Int J Mol Sci. 2016 Aug; 17(8): 1318.
Published online 2016 Aug 11. doi: 10.3390/ijms17081318
PMCID: PMC5000715
Shaker A. Mousa, Academic Editor
Shaker A. Mousa, Academic Editor
Abstract
The
risk of cardiovascular disease and atherosclerosis progression is
significantly increased after menopause, probably due to the decrease of
estrogen levels. The use of hormone replacement therapy (HRT) for
prevention of cardiovascular disease in older postmenopausal failed to
meet expectations. Phytoestrogens may induce some improvements in
climacteric symptoms, but their effect on the progression of
atherosclerosis remains unclear. The reduction of cholesterol
accumulation at the cellular level should lead to inhibition of the
atherosclerotic process in the arterial wall. The inhibition of
intracellular lipid deposition with isoflavonoids was suggested as the
effective way for the prevention of plaque formation in the arterial
wall. The aim of this double-blind, placebo-controlled clinical study
was to investigate the effect of an isoflavonoid-rich herbal preparation
on atherosclerosis progression in postmenopausal women free of overt
cardiovascular disease. One hundred fifty-seven healthy postmenopausal
women (age 65 ± 6) were randomized to a 500 mg isoflavonoid-rich herbal
preparation containing tannins from grape seeds, green tea leaves, hop
cone powder, and garlic powder, or placebo. Conventional cardiovascular
risk factors and intima-media thickness of common carotid arteries
(cIMT) were evaluated at the baseline and after 12 months of treatment.
After 12-months follow-up, total cholesterol decreased by 6.3% in
isoflavonoid-rich herbal preparation recipients (p = 0.011) and by 5.2% in placebo recipients (p = 0.020); low density lipoprotein (LDL) cholesterol decreased by 7.6% in isoflavonoid-rich herbal preparation recipients (p
= 0.040) and by 5.2% in placebo recipients (non-significant, NS); high
density lipoprotein (HDL) cholesterol decreased by 3.4% in
isoflavonoid-rich herbal preparation recipients (NS) and by 4.5% in
placebo recipients (p = 0.038); triglycerides decreased by 6.0%
in isoflavonoid-rich herbal preparation recipients (NS) and by 7.1% in
placebo recipients (NS). The differences between lipid changes in the
isoflavonoid-rich herbal preparation and placebo recipients did not
reach statistical significance (p > 0.05). Nevertheless, the
mean cIMT progression was significantly lower in isoflavonoid-rich
herbal preparation recipients as compared to the placebo group (6 μm, or
<1%, versus 100 μm, or 13%; p < 0.001 for the
difference). The growth of existing atherosclerotic plaques in
isoflavonoid-rich herbal preparation recipients was inhibited by
1.5-fold (27% versus 41% in the placebo group). The obtained results
demonstrate that the use of isoflavonoid-rich herbal preparation in
postmenopausal women may suppress the formation of new atherosclerotic
lesions and reduce the progression of existing ones, thus promising new
drug for anti-atherosclerotic therapy. Nevertheless, further studies are
required to confirm these findings.
Keywords: atherosclerosis, menopause, herbal preparation, prevention, intimal medial thickens, isoflavonoids, phytoestrogens
1. Introduction
Postmenopausal
status increases cardiovascular risk due to accelerated atherosclerosis
progression. Cardiovascular diseases remain the leading cause of
mortality and morbidity among postmenopausal women. The cardiovascular
risk related to postmenopausal status is predominately due to the rapid
decrease of estrogen levels, which are attributed to the indirect
protective effect on lipid and glycemic control, and to the direct
effect on endothelial function [1,2].
The use of hormone replacement therapy (HRT) in cardiovascular
prevention failed to meet expectations and it has been recognized that
long-term use of hormone therapy may actually increase the risk of
cardiovascular disease (CVD) in postmenopausal women, as shown in the
Heart and Estrogen/progestin Replacement Study (HERS) trial, which was
conducted in older postmenopausal women with established coronary heart
disease (CHD) [3,4].
The known side effect of HRT—that is, an increased risk of malignant
hormone-dependent tumors—also produced a negative impact on the
perspectives of such therapy [5,6].
The general outcome of several large-scale trials was that neither
estrogen nor estrogen/progestin decreased cardiovascular disease [7]. However, later analysis has shown that HRT started in early postmenopause provides cardiovascular benefit and no harm [8,9].
In spite of these findings, the expert opinion says that HRT should not
be used for the primary or secondary prevention of CHD; it should be
limited to the treatment of menopausal symptoms at the lowest effective
dosage over the shortest duration possible, and continued use should be
re-evaluated on a periodic basis [10,11].
Phytoestrogens,
mainly isoflavonoids, are believed to be an alternative to HRT in
postmenopausal women. Phytoestrogens comprise a rather heterogeneous
group of natural compounds of plant origin with structures similar to
estrogen E2. Three of the most active compounds are coumestans,
prenylflavonoids, and isoflavones. The hypothetical effects of
phytoestrogens are mediated via estrogen receptors (ERα and ERβ), and
the G protein-coupled estrogen receptor (GPER). It is also known that
phytoestrogens have high affinity for ERβ, which explains their
different action from endogenous estrogens [12].
Similar to endogenous estrogens, phytoestrogens may provide beneficial
effects on cardiovascular system through the effects on the vascular
endothelium [13], vascular smooth muscle cells [14,15], intracellular cholesterol metabolism [16,17,18], extracellular matrix synthesis [19], and vascular inflammation [20].
Dietary
supplementation with phytoestrogens may inhibit the development of
atherosclerotic lesions. It has been demonstrated that phytoestrogens
from grapes prevent cholesterol accumulation in blood-derived cultured
monocytes from postmenopausal women [17].
Animal studies support the anti-atherogenic properties of
phytoestrogens; for example, genistein inhibited atherogenesis in
hypercholesterolemic rabbits mostly via its beneficial effects on
endothelial dysfunction [21]. Resveratrol (stilbene with known estrogen-like activity) exhibited multiple anti-atherogenic effects [22], including inhibition of intimal hyperplasia [23] and inhibition of low density lipoprotein (LDL) oxidation [24].
The results of experimental studies demonstrate that phytoestrogens
have a potential in anti-atherosclerotic therapy, because they are able
to modulate several mechanisms of atherosclerosis progression.
Previously,
in an ex vivo model, we evaluated the anti-atherosclerotic effect of
phytoestrogen-rich plants and their combinations [17].
Based on the results of dose titration studies, qualitative
compositions of isoflavonoid-rich anti-atherosclerotic herbal
preparation—active ingredients: tannins from grape seeds, green tea
leaves, hop cone powder, and garlic powder—was developed. The aim of the
present study was to investigate the effect of this isoflavonoid-rich
herbal preparation on the progression of subclinical carotid
atherosclerosis in healthy postmenopausal women.
