=8.2 mg/dl; p=0.002). There were 388 E4 carriers and 173 women who were ApoE4
negative in total. Among E4 carriers, mean levels of triglyceride were 114.1 mg/dl in the HT
group and 105.1 mg/dl in the placebo group (p=0.04). Among women who were ApoE4
negative, mean levels were 111.7 mg/dl in the HT group and 104.3 mg/dl in the placebo group
(p=0.01).
In the early menopause group, mean levels of triglyceride were 114.3 mg/dl in the HT group
and 107.9 mg/dl in the placebo group. The difference of mean levels between HT and placebo
groups was 6.3 mg/dl (p=0.11). There were 170 participants who were ApoE4 negative, mean
levels of triglyceride were 113.9 mg/dl in the HT group and 105.4 mg/dl in the placebo group
(p=0.05).
In the late menopause group, mean levels of triglyceride were 114.0 mg/dl in the HT group
and 104.2 mg/dl in the placebo group. The mean level of triglyceride in the HT group was
significantly higher than in the placebo group (difference=9.8 mg/dl; p=0.005). For 102 E4
carriers, the mean levels of triglyceride were 116.1 mg/dl in the HT group and 102.8 mg/dl in the
placebo group (p=0.02).
32

Table 6 — Comparison of Triglyceride mean level (mg/dl) by treatment and ApoE4 genotype
HT (SE) Placebo (SE) Difference of means (SE) P-value
a
P-value for interaction
b

Total sample (n=561) 112.9 (2.3) 104.7 (2.2) 8.2 (2.6) 0.002 0.75
ApoE4- (n=173) 111.7 (2.3) 104.3 (2.4) 7.4 (2.9) 0.01
ApoE4+ (n=388) 114.1 (3.3) 105.1 (3.3) 9.0 (4.4) 0.04
Menopause < 6 years (n=241) 114.3 (4.8) 107.9 (5.1) 6.3 (3.9) 0.11 0.58
ApoE4 - (n=170) 113.9 (4.7) 105.4 (5. 1) 8.5 (4.3) 0.05
ApoE4 + (n=71) 114.6 (6.2) 110.5 (6.4) 4.2 (6.6) 0.53
Menopause >= 10 years (n=320) 114.0 (2.6) 104.2(2.5) 9.8 (3.5) 0.005 0.32
ApoE4 - (n=218) 111.9 (2.97) 105.6 (2.9) 6.3 (3.9) 0.11
ApoE4 + (n=102) 116.1 (4.2) 102.8 (4.0) 13.3 (5.8) 0.02
All values are expressed as P-value, significant values are showed in bold.
P-values were generated using mixed models with an unstructured covariance structure.
a
P-values are for the difference of LS means.
b
P-values are for interaction term treatment × ApoE4 genotype.

33

98
100
102
104
106
108
110
112
114
116
ApoE4 - (n=388) ApoE4 + (n=173)
111.69
114.14
104.34
105.13
Figure 4: Comparison of Triglyceride cholesterol mean level (mg/dl)
(n=561)
HT
Placebo
E4 - : p=0.01
E4+ : p=0.04
34

CHAPTER 4: DISCUSSION
We evaluated whether the effects of HT on lipid levels (total, LDL, HDL cholesterol, and
triglycerides) and progression rates of CIMT differed by ApoE4 genotypes. CIMT is a direct
measure of subclinical atherosclerosis, and CIMT was repeatedly measured during the ELITE
trial as the primary trial outcome. Post-randomization lipid levels including total, LDL, HDL
cholesterol and triglycerides were analyzed while considering the effects of HT and ApoE4
genotypes. Our results show that the effects of HT on lipids levels measured post-randomization
and CIMT progression did not differ by ApoE4 genotype after adjusting for time since
menopause, baseline CIMT stratum, hysterectomy status and baseline lipid levels measured
before randomization. When analyzed within early and late postmenopause strata, ApoE4
genotype also did not modify the effect of HT on CIMT progression and on-trial lipids.
Previous studies, as well as the primary results of the ELITE trial, have indicated that
hormone therapy reduces the rate of progression of subclinical atherosclerosis in postmenopausal
women [6, 27, 28]. A timing hypothesis for HT that predicted HT would benefit progression of
subclinical atherosclerosis when initiated in early postmenopausal women, but not in late
postmenopausal women, was specifically tested and supported in ELITE [26]. In ELITE, the HT-
placebo difference on average rates of CIMT progression was significant among early
postmenopausal women, but not in late postmenopausal women. In women within 6 years of
menopause who were randomized to HT, the average rate of CIMT progression was 0.0034
mm/year slower than women who were randomized to placebo (p=0.02). Among women who
were 10 or more years past menopause at the time of randomization, the rates of CIMT
progression in the placebo and treatment groups were similar (0.0104 and 0.0088 mm/year,
respectively; p=0.15).
35

