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Effect of estradiol on circulating levels of inflammatory cytokines in postmenopausal women

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Effect of estradiol on circulating levels of inflammatory cytokines in postmenopausal women

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Effect of Estradiol on Circulating Levels of Inflammatory Cytokines in
Postmenopausal Women

by

Xiaofu Dai

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOSTATISTISC)

May 2020

Copyright 2020 Xiaofu Dai
ii

TABLE OF CONTENTS
ACKNOWLEDGMENTS ........................................................................................................................... iii
ABSTRACT ................................................................................................................................................. iv
INTRODUCTION ........................................................................................................................................ 1
METHODS ................................................................................................................................................... 3
Study Design ............................................................................................................................................. 3
Treatment and Follow-up .......................................................................................................................... 3
Statistical Analysis .................................................................................................................................... 4
RESULTS ..................................................................................................................................................... 7
Baseline Characteristics ............................................................................................................................ 7
Estradiol Effect by Visit ............................................................................................................................ 8
Average Effect of Estradiol over 36-month follow-up ............................................................................. 8
TABLES ..................................................................................................................................................... 10
Table 1. Baseline Characteristics ............................................................................................................ 10
Table 2. The Effect of Estradiol by Visit (IL-1α, IL-1β) ........................................................................ 11
Table 3. The Effect of Estradiol by Visit ................................................................................................ 12
Table 4.1. Average Effect of Estradiol over Follow-up .......................................................................... 13
Table 4.2. Average Effect of Estradiol over Follow-up (IL-1α, IL-1β) .................................................. 13
DISCUSSION ............................................................................................................................................. 14
REFERENCES ........................................................................................................................................... 17

iii

ACKNOWLEDGMENTS
Thanks to my great mentor Wendy Mack for showing me her research and letting me be
one member of the team. In addition, thanks for her always being supportive and helpful. Besides,
I would like to thank other committee members, Dr. Howard Hodis and Dr. Roksana Karim, for
their guidance.
Finally, I would like to thank other professors who have helped me, and my friends and
classmates who have always supported and encouraged me.

iv

ABSTRACT
BACKGROUND
ELITE and other studies have demonstrated that estrogen-containing hormone therapy (HT) may
exert beneficial effects on atherosclerosis and cardiovascular disease in women who are younger
or in the early postmenopause period. Such studies provide support for the hormone-timing
hypothesis, that hormone therapy may exert positive cardiovascular benefits when initiated in early,
but not in late postmenopause. However, the clinical and molecular mechanisms underlying the
age-related atheroprotective effects of HT intervention when administered early compared with
late after menopause remain unknown. In this thesis, ELITE trial data and stored samples were
used to test the effects of HT on blood markers of inflammation, as a possible mechanism for the
HT benefit on atherosclerosis in early postmenopausal women.
METHODS
The Early Versus Late Intervention Trial with Estradiol (ELITE) was a single-center, randomized,
double-blind, placebo-controlled trial (RCT). In this trial, the actual primary outcome was the rate
of change in carotid-artery intima-media thickness (CIMT), which was measured every 6 months.
ELITE showed that oral estradiol therapy was associated with less progression of subclinical
atherosclerosis (measured as CIMT) than was placebo when therapy was initiated within 6 years
after menopause but not when it was initiated 10 or more years after menopause, which validated
the timing hypothesis.
A total of 643 healthy postmenopausal women were randomly assigned to receive either oral 17β-
estradiol (1 mg per day) or placebo. To test the HT effects on inflammation as a possible
mechanism for the effects on CIMT progression noted above, the outcomes evaluated here were
v

the circulating levels of 14 inflammatory biomarkers; these markers were measured from stored
serum samples primarily from three trial visit times, baseline, 12 months and 36 months. Mixed
effects linear regression models were used to evaluate the effect of estradiol on the levels of these
inflammation biomarkers.
RESULTS
The average levels over the trial of circulating levels of inflammatory biomarkers was statistically
significantly different between estradiol- and placebo-treated participants for 4 biomarkers, E-
selectin (p = 0.0002), ICAM-1v(p = 0.03), IFNγ (p = 0.03) and IL-8 (p = 0.03). The average
circulating levels of these 4 biomarkers were significantly lower in the estradiol group. There was
no significant interaction between visit times and the estradiol treatment (almost all p > 0.05),
indicating that the HT effect on inflammation markers did not change over the trial.
CONCLUSIONS
Randomized administration of the sex hormone estradiol significantly altered proinflammatory
cytokine levels in the circulation. This provides a potential biological mechanism at the clinical
level that may explain the atheroprotective effects of HT as a function of time-since menopause.