The
intima-media thickness of common carotid arteries (cIMT) measured by
B-mode ultrasound is a widely accepted marker of subclinical
atherosclerosis; it is well correlated with the degree of coronary
atherosclerosis and is a significant predictor of clinical
manifestations of atherosclerosis. This instrumental marker is used in
clinical and epidemiological studies to assess the impact of
conventional and novel cardiovascular risk factors and treatment
regimens on atherosclerosis progression [25,26,27].
Several clinical trials were aimed at the assessment of the effects of
HRT or phytoestrogens on cIMT progression in postmenopausal women [28,29,30,31].
Thus, in this study we have used ultrasound examination of common
carotid arteries and cIMT measurement as a tool for quantitative
assessment of atherosclerosis, with annual cIMT progression as the
endpoint.
2. Results
2.1. Baseline Data
In
total, 157 asymptomatic postmenopausal women were included in the
study, 77 in the isoflavonoid-rich herbal preparation group, and 80 in
the placebo group. The groups did not differ in age, body mass index,
smoking status, family history of coronary artery disease, blood level
of triglycerides, and high density lipoprotein (HDL) cholesterol
(HDL-C), the prognostic risk of myocardial infarction, and sudden death.
No difference was found between groups in mean and maximum cIMT, and in
the size of asymptomatic carotid atherosclerotic plaques. However,
systolic and diastolic blood pressure levels were significantly higher
in placebo group, whereas total cholesterol and low density lipoprotein
(LDL) cholesterol (LDL-C) levels were significantly higher in
isoflavonoid-rich herbal preparation recipients at the baseline. The
proportion of patients with diabetes was also higher in
isoflavonoid-rich herbal preparation recipients. Baseline
characteristics of study participants are given in Table 1.
Baseline characteristics of study participants.
2.2. Follow-up
Of
the 157 enrolled study participants, 131 completed study protocol (57
isoflavonoid-rich herbal preparation recipients and 74 placebo
recipients). Among dropouts, 16 were lost for follow-up examination (12
in the isoflavonoid-rich herbal preparation recipients group, 4 in the
placebo group) and 10 refused to visit for personal reasons and withdrew
their informed consent (8 in the isoflavonoid-rich herbal preparation
recipients group, 2 in the placebo group). In participants available for
follow-up examination no adverse or side effects were registered in
both groups. Thus, the higher dropout rate observed in isoflavonoid-rich
herbal preparation recipients can hardly be explained by some adverse
or side effects of the study medication. The comparison of odds ratios
for dropout have shown that the observed dropout values are better
explained by chance, taking into account rather small sample size.
After
12-month follow-up, blood pressure, lipid profile, as well as
ultrasound characteristics of carotid atherosclerosis were determined in
both groups. Blood lipid levels decreased in both groups, and in the
placebo group these changes were statistically significant for total
cholesterol (from 252 to 239 mg/dL, or by 5.2% reduction, p =
0.020) and HDL-C (from 74 to 71 mg/dL, or by 4.5% reduction), in
isoflavonoid-rich herbal preparation recipients for total cholesterol
(from 271 to 254 mg/dL, or by 6.3% reduction) and LDL-C (from 170 to 157
mg/dL, or by 7.6% reduction; p = 0.040). The decrease in serum
triglyceride levels was statistically insignificant in both groups. The
difference between lipid changes in isoflavonoid-rich herbal
preparation and placebo recipients did not reach statistical
significance neither for total cholesterol, nor for LDL-C, HDL-C, and
triglycerides. Blood pressure levels and body mass index did not change
in either group. The changes of clinical and biochemical characteristics
from the baseline after 12-month follow-up are given in Table 2.
The changes of characteristics of study participants after 12-month follow-up.
In
isoflavonoid-rich herbal preparation recipients, no significant
increase of mean cIMT was observed; the increment accounted for 6 μm
(less than 1%), and the growth of atherosclerotic plaque growing
accounted for 27% of the baseline value. C, in the placebo group the
rate of atherosclerosis progression was higher (i.e., the increment of
mean cIMT accounted for more than 100 μm (13%) and the growth of
atherosclerotic plaques accounted for 40% of the baseline value) (Table 3).
There was a significant difference between the isoflavonoid-rich herbal
preparation and placebo recipients in mean cIMT increase over 12-month
follow-up (p < 0.001), but not in maximum cIMT increase (p = 0.89) or in the growth of existing atherosclerotic plaques (p
= 0.30). The samples of actual individual ultrasound images and the
mean values of cIMT at the baseline and after follow-up are shown in Figure 1.
Actual individual ultrasound images and cIMT values at the baseline and after 12-month follow-up. (a) Normal cIMT in apparently healthy postmenopausal women; (b) Abnormally increased cIMT in apparently healthy postmenopausal women; (c) Dynamics of cIMT ...
Carotid atherosclerosis progression.
3. Discussion
The
results of this study have demonstrated that mean cIMT progression was
slower in asymptomatic postmenopausal women who received
isoflavonoid-rich herbal preparation, as compared to women who received
the placebo. In addition, the herbal preparation decreased the total
cholesterol, LDL-C levels, and suppressed cIMT progression after 12
months of herbal preparation administration. It should be noted that in
our study, the baseline LDL-C and total cholesterol level, as well as
the prevalence of diabetes in isoflavonoid-rich herbal preparation
recipients were higher than in the placebo group; these risk factors of
atherosclerosis could suggest more pronounced cIMT and plaque
progression. However, we have seen the reverse effect; therefore, it may
be expected that the anti-atherosclerotic potency of isoflavonoid-rich
herbal preparation may even be underestimated in this study. On the
other hand, the reduction in total cholesterol and LDL-C after 12-month
follow-up in the isoflavonoid group could be due to regression towards
the mean, since they were higher at the baseline.
CVD
related to atherosclerotic process is responsible for the majority of
deaths in postmenopausal women. The prevention of the lipid accumulation
in cells is the key mechanistic factor for inhibition of
atherosclerotic plaque formation at the early stages of atherosclerosis
progression. Phytoestrogens have the capacity to affect plasma lipid
profile, but little is known regarding their effects on atherosclerosis
progression. In women undergoing coronary angiography for suspected
myocardial ischemia, beneficial association between blood levels of the
phytoestrogen daidzein and lipoproteins, particularly lower
triglycerides and higher HDL-C levels were previously reported [32].
