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Original Studies |
Joslin Diabetes Center (S.C.L., A.E.C., E.M.B., F.M.B., E.S.H.), Microcirculation Laboratory (P.S., F.W.L., A.V.), Department of Medicine (A.V.), and Division of Vascular Surgery (S.A., F.W.L.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Address correspondence and requests for reprints to: Aristidis Veves, M.D., Microcirculation Laboratory, Palmer 317, Beth Israel Deaconess Medical Center, West Campus, One Deaconess Road, Boston, Massachusetts 02215. E-mail: aveves{at}caregroup.harvard.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The biological mechanisms whereby estrogen exerts its cardiovascular protection is complex and not fully understood (4). A favorable change in lipoprotein profile associated with the use of estrogen was initially thought to be the major determining factor. However, it soon became clear that any alteration in lipoprotein profile could explain only 30 to 50% of the vascular benefit of estrogen (5, 6). Therefore, estrogen is likely to confer its cardioprotection via mechanisms beyond lipoprotein metabolism. These mechanisms include inhibition of platelet aggregation, vasodilation, antioxidation, antiproliferation of vascular smooth muscle cells (VSMC), and the enhancement of endothelial nitric oxide (NO) synthase expression and, hence, NO production (7). The endothelium plays a key role in the homeostasis of most of the above processes. Hence, it is likely that estrogen exerts its cardiovascular benefits through the regulation of endothelial function, which is now regarded as pivotal against atherogenesis (8).
Women with diabetes are at high risk for cardiovascular disease (9). Thus, it is important to know whether estrogen can confer on them the same cardioprotection as in nondiabetic women. Epidemiological data reveal that diabetic women have cardiovascular risk very similar to that of nondiabetic men (10). This would suggest that women with diabetes are denied the cardioprotection associated with estrogen. However, there are little data looking specifically at whether HRT can reduce the risk for cardiovascular disease in postmenopausal diabetic women. Our group had previously shown that in healthy women endothelial function deteriorates after menopause and HRT significantly ameliorates this dysfunction (11). We now undertake to investigate the effect of menopausal status and HRT on the endothelial function of healthy women and women with type 2 diabetes.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We included a total of 97 subjects, age 38 to 70 yr, who were
divided in six groups (Table 1
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first group consisted of 28 healthy premenopausal women, and the second
group had 16 premenopausal women with type 2 diabetes mellitus. A
premenopausal status was defined as having regular menstrual cycles
without taking any form of hormonal treatment in the preceding 6 months
of the study. The third group consisted of 12 healthy postmenopausal
women, and the fourth group had 17 postmenopausal women with type 2
diabetes mellitus. A postmenopausal status was defined as having
complete cessation of menstrual cycle for at least a year before entry
into the study. The fifth group included 13 healthy postmenopausal
women on HRT, and the sixth group had 11 postmenopausal women with type
2 diabetes mellitus receiving HRT. Women in the fifth and sixth groups
were considered postmenopausal if they had complete cessation of menses
for at least a year before initiation of HRT and had remained on HRT
for at least a year. Alternatively, they might not have a history of
complete cessation of menses but had been on HRT for at least a year
for significant peri-menopausal symptoms. All the healthy subjects had
a normal oral glucose tolerance test before entry into the study. The
diabetic individuals had the diagnosis of type 2 diabetes established
before they were screened for the study and did not require insulin
therapy for glucose control. Diabetes and normal glucose tolerance were
defined according to the recommendations of the American Diabetes
Association Expert Committee on the Classification and Diagnosis
of Diabetes (12).
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To avoid confounding factors known to affect endothelial function and/or glucose metabolism, the following strict exclusion criteria were applied to subjects in all groups: smoking any amount of cigarettes during the previous 6 months, subjects with a past history of cardiovascular disease (CAD, arrhythmia, CHF), stroke or transient ischemic attack, peripheral vascular disease, chronic renal disease (serum creatinine >1.5 mg/dL), severe dyslipidemia [serum triglyceride (TG) >600 mg/dL or cholesterol >300 mg/dL], or any other serious chronic disease requiring active treatment. Subjects were also excluded if they were on any of the following medications: any type of antihypertensives, lipid-lowering agents, glucocorticoids, antineoplastic agents, psychoactive agents, or bronchodilators. Premenopausal women on birth control pills were also excluded from the study. In addition, diabetic patients with proliferative retinopathy, peripheral somatic neuropathy, and/or macroalbuminuria (expressed as albumin to creatinine ratio, >300 µg/mg) were excluded from the study.