1

INTRODUCTION
Cardiovascular disease (CVD), as an age-related process, remains the number one cause of
death in women
1
. Female CVD mortality rates have increased in 43% of the 3,140 United States
counties since 2002, while the comparable CVD mortality rate for males only increased in 3.4%
2
.
As women age, the medical and financial burden of cardiovascular disease continues to increase.
It has been long assumed that the results of CVD intervention trials conducted primarily if not
exclusively among males are universal for women, thus providing a framework for primary
prevention of CVD in women. Over the past decade, accumulated data have refuted this
assumption
3-10
. Specifically, there is evidence for sex-specific efficacy of CVD primary prevention
therapies and the timing of initiation of postmenopausal hormone therapy (HT) according to age
and time-since-menopause as possible regulators of efficacies of CVD interventions
11
. Although
established primary prevention with statins, aspirin, and ACE-inhibitors reduces the risk of CVD
in men, the effects in women under primary prevention conditions are less certain and have been
found to not reduce all-cause mortality
3-10
. However, research studies support the observation that
HT significantly reduces CVD and all-cause mortality in primary prevention when initiated in
women who are younger than 60 years or are less than 10 years-since-menopause compared to
older women and women distant from menopause
11
. These differential associations with CVD and
mortality led to the menopause HT timing hypothesis
12
. This concept posits that HT benefits and
risks depend on the temporal initiation of therapy relative to age, time-since menopause or both,
which are in turn related to the health of the underlying tissues (i.e., healthy vascular endothelium
and extent of atherosclerosis burden) and their responsiveness to HT
13,14
.
We have validated the timing hypothesis in a recently completed randomized controlled
trial, namely the Early Versus Late Intervention Trial with Estradiol (ELITE). ELITE showed that

2

administration of HT within 6 years after menopause significantly reduced the progression of
subclinical atherosclerosis compared with placebo, measured by changes in carotid intima-media
thickness (CIMT); in contrast, HT showed no effect in women who received HT 10 years or more
after menopause (1)
15
. These data clearly indicate that there is a sex-specific treatment and perhaps
age-related opportunity to reduce CVD and all-cause mortality in women. However, the biological
mechanism of the age-related atheroprotective effects of HT on atherosclerosis progression
observed in early menopause in ELITE is unknown.
Based on these ELITE data, it can be hypothesized that early initiation of HT within 6 years
of menopause has a positive effect on circulating bioavailability and signaling of sex hormones.
Indeed, a post hoc analysis of ELITE trial data showed that higher achieved blood levels of
estradiol (E2) were significantly associated with reduced atherosclerosis progression in early
postmenopause, but with increased atherosclerosis progression in late postmenopause. A possible
mechanism for the beneficial benefit of HT and higher estradiol levels in early postmenopause
may be related to HT effects on atherosclerosis-associated inflammatory biomarkers, leading to a
reduction in CIMT progression in early postmenopause. In contrast, late initiation of HT (10 years
after menopause) may have no or adverse effects on these same inflammation pathways and
downstream consequences at the level of the arterial wall
15
. We used stored serum samples from
the ELITE trial to measure clinical biomarkers of inflammation including inflammatory cytokines,
chemokines and cell adhesion molecules associated with atherosclerosis (IL-1α, IL-1β, IL-6, IL-
8, IL-10, INF-γ, MCP-1, MIP-1α, TNF-α, sICAM-1, sVCAM-1, VEGF and E- and P-selectins).
We tested whether markers of inflammatory pathways potentially regulated by HT might explain
the effects of HT on atherosclerosis; specifically, herein we test the HT effects on inflammation
markers in ELITE.