The main association of phytoestrogens with lipoprotein levels was
incrementally related to diadzein, but not with other lipoprotein
modulators. In another clinical study it has been shown that isoflavones
induce the reduction of total cholesterol and LDL-C plasma levels
without affecting triglycerides or HDL-C levels [33].
Our results are in line with previous findings; however, in our study
blood lipid levels were decreased in both groups: in the placebo group,
these changes were significant for total cholesterol and HDL-C; and in
the isoflavonoid-rich herbal preparation recipients, for total
cholesterol and LDL-C. The ability of phytoestrogens to reduce the
accumulation of cholesterol in cells is a possible mechanism to explain
the effects on mean cIMT. Previously, we have evaluated the
anti-atherogenic effect of phytoestrogen-rich plants using an ex vivo
model based on primary cultures of monocytes isolated from the blood of
healthy donors. In this model, the ability of human serum to induce
accumulation of cholesterol in cultured cells (serum atherogenicity) was
measured, as well as the effect of single dose oral administration of
plant extract on serum atherogenicity [17,34].
Grape seeds extract (100 mg) lowered serum atherogenicity by 71%, 78%,
and 81% at 2, 4, and 6 h after oral intake of a single dose. Similar
effects were observed for hop cones (250 mg), garlic powder (150 mg),
sage leaves (100 mg), green tea leaves (250 mg), sea kelp (500 mg),
fucus (250 mg), and carrot (1000 mg). The ability of soya beans extract
(35 mg) to lower serum atherogenicity by 28%, 38%, and 30% at 2, 4, and 6
h after a single dose administration, respectively, was demonstrated [17,35].
The
main endpoint of the current study was to identify the annual changes
in cIMT progression. Several studies demonstrated that cIMT is a
significant and independent predictor of cardiovascular events, and
allows for non-invasive evaluation of early atherosclerosis progression
in asymptomatic patients [36].
Only a few clinical trials investigated the effect of phytoestrogens on
atherosclerosis progression in postmenopausal women. In the recent
long-term intervention trial (2.7 years) with soy isoflavones, the
inhibition of subclinical atherosclerosis progression evaluated by cIMT
was demonstrated. Healthy postmenopausal women were randomized in two
groups; the first group who received daily supplement with 25 g soy
protein containing 91 mg of isoflavones, and the second group who
received a placebo. In both groups, the increment of cIMT was observed.
However, in the soy group the cIMT progression was not statistically
significant (p = 0.35), and was 16% lower than in the placebo
group. That study has enrolled 350 participants, and the duration was
more than two years. The authors suggested that further use of
isoflavone-rich dietary supplements would allow achieving the
significant difference in the rate of atherosclerosis progression
between the two groups [28].
In
our study, mean cIMT changes in both groups were observed. However, in
herbal preparation recipients this increase was negligible, but in the
placebo group the increment was significantly higher than in herbal
preparation recipients, and accounted for 111 μm, or 13% increase. This
fact indicates that in postmenopausal women the rate of carotid
atherosclerosis progression is notably high. In the study by Rossi et
al. [37],
the mean cIMT progression accounted for 103 μm (range from −250 to 567
μm; IQR from 0 to 200 μm) per year in hypertensive postmenopausal women,
and this progression rate is very close to our data. It should be noted
that in our study the difference between the two groups in systolic and
diastolic blood pressure at the baseline was statistically significant
(135/83 versus 127/79 in placebo group and herbal preparation group,
respectively), and this fact may partly explain the rather high
progression of cIMT in the placebo group, which has not been replicated
in any other studies [29,30]. On the other hand, Colacurci et al. have demonstrated rather similar cIMT progression rates in non-hypertensive women [31].
The limitations of our study, such as the duration of the follow-up and
rather limited sample size, did not allow defining the proportion of
cIMT progression rates in placebo recipients explained by the higher
blood pressure.
It should be noted that the time since
menopause is of the essence when studying atherosclerosis progression
and medical intervention. There exists an opinion that timely HRT may
offer protection against CVD, whereas in older women there may be
cardiovascular harm associated with HRT use [8,9].
Hodis et al. have recently demonstrated that anti-atherosclerotic
effects of HRT on cIMT progression differed between early and late
postmenopause. Oral estradiol therapy was associated with less
progression of subclinical atherosclerosis measured as cIMT dynamics
than was placebo when therapy was initiated within six years after
menopause, but not when it was initiated ten or more years after
menopause [29].
Thus, estradiol was shown to be effective in reducing cIMT progression.
On the other hand, in the Kronos Early Estrogen Prevention Study
(KEEPS) performed in more than 700 healthy women aged 42 to 59 within
three years after menopause, the carotid ultrasound studies showed
similar rates of progression of cIMT in all three treatment groups (0.45
mg a day of Premarin—an oral conjugated equine estrogen (o-CEE)—or 50
µg a day of transdermal estradiol via a Climara patch, or placebo) over
the four years of study. However, these changes were reported to be
generally small; therefore, slow cIMT progression limited the
statistical power to detect any differences among the groups [30].
According to the results of our study, isoflavonoid-rich herbal
preparations may provide a direct anti-atherosclerotic effects, but no
direct comparisons with estrogens were performed. In general, it may be
proposed that anti-atherosclerotic action of drugs should be realized
via prevention of intracellular cholesterol accumulation in vascular
wall cells, but it is unclear if estrogens may possess the same
mechanistic effect at the cellular level. In our previous studies on
prevention of intracellular cholesterol accumulation, modified LDL were
used to induce intracellular lipid deposition, and the effects of drugs
or chemical compounds mainly related to LDL binding, uptake,
internalization, and metabolism in cells were in focus. In contrast,
Wang et al. explored the alternative way of preventing foam cell
formation via cholesterol efflux modulation. They have demonstrated that
17β-estradiol promotes cholesterol efflux from vascular smooth muscle
cells and reduces foam cell formation via ERβ- and liver X receptor
(LXR) α-dependent upregulation of ABCA1 and ABCG1 [38].
Another mechanism of atherosclerosis prevention may be related to
anti-inflammatory effects, and it was shown that estradiol can regulate
monocyte chemotactic protein-1 (MCP-1) in human coronary artery smooth
muscle cells [39], increase prostacyclin synthesis in cells from atherosclerotic lesions [40], impair endothelial function in postmenopausal women [41], transform growth factor activity [42], and attenuate atherogenesis via selective estrogen receptor beta modulator 8β-VE2 [43]. On the other hand, anti-inflammatory effects of phytoestrogens are also known [44,45,46,47].
Therefore, anti-atherogenic effects of both estrogens and isoflavonoids
are not limited to the inhibition of direct accumulation of cholesterol
in cells only.