This study was a collaborative effort between the Clinical Research Center of the Joslin Diabetes Center and the Department of Medicine and Division of Vascular Surgery, Beth Israel Deaconess Medical Center (Boston, MA). The protocol was approved by the ethics committee or institutional review board at each center, and all participants gave written informed consent. Volunteers for the study were recruited through local advertisement at the Joslin Diabetes Center and The Beth Israel Deaconess Medical Center.
Methods
Subjects were studied after an overnight fast and a 24-h period of abstinence from alcohol and vigorous exercise. Women on HRT were requested to take their regular dose of HRT on the morning of vascular reactivity examination. A standard 75-g oral glucose tolerance test was performed in those individuals without a known history of diabetes to evaluate their glucose status. Eligible individuals were asked to come back for a second visit to the Joslin Clinical Research Center after an overnight fast of 12 h to perform the clinical and laboratory evaluations. A general physical examination was performed by a physician in the study. The diagnosis of proliferative retinopathy was made on the basis of clinical examination or a history of previous retinal laser treatment. The SBP and DBP readings were recorded to the nearest 2 mm Hg as the mean of two measurements with the subjects seated. The subjects’ weight, height, and waist to hip ratio (WHR) were also obtained. The body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters.
Blood samples were drawn from an antecubital vein with a 19-gauge needle without venous stasis. Plasma glucose, total serum cholesterol, and TG were measured using the Synchron CX analyzer (Beckman Coulter, Inc., Systems), whereas high-density lipoprotein (HDL) serum cholesterol was measured directly (Sigma Diagnostics). Low-density lipoprotein (LDL) cholesterol levels were calculated using the Friedwald formula. The HbA1c (normal range, 4–6%) was determined in whole blood using ion-exchange high-performance liquid chromatography. Plasma insulin and estradiol were measured using the RIA method. Soluble intercellular adhesion molecule (sICAM; R&D Systems, Minneapolis, MN) was measured in the plasma of those women with type 2 diabetes in duplicate using the enzyme-linked immunoassay method.
All vascular reactivity measurements were performed on the same morning as the clinical evaluation while the subjects were still fasting. The investigators (PS and SCL) who performed the measurements were blinded to the medical history of the subjects. The endothelial function of the superficial cutaneous circulation at the forearm level was evaluated by performing laser Doppler perfusion imaging measurements before and after the iontophoresis of acetylcholine (Ach) and sodium nitroprusside (NaNP). This is a noninvasive technique that avoids any systemic effects of the used drugs. Ach chloride was used to assess endothelium-dependent vasodilation because its main effect is to stimulate endothelial cell production and/or release of NO. NaNP was used to assess endothelium-independent vasodilatation because it directly relaxes VSMC, "bypassing" endothelial cell generation of NO.
We used the MIC1 iontophoresis system (Moor Instruments Ltd., Millwey, Devon, England). Specifically, the iontophoresis chamber was filled with a small quantity (<1 mL) of 1% Ach chloride solution and a constant current of 200 microampere was applied for 60 sec, achieving a dose of 6 mC/cm+2. The changes in the superficial cutaneous blood vessel perfusion were assessed before and after the iontophoresis of Ach by a laser Doppler Perfusion Imager (Lisca PIM 1.0; Lisca Development AB, Linkoping, Sweden). The imager uses a 1 mW Helium-Neon laser beam of 633 nm wavelength, which sequentially scans the skin area where iontophoresis is performed. The maximum number of measured spots is 4096, and the apparatus produces a color-coded image of skin erythrocyte flux on a computer monitor. The same procedure was performed using nitroprusside to assess endothelium-independent vasodilatation. The reproducibility of the technique has been reported previously by our group (13). The coefficient of variation of the baseline measurement was 14.1% and during maximal hyperemic response after the iontophoresis 13.7%.
Data analysis
The Minitab statistical package (version 12.0; Minitab Inc., State College, PA) for personal computers was used for the statistical analysis. For parametric data, the ANOVA test was used, followed by the Fisher’s exact test for pair-wise comparison among the various groups. For nonparametric data, the Kruskal-Wallis test was used. Pearson correlation was calculated for the microvascular reactivity and the following parameters: age, SBP and DBP, BMI, WHR, fasting blood glucose (FBG), fasting insulin, HBA1c, total cholesterol, HDL, LDL, and TG. Stepwise regression analysis was performed to determine the relationship between vascular response to Ach and NaNP and all the above-mentioned parameters. Subsequently, multiple linear regression analysis was carried out to assess the overall contributions of these parameters toward the variation in microvascular reactivity. A P = 0.05 or less is considered statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. The duration of diabetes was similar
in all three diabetic groups. BMI was higher in the premenopausal
diabetic women and postmenopausal diabetic women on HRT when compared
with the premenopausal healthy women and postmenopausal healthy women
on HRT, respectively (P < 0.001). The SBP was higher
in premenopausal diabetic women and postmenopausal diabetic women when
compared with the premenopausal healthy women and postmenopausal
healthy women, respectively (P < 0.001). The DBP was
higher in the premenopausal diabetic women when compared with the
premenopausal healthy women respectively (P < 0.001).