3

METHODS
Study Design
ELITE was a single-center, randomized, double-blind, placebo-controlled trial in which
serial carotid arterial measurements were obtained noninvasively
15
. The primary trial outcome was
the rate of change in intima–media thickness of the far wall of the right distal common carotid
artery, assessed by means of computer image processing of B-mode ultrasonograms that were
obtained at two baseline examinations (averaged to obtain the baseline CIMT value) and every 6
months during trial follow-up
16
. Participants were healthy postmenopausal women, ranging in age
from 40 to 80, without diabetes and without clinical evidence of cardiovascular disease. Women
reported no regular menses for at least 6 months or had surgically induced menopause, as well as
a serum estradiol level lower than 25 pg per milliliter (92 pmol per liter).
In this post hoc analysis of ELITE trial data and stored samples, serial circulating levels of
14 inflammatory biomarkers (IL-1α, IL-1β, IL-6, IL-8, IL-10, INF-γ, MCP-1, MIP-1α, TNF-α,
sICAM-1, sVCAM-1, VEGF and E- and P-selectins) were measured over three visits, at baseline
prior to randomization, and at 12 and 36 months post-randomization.

Treatment and Follow-up
Participants were randomly assigned to receive oral 17β-estradiol (E2, 1 mg daily), plus
progesterone (45 mg) vaginal gel administered sequentially (once daily for 10 days of each 30-day
cycle) for women with a uterus, or matching placebo (plus sequential placebo vaginal gel for
women with a uterus) in a 1:1 ratio. Randomization was stratified on baseline carotid-artery
intima–media thickness (CIMT) (<0.75 or ≥0.75 mm), hysterectomy status (yes or no), and time

4

postmenopause (early, <6 year; late ≥10 years). Initial recruitment was based on a 5-year trial (3-
year recruitment, and 2 to 5 years of randomly assigned treatment or placebo). During the fifth
year of follow-up, the trial was extended by 2.5 years with supplemental funding from the NIH.
Participants were evaluated monthly in theresearch clinic for the first 6 months, and then every
other month until the trial was completed. The investigators, participants as well as other staff were
blinded to the treatment assignments.
In this post hoc trial analysis, the trial outcomes were the circulating levels of inflammatory
biomarkers. The design of this post hoc analysis defined three measurement times for the
inflammation outcomes, baseline, 12 months and 36 months. For each participant, inflammatory
biomarkers were measured at each of these visits.

Statistical Analysis
Data cleaning was performed to make visit times, baseline, 12 months and 36 months as
clean, as separate as possible. In order to concentrate observations in three visit times, a total of 11
participants were removed as the visits at which their inflammation measures were obtained were
far from the set visit times. Due to missed clinic visits and early termination from the trial and
subsequent sample availability, all participants did not have the three measurement times at
baseline, 12 months and 36 months. Due to these considerations, a total of 641 of the 643
randomized participants provided measurements for the baseline visit, 532 participants provided
measurements for the 12-month visit, and 525 participants provided measurements for the 36-
month visit. Participants whose 12-month measurement occurred at 14 or 18 months were retained
in the analysis, as these visit times were close to 12 months. Some participants completed the trial
prior to 36 months (the third inflammation measurement time); such participants whose

5

inflammation measures were obtained at 30 months were retained in the analysis while those
whose inflammation measures were obtained at 18 months (1 participant), 22 months (3
participants), 24 months (6 participants) and 26 months (1 participant) were deleted, as these visit
times were far away from 36 months.
Demographic characteristics, baseline CIMT, years since menopause and hysterectomy
status were compared between the estradiol and placebo groups with the use of independent t-test
for continuous variables and chi-square tests for categorical variables. The distributions of
inflammation biomarkers were graphically evaluated, and normalizing (log) transformations were
performed for skewed biomarkers. A normalizing transformation was not achievable for IL-1α and
IL-1β, due to a large number of zeros. The continuous variables IL-1α and IL-1β were therefore
transformed into 4-level categorical variables using the 25
th
, 50
th
, and 75
th
quantiles as the cut
points.
The HT (E2) effect on biomarkers over visit times were tested in the total sample.
The main tests of interest were HT (estradiol) treatment effects on inflammation
biomarkers. The circulating levels of inflammatory biomarkers were compared between the
estradiol and placebo groups. Two methods to compare the treatment groups on these biomarkers
were performed. The first approach evaluated whether the effect of estradiol treatment on
biomarkers differed over visit times. Mixed effects linear models were used for the normally
distributed biomarkers (some of which were log transformed). The primary independent variable
was the randomized treatment (E2, placebo), with each biomarker as the dependent variable. A
class-level covariate was the three visit times. Effect modification was evaluated by adding
interaction terms of visit times and treatment, i.e., testing if the treatment effect differed over the
three measurement times. A random effect was specified for the participant-specific intercept. The