Finally, the findings of our study are
in line with the results obtained from the study aimed to evaluate the
effect of selective estrogen receptor modulator Raloxifene on
atherosclerosis progression in postmenopausal clinically healthy women.
In a prospective study enrolling 155 postmenopausal women without
clinical manifestations of CVD, study participants were randomized in
two groups, receiving Raloxifene 60 mg daily or placebo for 18 months.
The cIMT progression for 18 months was 11.2 μm in Raloxifene group
versus 85.7 μm in the placebo group (p < 0.004). Thus, the
lower risk of cIMT progression was demonstrated in Raloxifene recipients
(odds ratio = 0.41; 0.32–0.70 at a 95% confidence interval) [31].
Nevertheless,
our study has certain limitations. The main one is the duration of the
follow-up, only 12 months. Long-term effects of isoflavonoid-rich
dietary supplement Karinat need to be further studied in order to
evaluate its effects on main cardiovascular risk factors and long-term
outcomes, such as myocardial infarction and stroke [48].
Indeed, longer observation may help to better understand the effects of
isoflavonoid-rich herbal preparations on main outcomes of CVD, but in
this study that was not the primary endpoint. The second notable
limitation is a rather small sample size. To interpret the results from
our study, the limited number of enrolled subjects needs to be taken
into consideration, as it may lead to confounding results, despite
randomization. Finally, it should be noted that the effect of
isoflavonoids or other estrogen-like molecules on cardiovascular health
may be realized more through endothelial function/dysfunction. In our
study we have evaluated only the effects on lipids, and the effect of
treatment on the arterial wall that reflects atherosclerotic profile. It
should be expedient to study the effects of isoflavonoid-rich herbal
medications also on endothelial function using, for example,
flow-mediated dilatation.
4. Materials and Methods
4.1. Study Medication
Isoflavonoid-rich herbal preparation contained tannins from grape seeds (Vitis vinifera L.), green tea leaves (Camellia sinensis L.), hop cone powder (Hunulus lupulus), and garlic powder (Allium sativum
L.). Commercially available purified compounds were used. This
preparation was officially registered as a dietary supplement “Karinat”
and was manufactured by INAT-Farma (Moscow, Russia). The quantified
chemical constituents are provided in Table 4.
The content of toxic elements, pesticides,
dichlorodiphenyltrichloroethane (DDT), and its metabolites and
microbiological purity have been controlled. The measurement of
cathechines and allicin contents was performed by high performance
liquid chromatography (HPLC). Based on previous dose titration studies,
the dosage regimen for isoflavonoid-rich herbal preparation was
determined [17].
The quantity of herbal constituents was 500 mg per capsule; a total of
three capsules were given daily, independently of meals, for 12 months.
The dosage regimen of Karinat (three capsules daily) provides for
estimated daily intake of 27.3 mg procyanidin, 2.5 mg genistein, 11.8 mg
daidzein, 4.6 mg flavones, 3.5 mg resveratrol, and 44.6 mg of other
polyphenolic compounds [49].
Herbal and technological composition of isoflavonoid-rich herbal preparation “Karinat”.
4.2. Study Design
The
study was performed in the Outpatient Clinic Nº 202 at Moscow State
University. In total, 157 asymptomatic postmenopausal women were
included in double-blind, placebo-controlled clinical study
(ClinicalTrials.gov Identifier, NCT01742000).
The inclusion criteria were as follows: the menopausal state
(physiological or surgical) at least for the last five years; maximum
cIMT more than 0.80 mm as determined by ultrasound B-mode examination of
carotid arteries; the absence of climacteric syndrome (no more than two
points by the Blatt-Kupperman score [50]).
Exclusion criteria were as follows: the use of HRT during the peri- and
postmenopausal period; the use of the lipid-lowering drugs for at least
six months prior to inclusion; the absence of signed informed consent;
the permanent use of sugar-lowering drugs (more than two months per
year); the history of myocardial infarction, stroke, heart failure,
uncontrolled hypertension (blood pressure above 145/95 mm·Hg in patients
receiving antihypertensive treatment); cancer; chronic kidney disease;
chronic liver disease; intolerability of the components of
isoflavonoid-rich herbal preparation; and/or adverse reactions and/or
side effects revealed during the follow-up. Some inclusion and exclusion
criteria were intentionally defined to be compatible in general with
those used in the KEEPS [51],
in order to allow the possibility of tentative comparison of the rate
of cIMT progression in early menopausal women. The use of lipid- and
sugar-lowering medications was considered as a limitation for the
inclusion in the study, since they may provide their own effects on cIMT
progression [52,53,54,55].
The study participants were randomized into two groups: the first group
who received isoflavonoid-rich herbal preparation (Karinat,
INAT-Farma), three capsules daily for 12 months, and the second group
who received a placebo. Karinat and placebo capsules looked identical.
4.3. Baseline Examination
Clinical
and laboratory examinations were performed at the inclusion to the
study and included anthropometric parameters (i.e., age, body mass
index, blood pressure); personal and family history of arterial
hypertension, diabetes mellitus, and coronary heart disease; lipid
profile (i.e., cholesterol, triglycerides, LDL-C, HDL-C), B-mode
ultrasound examination of common carotid arteries, as well as evaluation
of the severity of menopausal symptoms by the Blatt-Kupperman score [50].
4.4. Follow-up Examination
Follow-up
examination was performed after 12 months of treatment and included the
same clinical and laboratory examinations, as at the baseline. The rate
of cIMT progression was the primary endpoint of the study, since it is
conventionally used as an intermediate outcome for vascular risk
estimation. It was demonstrated that cIMT progression may be rather slow
[56].
We have investigated the cIMT progression in healthy postmenopausal
women after five years of menopause, and in this age the cIMT
progression was expected to be accelerated. Therefore, 12-month
follow-up was considered to be sufficient to detect significant changes
in cIMT in this cohort. On the other hand, the studies aimed to
investigate the atherosclerosis progression and/or the role of
anti-atherosclerotic therapy in postmenopausal women employed a one year
(12-month) follow-up [37,57]. These considerations prevented us from evaluating lipid results and carotid arteries earlier than 12-month intervals.
4.5. Blood Sampling and Lipid Measurements
Venous
blood was taken after overnight fasting. Commercially available
enzymatic kits (Fluitest CHOL, Fluitest TG, Fluitest HDL-CHOL,
Analyticon, Potsdam, Germany) were used for total cholesterol,
triglycerides, and HDL-C measurements in blood serum. LDL-C was
calculated with the Friedewald formula.
4.6. Calculation of Prognostic Cardiovascular Risk
The
calculation of ten-year prognostic risk of fatal and non-fatal
myocardial infarction and sudden death was performed in accordance with
PROCAM Study-derived Cox proportional hazards model [58].