However, all the subjects studied were normotensive.
The results of biochemical tests are shown in Table 2. The HBA1c, fasting plasma glucose and
TG levels, were higher in the premenopausal diabetic women,
postmenopausal diabetic women and postmenopausal diabetic women on HRT
when compared with the premenopausal healthy women, postmenopausal
healthy women and post-menopausal healthy women on HRT, respectively
(P < 0.001). Total cholesterol and LDL cholesterol
were higher in the premenopausal diabetic women compared with the
premenopausal healthy women (P < 0.001).
Microalbuminuria (albumin to creatinine ratio, 30–300 µg/mg) was
present in five diabetic patients, and their distribution in each group
is as shown in Table 2.
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and Fig. 1, A and B. The percentage increase in
blood flow over baseline after the iontophoresis of Ach (which causes
endothelium-dependent vasodilation) was higher in the premenopausal
healthy women and postmenopausal healthy women on HRT when compared
with the premenopausal diabetic women and postmenopausal diabetic women
on HRT, respectively (P < 0.001; Fig. 1A). Similarly,
the percentage increase in perfusion after the iontophoresis of NaNP
(which causes endothelium-independent vasodilation) was higher in the
premenopausal healthy women and postmenopausal healthy women on HRT
when compared with the premenopausal diabetic women and postmenopausal
diabetic women on HRT, respectively (P < 0.001; Fig. 1B). Among the healthy women, the premenopausal group and
postmenopausal on HRT group had significantly better
endothelium-dependent and -independent vasodilation when compared with
the group that was postmenopausal without HRT (P <
0.001; Fig. 1, A and B). However, among the diabetic women, the
endothelium-dependent and -independent vasodilation of those who were
premenopausal and those who were postmenopausal on HRT were not
significantly different from those who were postmenopausal without
HRT.
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shows the relationship between
microvascular reactivity and various clinical and metabolic parameters.
The response to Ach was significantly and inversely correlated with
age, SBP, DBP, BMI, FBG, fasting insulin, HBA1c, total cholesterol, LDL
and TG whereas the response to NaNP was significantly and inversely
correlated with age, SBP, BMI, WHR, FBG, fasting insulin, HBA1c, and
LDL. In addition, response to NaNP was positively correlated with HDL
(P < 0.05). On stepwise regression analysis, HBA1c and
age contributed to the variation in the response to Ach by 16% and
7%, respectively, whereas HBA1c, HDL, DBP, and age contributed to the
variation in the response to NaNP by 27%, 8%, 7% and 6%,
respectively. The addition of all of these factors (age, SBP, DBP, BMI,
WHR, FBG, fasting insulin, HBA1c, total cholesterol, HDL, LDL, and TG)
in the model increased the prediction of variation in response to Ach
and NaNP to 32.2% and 55.1%, respectively.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our first finding is in agreement with the epidemiological studies that have demonstrated that women with diabetes are at similar risk for cardiovascular diseases compared to men, thereby suggesting that the diabetic women are denied of the cardioprotection associated with estrogen (10, 17). The reason whereby women with diabetes lose their gender advantage is unclear. Diabetes is associated with dyslipidemia, increased oxidative stress, diminished NO production, enhanced monocyte adhesion, and increased platelet aggregation (18, 19, 20, 21, 22) Moreover, hyperglycemia and obesity/insulin resistance (which are hallmarks of type 2 diabetes) both have been associated with endothelial dysfunction (23, 24).
In the present study, the healthy women and diabetic women were not exactly matched in various aspects of clinical and metabolic parameters. We felt that by matching the healthy subjects and diabetic women in parameters, such as degree of obesity or blood pressure, we may inadvertently include in the control group some subjects who were not strictly normal but had some features of the insulin-resistant syndrome, although they were not diabetic as yet. However, to assess the importance of these metabolic factors toward the variation in microvascular reactivity, we have used stepwise regression analysis. Our results showed that only HBA1c and age contributed significantly (16% and 7%, respectively) to the variation in vascular response to Ach. Similarly, only HBA1c, HDL, DBP, and age contributed significantly (27%, 8%, 7%, and 6%, respectively) to the variation in vascular response to NaNP. Moreover, when we included all the relevant parameters in a multiple linear regression model, only up to 32.2% and 55.1% of the variation in vascular response to Ach and NaNP, respectively, could be accounted for. Therefore, these data indicate that difference in BMI, lipids, and other metabolic factors were not responsible for the observed results and that the presence of diabetes was probably the main factor responsible, whereas the existence of other contributing factors, including genetic variability, cannot be excluded (25).