6

(log) mean circulating levels of each biomarker were compared between the two treatment groups
at each visit time. Adjusting the 12-month and 36-month treatment comparisons for the baseline
value was performed for P-selectin, because the baseline P-selectin levels statistically significantly
differed between treatment groups (p = 0.004). The baseline values of the P-selectin measure were
carried forward into the 12-month and 36-month observations to allow adjustement for the baseline
level. In the mixed effects linear model for P-selectin, the dependent variables just included the
12-month and 36-month measures, and the baseline P-selectin values were added as a covariate.
Ordinal logistic regression mixed effect models were used for the two 4-level categorical variables
of IL-1α and IL-1β. These models contained the same interaction term and random effect as the
linear models. Odds ratios for treatment were obtained in each visit stratum; the odds represented
the odds of having a higher quartile level of IL-1α or IL-1β in estradiol compared to placebo.
Therefore, odds ratios < 1.0 indicated estradiol participants were more likely to be in lower quartile
levels of IL-1α or IL-1β, and odds ratios > 10 indicated estradiol participants were more likely to
be in higher quartiles of IL-1α or IL-1β compared to placebo.
In the second analytic approach, an overall post-randomization effect of estradiol treatment
on inflammatory biomarkers (i.e., at 12 and 36 months) was evaluated. Dependent variables were
the 12- and 36-month (not baseline) inflammation measures. Linear mixed effects models were
used for normally distributed markers and ordinal logistic regression mixed effect models were
used for the 4-level categorical inflammation markers. The same adjustment was performed for P-
selectin as detailed above. Visit times were modeled as class-level independent variables; a random
effect was specified for the participant-specific intercept. The (log) mean circulating levels of
inflammatory biomarkers were compared between the estradiol and placebo groups for continuous
variables and odds ratios were calculated for categorical variables.

7

Numerical data are expressed as means (±SD). A P-value<0.05 was considered statistically
significant. Statistical analysis was performed using Statistical Analysis System software version
9.4 (SAS institute, Inc., Cary, North Carolina).

RESULTS
Baseline Characteristics
A total of 643 women underwent randomization, of whom 323 were assigned to receive
17β-estradiol, and 320 were assigned to receive matching placebo. Based on the above data
cleaning standards, 11 observations (7 in the placebo group and 4 in the estradiol group) were
removed from the analysis sample in the 36 months measure. A total of 641 (322 in the estradiol
group and 319 in the placebo group) of the 643 randomized participants provided measurements
for the baseline visit that were included in the final analysis.
Table 1 shows the baseline characteristics of the participants. The mean (SD) age of
participants was 60.2 (7.0) in the placebo group and 61.0 (6.8) in the estradiol group. Participants
were primarily non-Hispanic whites (65.2% and 71.7% for the placebo and estradiol groups,
respectively). Most of the participants were well educated; in the placebo group, 26.6% had a
bachelor’s degree and 36.1% had a graduate or professional education; the corresponding
proportions were 27.6% and 42.5% in the estradiol group. About half of the participants’ CIMT
values were smaller than 0.75 mm in both the placebo and estradiol groups. The number of
participants in late postmenopause ( ≥10 years) (58.3% and 57.8% for the placebo and estradiol
groups, respectively) was only a little larger than that of participants in early postmenopause (<6

8

years). Most participants had not had a hysterectomy (82.5% and 81.1% for the placebo and
estradiol groups, respectively).

Estradiol Effect by Visit
Tables 2 and 3 show the estradiol effect on the circulating levels of inflammatory
biomarkers over visit. For E-selectin and IL-8, the (log) mean levels of circulating were
statistically significantly different between the estradiol and placebo groups at both the 12-month
and the 36-month visits (for E-selectin: p < 0.0001 at 12 months and p = 0.0002 at 36 months; for
IL-8: p = 0.04 at 12 months and p = 0.048 at 36 months). The circulating levels of E-selectin and
IL-8 decreased significantly in the estradiol group compared to placebo. The (log) mean circulating
levels significantly differed between two treatment groups for ICAM-1 at 12 months (estradiol
minus placebo difference, -0.034 log(ng/mL); 95% CI, -0.067 to -0.001; p = 0.045) and for IFNγ
at 36 months (estradiol minus placebo difference, -0.18 log(pg/mL); 95% CI, -0.32 to -0.04; p =
0.012) (Table 2). The circulating levels of both of these measures decreased significantly in the
estradiol group compared to placebo. Other inflammatory biomarkers showed no significant
differences between treatment groups (p > 0.05). Effect modification of estradiol treatment by visit
was evaluated by the visit-by-treatment interaction terms; there were no significant interaction
between visit times and the estradiol treatment (p > 0.05) except for TNF-α (p = 0.049).