Such variables as female gender, age, blood pressure, smoking, diabetes
mellitus, total cholesterol, triglycerides, and family history of acute
myocardial infarction (first-grade relatives with the events occurred
before the age of 60 years), were used for risk calculation, and the
regional adjustment coefficient was applied [59].
4.7. Carotid Artery Ultrasound Examination
To
examine the carotid arterial wall, B-mode high-resolution
ultrasonography with a linear vascular 7.5 MHz probe (SSI-1000 scanner,
SonoScape, Shenzhen, China) was performed by three operators. The
examination included a scanning of the left and right common carotid
arteries, the carotid sinus area, as well as external and internal
carotid arteries, with a focus on the far wall of the artery in three
fixed projections (anterior, lateral, and posterior [60]).
The measurements were performed on distal 10 mm of common carotid
artery (the opposite site from carotid sinus of the common carotid
artery). Reproducibility of cIMT measurements was assessed according to
the protocol of the IMPROVE Study [61].
Within-operator coefficient of variation (CV) was 2.6%; reproducibility
coefficient accounted for 0.040. The frozen scans were digitized for
subsequent cIMT quantitative measurement using specialized software
package (M’Ath ver. 3.1, IMT, Paris, France). The cIMT far wall was
measured as the distance from the leading edge of the first echogenic
zone to the leading edge of the second echogenic zone. The measurements
were performed by an independent certified reader in a blinded manner.
The mean of all measurements in the anterior, posterior, and lateral
projections were considered as integral measurements of cIMT.
4.8. Statistical Analysis
The
significance of differences was analyzed with the IBM SPSS 21.0 program
package (IBM, Armonk, NY, USA). The Mann–Whitney statistics or t-test
were applied for between-group valuations, Wilcoxon statistics were
performed for within-group effect comparisons, and Pearson's chi-squared
was used for the assessment of nominal variables distributions.
Pearson's correlation analysis and regression analysis were applied for
the evaluation of the relationship between the values of risk changes
and clinical and biochemical variables. The data are reported as the
mean and standard deviation (SD). The differences were considered
statistically significant at the 0.95 level of confidence (p < 0.05).
5. Conclusions
Our
data suggest that the use of the isoflavonoid-rich herbal preparation
Karinat may play an important role in the prevention of atherosclerosis
progression in postmenopausal women, since it essentially suppressed the
formation of new atherosclerotic lesions approximately by 1.5-fold and
slowed the progression of existing ones. Further evaluation of the study
results should be based on the precise knowledge of cardioprotective,
metabolic, and anti-atherosclerotic effects of isoflavonoids, other
phytoestrogens and their combinations. The isoflavonoid-rich herbal
preparation used in our study provides intake of a mix of polyphenolic
compounds, including procyanidin, genistein, daidzein, flavones, and
resveratrol [49],
but the role of each compound in the inhibition of cIMT and plaque
progression remains to be unraveled. Our study unambiguously suggests
that there is the potential for this herbal supplement for the
prevention of atherosclerosis in postmenopausal women. However, it is
worth noting that the present study is preliminary in nature, and the
herbal preparations are still limited to prevention, but not treatment.
Thus,
the use of isoflavonoid-rich herbal preparations may be considered
nowadays as a promising approach for the development of
anti-atherosclerotic therapy. Nevertheless, further studies are required
to confirm this possibility.
Acknowledgments
This study was supported in part by the Ministry of Education and Sciences, Russian Federation (Project # RFMEFI61614X0010).
Author Contributions
All
authors contributed to the design and implementation of this study.
Veronika A. Myasoedova, Tatyana V. Kirichenko, Alexandra A. Melnichenko,
Varvara A. Orekhova and Alessio Ravani performed the examination of
study participants and analyzed the data obtained. Igor A. Sobenin and
Alexander N. Orekhov have elaborated the concept of the study and
supervised the project. All authors contributed to the writing of this
manuscript.
References
1. Rossi R., Grimaldi T., Origliani G., Fantini G., Coppi F., Modena M.G. Menopause and cardiovascular risk. Pathophysiol. Haemost. Thromb. 2002;32:325–328. doi: 10.1159/000073591. [PubMed] [Cross Ref]
2. Rossi
R., Cioni E., Nuzzo A., Origliani G., Modena M.G. Endothelial-dependent
vasodilation and incidence of type 2 diabetes in a population of
healthy postmenopausal women. Diabetes Care. 2005;28:702–707. doi: 10.2337/diacare.28.3.702. [PubMed] [Cross Ref]
3. Hulley
S., Grady D., Bush T., Furberg C., Herrington D., Riggs B., Vittinghoff
E. Randomized trial of estrogen plus progestin for secondary prevention
of coronary heart disease in postmenopausal women. Heart and
Estrogen/progestin Replacement Study (HERS) Research Group. J. Am. Med. Assoc. 1998;280:605–613. doi: 10.1001/jama.280.7.605. [PubMed] [Cross Ref]
4. Grady
D., Herrington D., Bittner V., Blumenthal R., Davidson M., Hlatky M.,
Hsia J., Hulley S., Herd A., Khan S., et al. HERS Research Group.
Cardiovascular disease outcomes during 6.8 years of hormone therapy:
Heart and Estrogen/progestin Replacement Study follow-up (HERS II) J. Am. Med. Assoc. 2002;288:49–57. doi: 10.1001/jama.288.1.49. [PubMed] [Cross Ref]
5. Gompel A., Santen R.J. Hormone therapy and breast cancer risk 10 years after the WHI. Climacteric. 2012;15:241–249. doi: 10.3109/13697137.2012.666456. [PubMed] [Cross Ref]
6. Justenhoven
C., Obazee O., Brauch H. The pharmacogenomics of sex hormone
metabolism: Breast cancer risk in menopausal hormone therapy. Pharmacogenomics. 2012;13:659–675. doi: 10.2217/pgs.11.144. [PubMed] [Cross Ref]
7. Rossouw
J.E., Anderson G.L., Prentice R.L., LaCroix A.Z., Kooperberg C.,
Stefanick M.L., Jackson R.D., Beresford S.A., Howard B.V., Johnson K.C.,
et al. Writing Group for the Women's Health Initiative Investigators.