In this thesis, we conducted a formal subgroup analysis to test whether the effect of HT on
CIMT progression in women differed by ApoE4 genotype, We found no significant interactions
of ApoE4 genotype with the HT effect on CIMT progression, either in the total sample
(interaction p = 0.95), in the early postmenopausal strata (interaction p = 0.56), or in the late
postmenopausal group (interaction p = 0.51). The rates of CIMT progression in the placebo and
estradiol groups were similar in the E4 negative group (p=0.41) and the E4 positive group
(p=0.54).
Beilby et al. [29] investigated the association between ApoE genotypes and CIMT levels in a
cross-sectional study with randomly selected community subjects. Horejsí et al. [30] investigated
the association between ApoE genotypes and CIMT in a cross-sectional study among 114
hyperlipoproteinaemia patients. Neither study observed significant associations between ApoE
genotypes and CIMT. However, in a community-based study, Wohlin et al. [31] found that men
homozygous with the E4 allele had thicker CIMT measures than non-e4 carriers after adjusting
for lipid variables and other CVD risk factors. In another community study, Volcik et al. [32]
reported that the E4 allele was associated with higher CIMT measures than carriers of E3 allele
in both whites and African Americans. In a cross-sectional study among Japanese men, Haraki et
al. [33] found that the presence of E4 allele was a higher risk for increased CIMT compared to
persons who were negative for E4. In all ELITE subjects, we did not observe significant
associations between ApoE4 genotype and CIMT level (p=0.60) or CIMT progression (p=0.32).
While the main effects of ApoE4 genotype on CIMT progression were not significant in the
early postmenopause group (p=0.77), ApoE4 carriers in the late postmenopause group showed
significantly greater CIMT progression than their ApoE4 negative comparators (p=0.04).
Moreover, for the total sample, we did not observe a significant difference of CIMT progression
36

rate between HT group and placebo group in either ApoE4 carriers (p=0.54) or noncarriers
(p=0.41).
ApoE is a multifunctional protein that is associated with the metabolism of cholesterol and
triglycerides to mediate clearance of chylomicrons and very low-density lipoproteins from the
bloodstream [34]. Some previous studies indicated that the ApoE4 allele was associated with
higher levels of total cholesterol [31, 32, 34-37]. In ELITE, the ApoE4 genotype was
significantly associated with higher mean total cholesterol levels than non-E4 carriers (p=0.04).
However, among all participants, the HT-placebo difference between estimated mean levels of
total cholesterol was not significant (p=0.33). Moreover, the effect of HT on total cholesterol
levels was not modified by ApoE4 genotype (interaction p=0.23).
Prior studies have showed that HT was associated with higher levels of HDL cholesterol [38-
40]. Our study supports this concept and showed that on-trial HDL cholesterol levels were
significantly elevated with HT compared to placebo in the total sample (p=0.0001). On-trial
HDL cholesterol levels were significantly raised with HT compared to placebo both in the
ApoE4-negative group (p=0.001) and in the total ApoE4-positive group (p=0.02). The effect of
HT on HDL cholesterol was not modified by the ApoE4 genotype among all participants
(interaction p=0.89) or in the early or late postmenopause groups (p=0.45 and p=0.28;
respectively).
With respect to LDL cholesterol, some studies have indicated that the serum LDL cholesterol
level is reduced with hormone treatment [36, 37, 39-41]. Others identified a significant
association between certain ApoE genotypes and LDL cholesterol levels [35, 42, 43].
Corroborating these studies, we observed that LDL cholesterol levels were significantly reduced
with HT among all participants in ELITE (p=0.0006) but this effect was not different by ApoE4
37