Average Effect of Estradiol over 36-month follow-up
The overall on-trial effect of estradiol on the circulating levels of inflammatory biomarkers
after controlling for visit are summarized in Tables 4.1 and 4.2. The biomarkers with significant
results in the previous section (by-visit analysis) remained significant in this evaluation. After

9

controlling for visit, the effects of estradiol on biomarkers were statistically significantly different
from placebo for 5 biomarkers: E-selectin (estradiol minus placebo difference (95% CI): -0.131 (-
0.200, -0.063) log(ng/mL), p = 0.0002), ICAM-1 (estradiol minus placebo difference (95% CI): -
0.037 (-0.070, -0.004) log(ng/mL), p = 0.027), IFNγ (estradiol minus placebo difference (95% CI):
-0.144 (-0.275, -0.014) log(pg/mL), p = 0.030) and IL-8 (estradiol minus placebo difference (95%
CI): -0.249 (-0.476, -0.021) log(pg/mL), p = 0.032). These circulating levels decreased
significantly in the estradiol group compared to placebo. For other inflammatory biomarkers, the
overall effect of estradiol was not statistically significantly different from the placebo over follow-
up (p > 0.05).

10

TABLES
Table 1. Baseline Characteristics.*

Variable
Placebo
(n = 319)
Estradiol
(n = 322)
Age, years 60.2 ± 7.0 61.0 ± 6.8
Race, n (%)

White non-Hispanic 208 (65.2) 231 (71.7)
Black non-Hispanic 32 (10.0) 27 (8.4)
Hispanic 49 (15.4) 41 (12.7)
Asian 30 (9.4) 23 (7.2)
Formal education

High School Graduate or Less
†
16 (5.0) 6 (1.9)
Trade/ Business School 11 (3.5) 5 (1.6)
Some College 92 (28.8) 85 (26.4)
Bachelor's Degree 85 (26.6) 89 (27.6)
Beyond Bachelor's Degree 115 (36.1) 137 (42.5)
CIMT, n (%)

< 0.75mm 161 (50.5) 163 (50.6)
≥ 0.75mm 158 (49.5) 159 (49.4)
Time since Menopause, n (%)

< 6yrs 133 (41.7) 136 (42.2)
≥ 10yrs 186 (58.3) 186 (57.8)
Hysterectomy Status, n (%)

No 263 (82.5) 261 (81.1)
Yes 56 (17.5) 61 (18.9)
* Included in the table are the 641 participants who were included in the final analyses. Comparisons
between the estradiol and placebo groups were conducted with the use of an independent t-test for age and
a chi-square test for categorical variables. The differences between two groups were not significant (all p >
0.05). All continuous variables are summarized by the mean ± SD. All categorical variables are summarized
by the frequency (percentage %).
† High School Graduate or Less refers to 8th graduate or less, some high school and high school graduate.

11

Table 2. The Effect of Estradiol by Visit (IL-1α, IL-1β).*
Biomarker Visit Sample
Size
‡

OR p-value
(OR)
p-value
†

(Interaction)
Estimation 95% CIs
IL-1α, pg/mL Baseline 322/319 0.788 (0.232, 2.673) 0.70
0.54
12months 266/266 0.636 (0.185, 2.187) 0.47
36months 258/256 0.906 (0.263, 3.122) 0.88
IL-1β, pg/mL Baseline 322/319 0.563 (0.153, 2.071) 0.39
0.45

12months 266/266 0.413 (0.110, 1.550) 0.19

36months 258/256 0.603 (0.157, 2.312) 0.46
* The ordinal logistic regression results for IL-1α and IL-1β were showed in the table. Odds ratio in each
visit stratum was calculated.
† Effect modification of estradiol effect by visit was evaluated by the interaction term.
‡ Sample size of participants at each visit is the number of estradiol participants/the number of placebo
participants.