Risks and benefits of estrogen plus progestin in healthy postmenopausal
women: Principal results from the Women’s Health Initiative randomized
controlled trial. J. Am. Med. Assoc. 2002;288:321–333. [PubMed]
8. Rossouw
J.E., Prentice R.L., Manson J.E., Wu L., Barad D., Barnabei V.M., Ko
M., LaCroix A.Z., Margolis K.L., Stefanick M.L. Postmenopausal hormone
therapy and risk of cardiovascular disease by age and years since
menopause. J. Am. Med. Assoc. 2007;297:1465–1477. doi: 10.1001/jama.297.13.1465. [PubMed] [Cross Ref]
9. Manson
J.E., Chlebowski R.T., Stefanick M.L., Aragaki A.K., Rossouw J.E.,
Prentice R.L., Anderson G., Howard B.V., Thomson C.A., LaCroix A.Z., et
al. Menopausal hormone therapy and health outcomes during the
intervention and extended poststopping phases of the Women's Health
Initiative randomized trials. J. Am. Med. Assoc. 2013;310:1353–1368. doi: 10.1001/jama.2013.278040. [PMC free article] [PubMed] [Cross Ref]
10. American
College of Obstetricians and Gynecologists Committee on Gynecologic
Practice ACOG Committee Opinion No. 420, November 2008: Hormone therapy
and heart disease. Obstet. Gynecol. 2008;112:1189–1192. [PubMed]
11. Mosca
L., Benjamin E.J., Berra K., Bezanson J.L., Dolor R.J., Lloyd-Jones
D.M., Newby L.K., Piña I.L., Roger V.L., Shaw L.J., et al.
Effectiveness-based guidelines for the prevention of cardiovascular
disease in women -2011 Update: A Guideline from the American Heart
Association. Circulation. 2011;123:1243–1262. doi: 10.1161/CIR.0b013e31820faaf8. [PMC free article] [PubMed] [Cross Ref]
12. Glazier
M.G., Bowman M.A. A review of the evidence for the use of
phytoestrogens as a replacement for traditional estrogen replacement
therapy. Arch. Intern. Med. 2001;161:1161–7112. doi: 10.1001/archinte.161.9.1161. [PubMed] [Cross Ref]
13. Sandoval
M.J., Cutini P.H., Rauschemberger M.B., Massheimer V.L. The soyabean
isoflavone genistein modulates endothelial cell behaviour. Br. J. Nutr. 2010;104:171–179. doi: 10.1017/S0007114510000413. [PubMed] [Cross Ref]
14. Finking
G., Wohlfrom M., Lenz C., Wolkenhauer M., Eberle C., Hanke H. The
phytoestrogens genistein and daidzein, and 17 beta-estradiol inhibit
development of neointima in aortas from male and female rabbits in vitro
after injury. Coron. Artery Dis. 1999;10:607–615. doi: 10.1097/00019501-199912000-00010. [PubMed] [Cross Ref]
15. Shen
J., White M., Husband A.J., Hambly B.D., Bao S. Phytoestrogen
derivatives differentially inhibit arterial neointimal proliferation in a
mouse model. Eur. J. Pharmacol. 2006;548:123–128. doi: 10.1016/j.ejphar.2006.07.050. [PubMed] [Cross Ref]
16. Safari M.R., Sheikh N. Effects of flavonoids on the susceptibility of low-density lipoprotein to oxidative modification. Prostaglandins Leukot. Essent. Fatty Acids. 2003;69:73–77. doi: 10.1016/S0952-3278(03)00085-1. [PubMed] [Cross Ref]
17. Nikitina
N.A., Sobenin I.A., Myasoedova V.A., Korennaya V.V., Mel’nichenko A.A.,
Khalilov E.M., Orekhov A.N. Antiatherogenic effect of grape flavonoids
in an ex vivo model. Bull. Exp. Biol. Med. 2006;141:712–715. doi: 10.1007/s10517-006-0260-7. [PubMed] [Cross Ref]
18. Guo
Y., Wu G., Su X., Yang H., Zhang J. Antiobesity action of a daidzein
derivative on male obese mice induced by a high-fat diet. Nutr. Res. 2009;29:656–663. doi: 10.1016/j.nutres.2009.09.005. [PubMed] [Cross Ref]
19. Cao
C., Li S., Dai X., Chen Y., Feng Z., Zhao Y., Wu J. Genistein inhibits
proliferation and functions of hypertrophic scar fibroblasts. Burns. 2009;35:89–97. doi: 10.1016/j.burns.2008.03.011. [PubMed] [Cross Ref]
20. Dharmappa
K.K., Mohamed R., Shivaprasad H.V., Vishwanath B.S. Genistein, a potent
inhibitor of secretory phospholipase A2: A new insight in down
regulation of inflammation. Inflammopharmacology. 2010;18:25–31. doi: 10.1007/s10787-009-0018-8. [PubMed] [Cross Ref]
21. Lee
C.S., Kwon S.J., Na S.Y., Lim S.P., Lee J.H. Genistein supplementation
inhibits atherosclerosis with stabilization of the lesions in
hypercholesterolemic rabbits. J. Korean Med. Sci. 2004;19:656–661. doi: 10.3346/jkms.2004.19.5.656. [PMC free article] [PubMed] [Cross Ref]
22. Wang
Z., Zou J., Cao K., Hsieh T.C., Huang Y., Wu J.M. Dealcoholized red
wine containing known amounts of resveratrol suppresses atherosclerosis
in hypercholesterolemic rabbits without affecting plasma lipid levels. Int. J. Mol. Med. 2005;16:533–540. [PubMed]
23. Zou
J., Huang Y., Cao K., Yang G., Yin H., Len J., Hsieh T.C., Wu J.M.
Effect of resveratrol on intimal hyperplasia after endothelial
denudation in an experimental rabbit model. Life Sci. 2000;68:153–163. doi: 10.1016/S0024-3205(00)00925-5. [PubMed] [Cross Ref]
24. Brito
P., Almeida L.M., Dinis T.C. The interaction of resveratrol with
ferrylmyoglobin and peroxynitrite; protection against LDL oxidation. Free Radic. Res. 2002;36:621–631. doi: 10.1080/10715760290029083. [PubMed] [Cross Ref]
25. Stein
J.H., Korcarz C.E., Post W.S. Use of carotid ultrasound to identify
subclinical vascular disease and evaluate cardiovascular disease risk:
Summary and discussion of the American Society of Echocardiography
consensus statement. Prev. Cardiol. 2009;12:34–38. doi: 10.1111/j.1751-7141.2008.00021.x. [PubMed] [Cross Ref]
26. Amato
M., Montorsi P., Ravani A., Oldani E., Galli S., Ravagnani P.M.,
Tremoli E., Baldassarre D. Carotid intima-media thickness by B-mode
ultrasound as surrogate of coronary atherosclerosis: Correlation with
quantitative coronary angiography and coronary intravascular ultrasound
findings. Eur. Heart J. 2007;28:2094–2101. doi: 10.1093/eurheartj/ehm244. [PubMed] [Cross Ref]
27. Chambless
L.E., Heiss G., Folsom A.R., Rosamond W., Szklo M., Sharrett A.R.,
Clegg L.X. Association of coronary heart disease incidence with carotid
arterial wall thickness and major risk factors: The Atherosclerosis Risk
in Communities (ARIC) Study, 1987–1993. Am. J. Epidemiol. 1997;146:483–494. doi: 10.1093/oxfordjournals.aje.a009302. [PubMed] [Cross Ref]
28. Hodis
H.N., Mack W.J., Kono N., Azen S.P., Shoupe D., Hwang-Levine J.,
Petitti D., Whitfield-Maxwell L., Yan M., Franke A.A., et al. Women’s
Isoflavone Soy Health Research Group. Isoflavone soy protein
supplementation and atherosclerosis progression in healthy
postmenopausal women: A randomized controlled trial. Stroke. 2011;42:3168–3175. doi: 10.1161/STROKEAHA.111.620831. [PMC free article] [PubMed] [Cross Ref]
29. Hodis
H.N., Mack W.J., Henderson V.W., Shoupe D., Budoff M.J., Hwang-Levine
J., Li Y., Feng M., Dustin L., Kono N., et al. ELITE Research Group.