Our study also showed that the use of HRT among the postmenopausal women with diabetes was associated with lower serum sICAM levels, thereby suggesting an attenuation in endothelial activation. In recent years, association between elevated sICAM levels and risk of future myocardial infarction has been demonstrated in apparently healthy men (26). Furthermore, investigators have also shown that the mean intimal-carotid thickness of the common carotid artery and carotid bifurcation are positively correlated with circulating sICAM levels among both men and women (27). Therefore, it is possible that HRT may have a potential benefit in reducing the cardiovascular risk of postmenopausal women with diabetes.
In the healthy women, postmenopausal status was associated with a reduction in vascular reactivity that could be improved with HRT. A number of studies have, likewise, demonstrated a positive effect of estrogens on the endothelial function of postmenopausal healthy women (11, 14). Therefore, an improvement in endothelial function may be one of the mechanisms whereby estrogen confers its cardiovascular benefits on postmenopausal healthy women. On the other hand, there was a trend to suggest that, to a lesser degree, HRT might also improve the vascular reactivity of postmenopausal diabetic women. However, this modest improvement did not reach statistical significance due to large interindividual variation in the measurement of vascular reactivity and inadequate sample size. A recent preliminary study suggested that a relative risk reduction for CHD in current users of HRT in the diabetic women are comparable with those seen in nondiabetic women (28). Therefore, additional studies would be required to investigate whether a less substantial degree of improvement in endothelial function with HRT could translate into a clinically meaningful reduction of cardiovascular risk in the diabetic women.
Impairment of both microvascular endothelium-dependent and -independent vasodilation was found in both postmenopausal healthy women and women with type 2 diabetes. Hence, vascular dysfunction in these women was not limited to the endothelium, but also involved the vascular smooth muscle cells. It has been suggested that the abnormal response to endogenous and exogenous NO donors implicate either increased inactivation of NO by reactive oxygen species or abnormalities of signal transduction in the guanylate cyclase pathway (29). This pattern of vascular dysfunction is also consistent with a recent study in which the macrovascular (brachial artery) response to flow-mediated endothelium-dependent dilation and sublingual nitroglycerin were both impaired in adults at risk for atherosclerosis (30). However, not all investigators have found impaired endothelium-independent vasodilation in postmenopausal women. For instance, Pinto et al. (31) reported selective impairment of endothelium-dependent vasodilation, which was restored with estrogen replacement in a group of women who received ovariectomy. In this group of women, endothelium-independent vasodilation was unaffected (31). Hence, additional studies would be necessary to clarify this issue.
There were three main limitations in this study. First, there was
considerable variability in the HRT regimens used by the participants.
Hence, our study was not able to ascertain whether a particular form of
HRT regimen was superior in terms of improving endothelial function.
Moreover, although the participants on HRT were requested to take their
regular HRT on the day of examination, we were unable to ascertain
whether the subjects adhered strictly to this instruction. These
factors might potentially confound our comparisons of vascular
reactivity among healthy and diabetic women on HRT. However, the
subjects on HRT were apparently estrogenized to a similar degree
because the serum estradiol levels among the healthy and diabetic
subjects on HRT were similar to each other and very close to those
subjects who were pre-menopausal (Table 2). Therefore, the variability
in HRT regimens did not affect the degree of estrogenization of the
participants to a large extent and, thus, would not invalidate our
observation. Second, it was appealing to assume that impaired vascular
reactivity would translate clinically into greater cardiovascular risk.
However, this relationship has not been firmly established by any
large-scale, long-term prospective study. Finally, the cross-sectional
design of this study could not rule out possible selection bias of
study subjects, which might account for some of our observations.
Therefore, a future prospective randomized study may provide further
insights into the effect of HRT on the vascular reactivity of healthy
women and diabetic women.
In summary, menopausal status and type 2 diabetes were associated with impaired endothelial-dependent and independent-vasodilation. HRT significantly improved the microvascular reactivity in postmenopausal healthy women. In contrast, the effect of HRT on the microvascular reactivity of postmenopausal diabetic women was less apparent, and the precise magnitude of the benefits requires further investigation. However, HRT seemed to be capable of attenuating endothelial activation in postmenopausal women with type 2 diabetes.
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Acknowledgments |
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Footnotes |
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Received March 10, 1999.
Revised August 5, 1999.
Accepted August 16, 1999.
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References |
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