12

Table 3. The Effect of Estradiol by Visit.*
Biomarker Visit Sample
size
Log(Mean) Difference p-value
†
(Treatment)
p-value
‡
(Visit)
E2 - Placebo
(SE) 95% CIs

E-Selectin
(ng/mL)
Baseline 322/319 -0.01 (0.03) (-0.08, 0.05) 0.69

12months 266/266 -0.14 (0.03) (-0.20, -0.07) <.0001 0.74
36months 258/256 -0.12 (0.03) (-0.19, -0.06) 0.0002 0.80
P-Selectin
§

(ng/mL)
12months 266/266 0.01 (0.02) (-0.02, 0.04) 0.42

36months 258/256 0.005 (0.016) (-0.026, 0.035) 0.77 0.997
ICAM-1
(ng/mL)
Baseline 322/319 0.01 (0.02) (-0.02, 0.04) 0.52

12months 266/266 -0.034 (0.017) (-0.067, -0.001) 0.045 0.29
36months 258/256 -0.031 (0.017) (-0.065, 0.002) 0.07 0.09
VCAM-1
(ng/mL)
Baseline 322/319 0.031 (0.019) (-0.007, 0.069) 0.11

12months 266/266 -0.002 (0.020) (-0.042, 0.038) 0.92 0.48
36months 258/256 0.001 (0.020) (-0.039, 0.041) 0.95 0.005
MIP -1α
(pg/mL)
Baseline 322/319 -0.02 (0.08) (-0.18, 0.14) 0.82

12months 266/266 -0.10 (0.08) (-0.26, 0.06) 0.21 0.20
36months 258/256 -0.03 (0.08) (-0.20, 0.13) 0.69 0.025
IFNγ
(pg/mL)
Baseline 322/319 -0.06 (0.07) (-0.19, 0.07) 0.38

12months 266/266 -0.11 (0.07) (-0.25, 0.02) 0.11 0.50
36months 258/256 -0.18 (0.07) (-0.32, -0.04) 0.012 0.01
MCP-1
(pg/mL)
Baseline 322/319 0.06 (0.03) (0.01, 0.11) 0.029

12months 266/266 -0.01 (0.03) (-0.06, 0.04) 0.73 0.03
36months 258/256 -0.02 (0.03) (-0.07, 0.03) 0.44 0.0002
TNF-α
(pg/mL)
Baseline 322/319 0.03 (0.03) (-0.02, 0.08) 0.18

12months 266/266 -0.04 (0.03) (-0.10, 0.01) 0.11 0.049
36months 258/256 0.01 (0.03) (-0.04, 0.06) 0.72 0.61
VEGF-A
(pg/mL)
Baseline 322/319 0.06 (0.05) (-0.04, 0.15) 0.27

12months 266/266 0.07 (0.05) (-0.03, 0.17) 0.16 0.42
36months 258/256 0.07 (0.05) (-0.03, 0.17) 0.18 0.06
IL-6
(pg/mL)
Baseline 322/319 -0.03 (0.06) (-0.14, 0.08) 0.59

12months 266/266 -0.09 (0.06) (-0.21, 0.03) 0.15 0.43
36months 258/256 -0.09 (0.06) (-0.21, 0.03) 0.15 0.04
IL-8
(pg/mL)
Baseline 322/319 -0.10 (0.11) (-0.32, 0.12) 0.37

12months 266/266 -0.23 (0.11) (-0.46, -0.01) 0.040 0.21
36months 258/256 -0.225 (0.114) (-0.448, -0.002) 0.048 0.06
IL-10
(pg/mL)
Baseline 322/319 -0.016 (0.112) (-0.233, 0.201) 0.89

12months 266/266 -0.13 (0.11) (-0.35, 0.10) 0.27 0.65
36months 258/256 -0.076 (0.113) (-0.298, 0.146) 0.50 0.99
* Treatment group comparisons on log-transformed biomarkers
† P-values for (log) mean comparisons between the treatment groups by visit.
‡ P-values for difference from baseline visit.
§ Baseline value adjustment was performed for P-selectin, as the baseline values statistically significantly
differed between the treatment groups (p = 0.004).