Vascular effects of early versus late postmenopausal treatment with
estradiol. N. Engl. J. Med. 2016;374:1221–1231. doi: 10.1056/NEJMoa1505241. [PMC free article] [PubMed] [Cross Ref]
30. Harman
S.M. Effects of oral conjugated estrogen or transdermal estradiol plus
oral progesterone treatment on common carotid artery intima media
thickness (CIMT) and coronary artery calcium (CAC) in menopausal women:
Initial results from the Kronos Early Estrogen Prevention Study (KEEPS) Menopause. 2012;19:1365.
31. Colacurci
N., Fornaro F., Cobellis L., de Franciscis P., Torella M., Sepe E.,
Arciello A., Cacciapuoti F., Paolisso G., Manzella D. Raloxifene slows
down the progression of intima-media thickness in postmenopausal women. Menopause. 2007;14:879–884. doi: 10.1097/gme.0b013e3180577893. [PubMed] [Cross Ref]
32. Bairey
Merz C.N., Johnson B.D., Braunstein G.D., Pepine C.J., Reis S.E.,
Paul-Labrador M., Hale G., Sharaf B.L., Bittner V., Sopko G., et al.
Phytoestrogens and lipoproteins in women. J. Clin. Endocrinol. Metab. 2006;91:2209–2213. doi: 10.1210/jc.2005-1853. [PubMed] [Cross Ref]
33. Crouse
J.R., 3rd, Morgan T., Terry J.G., Ellis J., Vitolins M., Burke G.L. A
randomized trial comparing the effect of casein with that of soy protein
containing varying amounts of isoflavones on plasma concentrations of
lipids and lipoproteins. Arch. Intern. Med. 1999;159:2070–2076. doi: 10.1001/archinte.159.17.2070. [PubMed] [Cross Ref]
34. Sobenin I.A., Chistiakov D.A., Bobryshev Y.V., Orekhov A.N. Blood atherogenicity as a target for anti-atherosclerotic therapy. Curr. Pharm. Des. 2013;19:5954–5962. doi: 10.2174/1381612811319330014. [PubMed] [Cross Ref]
35. Myasoedova
V.A., Sobenin I.A. Background, rationale and design of clinical study
of the effect of isoflavonoid-rich botanicals on natural history of
atherosclerosis in women. Atheroscler. Suppl. 2008;9:171. doi: 10.1016/S1567-5688(08)70689-5. [Cross Ref]
36. Lorenz
M.W., Markus H.S., Bots M.L., Rosvall M., Sitzer M. Prediction of
clinical cardiovascular events with carotid intima-media thickness: A
systematic review and meta-analysis. Circulation. 2007;115:459–467. doi: 10.1161/CIRCULATIONAHA.106.628875. [PubMed] [Cross Ref]
37. Rossi
R., Nuzzo A., Olaru A.I., Origliani G., Modena M.G. Endothelial
function affects early carotid atherosclerosis progression in
hypertensive postmenopausal women. J. Hypertens. 2011;29:1136–1144. doi: 10.1097/HJH.0b013e328345d950. [PubMed] [Cross Ref]
38. Wang
H., Liu Y., Zhu L., Wang W., Wan Z., Chen F., Wu Y., Zhou J., Yuan Z.
17β-estradiol promotes cholesterol efflux from vascular smooth muscle
cells through a liver X receptor α-dependent pathway. Int. J. Mol. Med. 2014;33:550–558. [PubMed]
39. Seli
E., Selam B., Mor G., Kayisli U.A., Pehlivan T., Arici A. Estradiol
regulates monocyte chemotactic protein-1 in human coronary artery smooth
muscle cells: A mechanism for its antiatherogenic effect. Menopause. 2001;8:296–301. doi: 10.1097/00042192-200107000-00013. [PubMed] [Cross Ref]
40. O’Sullivan
M.G., Goodrich J.A., Adams M.R. Increased prostacyclin synthesis by
atherosclerotic arteries from estrogen-treated monkeys. Life Sci. 2001;69:395–401. doi: 10.1016/S0024-3205(01)01131-6. [PubMed] [Cross Ref]
41. Honisett
S.Y., Stojanovska L., Sudhir K., Kingwell B.A., Dawood T., Komesaroff
P.A. Hormone therapy impairs endothelial function in postmenopausal
women with type 2 diabetes mellitus treated with rosiglitazone. J. Clin. Endocrinol. Metab. 2004;89:4615–4619. doi: 10.1210/jc.2003-031414. [PubMed] [Cross Ref]
42. Gourdy
P., Schambourg A., Filipe C., Douin-Echinard V., Garmy-Susini B.,
Calippe B., Tercé F., Bayard F., Arnal J.F. Transforming growth factor
activity is a key determinant for the effect of estradiol on fatty
streak deposit in hypercholesterolemic mice. Arterioscler. Thromb. Vasc. Biol. 2007;27:2214–2221. doi: 10.1161/ATVBAHA.107.150300. [PubMed] [Cross Ref]
43. Sun
J., Ma X., Chen Y.X., Rayner K., Hibbert B., McNulty M., Dhaliwal B.,
Simard T., Ramirez D., O’Brien E. Attenuation of atherogenesis via the
anti-inflammatory effects of the selective estrogen receptor beta
modulator 8β-VE2. J. Cardiovasc. Pharmacol. 2011;58:399–405. doi: 10.1097/FJC.0b013e318226bd16. [PubMed] [Cross Ref]
44. Chacko
B.K., Chandler R.T., Mundhekar A., Khoo N., Pruitt H.M., Kucik D.F.,
Parks D.A., Kevil C.G., Barnes S., Patel R.P. Revealing
anti-inflammatory mechanisms of soy isoflavones by flow: Modulation of
leukocyte-endothelial cell interactions. Am. J. Physiol. Heart Circ. Physiol. 2005;289:H908–H915. doi: 10.1152/ajpheart.00781.2004. [PubMed] [Cross Ref]
45. Simoncini
T., Garibaldi S., Fu X.D., Pisaneschi S., Begliuomini S., Baldacci C.,
Lenzi E., Goglia L., Giretti M.S., Genazzani A.R. Effects of
phytoestrogens derived from red clover on atherogenic adhesion molecules
in human endothelial cells. Menopause. 2008;15:542–550. doi: 10.1097/gme.0b013e318156f9d6. [PubMed] [Cross Ref]
46. Andrade C.M., Sá M.F., Toloi M.R. Effects of phytoestrogens derived from soy bean on expression of adhesion molecules on HUVEC. Climacteric. 2012;15:186–194. doi: 10.3109/13697137.2011.582970. [PubMed] [Cross Ref]
47. Liu
T., Hou D.D., Zhao Q., Liu W., Zhen P.P., Xu J.P., Wang K., Huang H.X.,
Li X., Zhang H., et al. Phytoestrogen α-Zearalanol attenuates
homocysteine-induced apoptosis in human umbilical vein endothelial
cells. BioMed Res. Int. 2013;2013:813450. [PMC free article] [PubMed]
48. Orekhov
A.N., Sobenin I.A., Korneev N.V., Kirichenko T.V., Myasoedova V.A.,
Melnichenko A.A., Balcells M., Edelman E.R., Bobryshev Y.V.