genotypes (p=0.23). Moreover, Heikkinen, et al [44] reported that, in early postmenopausal
women, ApoE4-negative subjects respond more favorably to hormone replacement therapy with
respect to LDL cholesterol levels than those in ApoE4-positive subjects. This result was
supported in ELITE, where LDL cholesterol levels were lower in HT-treated compared to
placebo-treated women who were E4 negative in the early postmenopause group (p=0.001).
Among all participants, the LDL cholesterol levels were significantly reduced in HT-treated
compared to placebo-treated women who were E4 negative (p<.0001). However, in the ApoE4
positive group, the LDL cholesterol levels were not significantly changed with HT (p=0.18).
LDL cholesterol levels were also reduced with HT in both early and late postmenopause groups
(p=0.05 and 0.003; respectively). However, the effect of HT on LDL cholesterol levels was not
different by ApoE4 genotypes in either early or late postmenopause groups (interaction p=0.10
and 0.90; respectively).
In ELITE, triglycerides levels were significantly increased with HT in the total sample
(p=0.002), which is in accordance with numerous other reports [39-41, 45, 46]. The effect of HT
on triglycerides levels was not modified by ApoE4 genotype in the total sample (p=0.75), in the
early postmenopause group (p=0.58) or in the late postmenopause group (p=0.32).
One limitation of our study is that the number of participants in the early postmenopausal
ApoE4 positive group was not large (n=74), reducing power to detect interactions. However, it
did not hamper our observation in the total sample as well as the total ApoE4 positive strata.
In conclusion, while HT showed benefits on CIMT (in early postmenopause) and on lipids,
and lipid levels also differed by ApoE4 genotype, the effects of HT on serum lipid levels (total,
LDL, HDL cholesterol and triglycerides) and CIMT progression were not modified by ApoE4
genotype.
38

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Abstract (if available)

Abstract Background: Apolipoprotein E (APOE) plays an important role in cholesterol metabolism, which is related to the development of atherosclerosis. We evaluated whether the effects of hormone therapy (HT) on serum lipid levels and carotid artery intima-media thickness (CIMT) were modified by ApoE4 genotype. Using the longitudinal data from the Early vs. Late Intervention Trial with Estradiol (ELITE) trial, we conducted subgroup analyses by ApoE4 in relation to HT, CIMT progression, and serum lipid levels. ❧ Methods: A total of 643 postmenopausal women, with no clinical evidence of cardiovascular disease, were randomly assigned to receive either 17β-estradiol or placebo. Trial outcomes were the rate of change in CIMT and lipid mean levels. Mixed effect models were used to test whether the effect of HT on the change rate of CIMT or lipid mean levels differed by ApoE4 genotype strata (positive or negative for at least one E4 allele). The progression rate of CIMT and lipid mean levels in ApoE4 subgroups were estimated and statistically compared. ❧ Results: Combining women in the early and late menopause strata, effects of HT on the CIMT progression and on-trial mean levels serum lipid were not modified by ApoE4 genotype strata (interaction: CIMT progression p=0.95

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Asset Metadata

Creator Liu, Huaxin (author)

Core Title The effects of hormone therapy on carotid artery intima-media thickness and serum lipids by ApoE4 genotype

School Keck School of Medicine

Degree Master of Science

Degree Program Applied Biostatistics and Epidemiology

Publication Date 06/20/2016

Defense Date 08/09/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ApoE4,cardiovascular disease,hormone therapy,OAI-PMH Harvest,postmenopause,randomized trials,Women

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mack, Wendy (committee chair), Allayee, Hooman (committee member), Hodis, Howard (committee member)

Creator Email huaxin1990@gmail.com,huaxinli@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-255491

Unique identifier UC11280617

Identifier etd-LiuHuaxin-4455.pdf (filename),usctheses-c40-255491 (legacy record id)

Legacy Identifier etd-LiuHuaxin-4455-0.pdf

Dmrecord 255491

Document Type Thesis

Format application/pdf (imt)

Rights Liu, Huaxin

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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