13

Table 4.1. Average Effect of Estradiol over Follow-up.*
Biomarker Difference (SE)
‡
(n = 535)
95%CIs p-value
†

E-Selectin, ng/mL -0.131 (0.035) (-0.200, -0.063) 0.0002
P-Selectin
§
, ng/mL 0.009 (0.013) (-0.018, 0.035) 0.52
ICAM-1, ng/mL -0.037 (0.017) (-0.070, -0.004) 0.03
VCAM-1, ng/mL -0.004 (0.020) (-0.043, 0.036) 0.86
MIP -1α, pg/mL -0.095 (0.087) (-0.265, 0.076) 0.28
IFNγ, pg/mL -0.144 (0.066) (-0.275, -0.014) 0.03
MCP-1, pg/mL -0.002 (0.027) (-0.056, 0.052) 0.94
TNF-α, pg/mL -0.027 (0.024) (-0.075, 0.020) 0.26
VEGF-A, pg/mL 0.042 (0.054) (-0.063, 0.148) 0.43
IL-6, pg/mL -0.110 (0.060) (-0.228, 0.008) 0.067
IL-8, pg/mL -0.249 (0.116) (-0.476, -0.021) 0.03
IL-10, pg/mL -0.084 (0.117) (-0.313, 0.145) 0.47
* Treatment group comparisons on log-transformed biomarkers.
† P-values for (log) mean comparisons between the treatment groups.
‡ Differences are the (log) mean circulating level in the estradiol group minus that in the placebo group.
§ Baseline value adjustment was performed for P-selectin.

Table 4.2. Average Effect of Estradiol over Follow-up (IL-1α, IL-1β).*
Biomarkers OR p-value
Estimation
(n = 535)
95% CIs
IL-1α, pg/mL 0.773 (0.245, 2.443) 0.66
IL-1β, pg/mL 0.582 (0.150, 2.261) 0.43
* The ordinal logistic regression results for IL-1α and IL-1β were showed in the table. Odds
ratios were calculated.

14

DISCUSSION
A large body of evidence suggests that the decrease of ovarian function with menopause is
associated with an increase in pro-inflammatory biomarkers and inflammatory processes that are
integral to the formation of atherosclerosis
17-20
. Use of ELITE data provided the opportunity to test
possible mechanisms for an HT effect on atherosclerosis progression. We hypothesized that the
protective effect of HT on atherosclerosis may be mediated by the bioavailability of sex hormones
upstream of inflammatory pathways, and these inflammatory pathways are downstream of
estrogen receptor (ESR) signals directly at the level of the arterial wall
13,21
. From the perspective
of molecular biology, genetic variation in estrogen receptor-α (ESR1) is associated with decreased
levels of E-selectin in the circulation, which is consistent with our results
22-24
. In vitro experiments
in uterine arteries from women who were within 5 years of menopause showed decreased
inflammatory cytokine production in response to estradiol, whereas arteries from women who were
more than 5 years post-menopause exhibited a proinflammatory profile with estradiol treatment
25
.
This randomized controlled trial focused on 14 inflammatory biomarkers to evaluate the
effect of estradiol on the circulating levels of markers. We hypothesized that comparison of these
biomarkers between estradiol- and placebo-treated participants would provide insights on possible
molecular mechanisms of the beneficial HT effect on atherosclerosis progression. In the study, the
circulating levels of E-selectin, ICAM-1, IFNγ and IL-8 were statistically significantly lower in
the estradiol group compared to the placebo group. These results are consistent with results of
other studies that have suggested that there are significant negative associations between markers
of inflammation and pharmacological levels of circulating SHBG and sex hormones (estradiol)
26
.
E-selectin is a cell adhesion molecule expressed on the surface of activated endothelial
cells. E-selectin is expressed during inflammation and functions as an endothelial-leukocyte