Anti-atherosclerotic therapy based on botanicals. Recent Pat. Cardiovasc. Drug Discov. 2013;8:56–66. doi: 10.2174/18722083113079990008. [PMC free article] [PubMed] [Cross Ref]
49. Sobenin
I.A., Myasoedova V.A., Orekhov A.N. Phytoestrogen-rich dietary
supplements in anti-atherosclerotic therapy in postmenopausal women. Curr. Pharm. Des. 2016;22:152–163. doi: 10.2174/1381612822666151112150520. [PubMed] [Cross Ref]
50. Alder E. The Blatt-Kupperman menopausal index: A critique. Maturitas. 1998;29:19–24. doi: 10.1016/S0378-5122(98)00024-3. [PubMed] [Cross Ref]
51. Harman
S.M., Brinton E.A., Cedars M., Lobo R., Manson J.E., Merriam G.R.,
Miller V.M., Naftolin F., Santoro N. KEEPS: The Kronos Early Estrogen
Prevention Study. Climacteric. 2005;8:3–12. doi: 10.1080/13697130500042417. [PubMed] [Cross Ref]
52. Artom N., Montecucco F., Dallegri F., Pende A. Carotid atherosclerotic plaque stenosis: The stabilizing role of statins. Eur. J. Clin. Investig. 2014;44:1122–1134. doi: 10.1111/eci.12340. [PubMed] [Cross Ref]
53. Huang
Y., Li W., Dong L., Li R., Wu Y. Effect of statin therapy on the
progression of common carotid artery intima-media thickness: An updated
systematic review and meta-analysis of randomized controlled trials. J. Atheroscler. Thromb. 2013;20:108–121. doi: 10.5551/jat.14001. [PubMed] [Cross Ref]
54. Papanas N., Maltezos E. Oral antidiabetic agents: Anti-atherosclerotic properties beyond glucose lowering? Curr. Pharm. Des. 2009;15:3179–3192. doi: 10.2174/138161209789057995. [PubMed] [Cross Ref]
55. Katakami
N., Kaneto H., Matsuhisa M., Shimomura I., Yamasaki Y. Effects of
glimepiride and glibenclamide on carotid atherosclerosis in type 2
diabetic patients. Diabetes Res. Clin. Pract. 2011;92:e20–e22. doi: 10.1016/j.diabres.2010.12.023. [PubMed] [Cross Ref]
56. Mackinnon
A.D., Jerrard-Dunne P., Sitzer M., Buehler A., von Kegler S., Markus
H.S. Rates and determinants of site-specific progression of carotid
artery intima-media thickness: The carotid atherosclerosis progression
study. Stroke. 2004;35:2150–2154. doi: 10.1161/01.STR.0000136720.21095.f3. [PubMed] [Cross Ref]
57. Koyasu
M., Ishii H., Watarai M., Takemoto K., Inden Y., Takeshita K., Amano
T., Yoshikawa D., Matsubara T., Murohara T. Impact of acarbose on
carotid intima-media thickness in patients with newly diagnosed impaired
glucose tolerance or mild type 2 diabetes mellitus: A one-year,
prospective, randomized, open-label, parallel-group study in Japanese
adults with established coronary artery disease. Clin. Ther. 2010;32:1610–1617. [PubMed]
58. Odell P.M., Anderson K.M., Kannel W.B. New models for predicting cardiovascular events. J. Clin. Epidemiol. 1994;47:583–592. doi: 10.1016/0895-4356(94)90206-2. [PubMed] [Cross Ref]
59. Tunstall-Pedoe
H., Kuulasmaa K., Mähönen M., Tolonen H., Ruokokoski E., Amouyel P.
Contribution of trends in survival and coronary-event rates to changes
in coronary heart disease mortality: 10-year results from 37 WHO MONICA
project populations. Monitoring trends and determinants in
cardiovascular disease. Lancet. 1999;353:1547–1557. doi: 10.1016/S0140-6736(99)04021-0. [PubMed] [Cross Ref]
60. Salonen
R., Nyyssönen K., Porkkala E., Rummukainen J., Belder R., Park J.S.,
Salonen J.T. Kuopio Atherosclerosis Prevention Study (KAPS). A
population-based primary preventive trial of the effect of LDL lowering
on atherosclerotic progression in carotid and femoral arteries. Circulation. 1995;92:1758–1764. doi: 10.1161/01.CIR.92.7.1758. [PubMed] [Cross Ref]
61. Baldassarre
D., Nyyssönen K., Rauramaa R., de Faire U., Hamsten A., Smit A.J.,
Mannarino E., Humphries S.E., Giral P., Grossi E., et al. IMPROVE study
group. Cross-sectional analysis of baseline data to identify the major
determinants of carotid intima-media thickness in a European population:
The IMPROVE study. Eur. Heart J. 2010;31:614–622. doi: 10.1093/eurheartj/ehp496. [PubMed] [Cross Ref]
Articles from International Journal of Molecular Sciences are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)