15

adhesion molecule. E-selectin plays a role in recruiting leukocytes to the site of injury that
putatively occurs during the development of atherosclerosis specifically through cell surface
attachment
27
.
IFNγ (Interferon gamma) is an interferon cytokine, which is a product of human leukocytes
stimulated with phytohemagglutinin
28
. IFNγ is produced predominantly by natural killer (NK) and
natural killer T (NKT) cells as part of the innate immune response, and by CD4 Th1 and CD8
cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops
29
. IFNγ
has antiviral, immunoregulatory, and anti-tumor properties, which can promote adhesion and
binding required for leukocyte migration
30
. Also, IFNγ appears to stabilize E-selectin surface
expression without prolonging its duration of synthesis
31
. Results from this study suggest that in
part, HT may reduce atherosclerosis progression by reducing the expression of E-selectin and IFNγ
and attachment of leukocytes to the endothelium during the development of atherosclerosis.
ICAM-1 (Intercellular Adhesion Molecule 1) is a member of the immunoglobulin
superfamily and is typically expressed on endothelial cells and cells of the immune system. ICAM-
1 is an endothelial- and leukocyte-associated transmembrane protein which is important in
stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration
32
. ICAM-1
ligation produces proinflammatory effects such as inflammatory leukocyte recruitment by
signaling through cascades involving a number of kinases
33
. HT may reduce atherosclerosis
progression by reducing the expression of ICAM-1 and leukocyte endothelial transmigration
during the development of atherosclerosis.
IL-8 (Interleukin 8) is a chemokine mainly produced by macrophages. IL-8 is a potent
promoter of angiogenesis. Il-8 and its receptors, CXCR1 and CXCR2, have been observed on
endothelial cells
34,35
and play a role in endothelial cell proliferation
36
which is directly associated

16

with atherosclerosis progression. HT may reduce atherosclerosis progression directly by reducing
the expression of IL-8. These results in total indicate that HT affects endothelial cell adhesion
molecules and cytokines released by macrophages; the anti-atherosclerosis effect of HT may be
based on these two basic contributors to atherogenesis, cell adhesion molecules (ligands that
capture leukocytes that roll along the endothelial surface) and cytokines that attract leukocytes to
sites of injury/atherosclerosis (generally through the endothelium via leukocyte transmigration).
Thus, our results pave the way for investigating the molecular mechanisms of the age-
related atheroprotective effects of HT, and prepare us for further studies investigating the
association between the effect of HT on the inflammatory process and the timing of HT initiation
in relation to menopause, the association of changes in these HT-associated inflammation
measures with atherosclerosis progression measured by CIMT, and methylation status of ESRs as
well as inflammatory pathway genes in postmenopausal women.
In conclusion, these data suggest that the sex hormone estradiol regulates proinflammatory
cytokine levels in the circulation, which provides one inter-related biological mechanism at the
clinical level that may explain the atheroprotective effects of HT as a function of time-since
menopause.

17

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Abstract (if available)

Abstract BACKGROUND: ELITE and other studies have demonstrated that estrogen-containing hormone therapy (HT) may exert beneficial effects on atherosclerosis and cardiovascular disease in women who are younger or in the early postmenopause period. Such studies provide support for the hormone-timing hypothesis, that hormone therapy may exert positive cardiovascular benefits when initiated in early, but not in late postmenopause. However, the clinical and molecular mechanisms underlying the age-related atheroprotective effects of HT intervention when administered early compared with late after menopause remain unknown. In this thesis, ELITE trial data and stored samples were used to test the effects of HT on blood markers of inflammation, as a possible mechanism for the HT benefit on atherosclerosis in early postmenopausal women. ❧ METHODS: The Early Versus Late Intervention Trial with Estradiol (ELITE) was a single-center, randomized, double-blind, placebo-controlled trial (RCT). In this trial, the actual primary outcome was the rate of change in carotid-artery intima-media thickness (CIMT), which was measured every 6 months. ELITE showed that oral estradiol therapy was associated with less progression of subclinical atherosclerosis (measured as CIMT) than was placebo when therapy was initiated within 6 years after menopause but not when it was initiated 10 or more years after menopause, which validated the timing hypothesis. ❧ A total of 643 healthy postmenopausal women were randomly assigned to receive either oral 17β-estradiol (1 mg per day) or placebo. To test the HT effects on inflammation as a possible mechanism for the effects on CIMT progression noted above, the outcomes evaluated here were the circulating levels of 14 inflammatory biomarkers

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Asset Metadata

Creator Dai, Xiaofu (author)

Core Title Effect of estradiol on circulating levels of inflammatory cytokines in postmenopausal women

School Keck School of Medicine

Degree Master of Science

Degree Program Biostatistics

Publication Date 04/21/2020

Defense Date 03/20/2020

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

Tag ELITE trial,estradiol,inflammatory cytokines,OAI-PMH Harvest,postmenopausal

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mack, Wendy (committee chair), Hodis, Howard N. (committee member), Karim, Roksana (committee member)

Creator Email xiaofuda@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-285019

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Document Type Thesis

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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...

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