help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1357
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajasingh, J.
Right arrow Articles by Kishore, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajasingh, J.
Right arrow Articles by Kishore, R.
Endocrinology Vol. 148, No. 8 3618-3624
Copyright © 2007 by The Endocrine Society

BRIEF COMMUNICATION

Enhanced Voluntary Alcohol Consumption after Estrogen Supplementation Negates Estrogen-Mediated Vascular Repair in Ovariectomized Mice

Johnson Rajasingh, Evelyn Bord, Gangjian Qin, Masaaki Ii, Marcy Silver, Hiromichi Hamada, Deepali Ahluwalia, David Goukassian, Yan Zhu, Douglas W. Losordo and Raj Kishore

Division of Cardiovascular Research, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135

Address all correspondence and requests for reprints to: Raj Kishore, Ph.D., Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: r-kishore{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Preclinical and observational studies in ovariectomized (OVX) animals and pre- and postmenopausal women, respectively, have suggested the cardioprotective effects of estrogen replacement therapy. However, randomized clinical trials have not confirmed estrogen-mediated cardioprotection. Although uncertainties about the duration and optimal type of estrogen replacement regimen might explain the disparity, other factors that may mask the protective effects of 17ß-estradiol (E2) on cardiovascular outcome need scrutiny. Increased ethanol consumption may be one such factor. We examined the effect of E2 supplementation on ethanol consumption in OVX mice and the effect of ethanol consumption on E2-mediated vascular repair, in vivo. OVX mice implanted with E2 pellets consumed significantly more ethanol, compared with those receiving placebo pellets. E2-induced increase in ethanol consumption was not affected by the absence of either estrogen receptor-{alpha} or -ß. Reendothelialization after carotid artery denudation was repressed, and neovascularization in ischemic hind limbs was blunted in mice consuming ethanol, despite E2 supplementation. In vitro, ethanol dose-dependently attenuated E2-induced endothelial cell (EC) proliferation and tube formation activity and enhanced EC apoptosis, suggesting that ethanol blocks E2-induced EC survival and function. Taken together our data suggest that increased ethanol consumption after E2 supplementation blunts the beneficial effects of E2 on EC function and that novel approaches to estrogen replacement for cardioprotection may benefit from the control of alcohol consumption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVER THE LAST four decades, epidemiological and case-controlled studies, showing favorable biological changes in cardiovascular risk factors with estrogen supplementation, have suggested that estrogen replacement therapy in postmenopausal women might be beneficial in terms of prevention of adverse cardiovascular events (1, 2). A large body of stringent, experimentally controlled animal studies has irrefutably shown the beneficial effect of estrogen [17ß-estradiol (E2)] in several cardiopathological models (3, 4, 5). However, recent data from large randomized clinical trials of primary Women’s Health Initiative or secondary Heart and Estrogen-Progestin Replacement Study prevention (6, 7) have not confirmed cardioprotection with hormone replacement therapy.

Although uncertainties about the timing of initiation, duration, and optimal type of hormone replacement therapy regimen might explain some of the disparity between randomized clinical trials vs. the clear benefits of estrogen in observational and animal studies, other factors that may mask the beneficial, protective effect of estrogen on cardiovascular outcome need to be considered. Interestingly, several studies in pre- and postmenopausal women have reported a correlation between levels of circulating estradiol to increased ethanol consumption (8, 9, 10). Data from well-controlled animal studies are even more striking. A number of experimental studies have shown that E2 dramatically increases the appetite for alcoholic beverages in both ovariectomized (OVX) and intact animals (8, 11, 12, 13, 14, 15). Whether observed increase in OVX animals, supplemented with exogenous estrogen, interferes with well-reported positive effects of estrogen on ischemic neovascularization in many animal models has not been studied and warrants further inquiry. In the present study, we report that increased intake of ethanol in OVX mice, which occurs after E2 replacement, results in significant attenuation of the beneficial effects of E2 on endothelial recovery after carotid artery injury. We also report that enhanced ethanol consumption leads to the inhibition of physiological blood flow recovery and neovascularization in a critical hind limb ischemia model. Additionally, we report that simultaneous exposure of endothelial cells (ECs) to ethanol and E2, in vitro, blunts E2-induced EC proliferation and tubulogenesis and enhances EC apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Eighty female BALB/c (Jackson Laboratory, Bar Harbor, ME) mice 8–10 wk of age were used in the initial part of the study. We started with BALB/c strain due to our previous observations that C57BL/6J mice have an accelerated recovery in hind limb ischemic model and that the physiological effect of E2 replacement and/or ethanol consumption might be too small to measure in B6 mice. Additionally, 20 estrogen receptor (ER)-{alpha} and 20 ER-ß null mice and 20 age- and sex-matched wild-type (WT) mice of the same genotype (C57BL/6J) were also used. Both ER-{alpha} and ER-ß mice are available and bred at our animal facilities. All animal procedures were performed in accordance with Caritas St. Elizabeth’s Institutional Animal Care and Use Committee.

Ovariectomy, E2 supplementation, and ethanol feeding
Mice were anesthetized by ip injection of 150 mg/kg ketamine and ovariectomized and received either 90-d-release E2 pellets (1.7 mg E2, release rate of approximately 188 pg/d; Innovative Research of America, Sarasota, FL) or placebo-containing pellets, implanted sc into the backs of the animals 1 wk after the ovariectomy. This dose was used to achieve levels in the upper range of those occurring in nonpregnant premenopausal humans at midcycle and in the luteal phase of the normal menstrual cycle. The circulating estrogen levels achieved by this dose in experimental mice were therefore 4 times higher than physiological levels in mice (4, 5). Animals were divided into four groups: 1) placebo with no access to ethanol; 2) E2 and no ethanol access; 3) placebo and choice of 10% ethanol solution or water in a two-bottle system; and 4) E2 and choice of 10% ethanol solution or water in a two bottle system. One group of mice implanted with either placebo or E2 pellets was given immediate access to 10% ethanol for 6 wk and consumption was recorded every 24 h. Body weights of each mouse in the two groups were recorded twice per week. Each mouse was housed in separate cage for 12 h each of light/dark cycle. Bottles were weighed before and after a 24-h drinking cycle to calculate the grams of ethanol consumed in preceding 24 h (to the one decimal point). Control bottles (water) were also weighed similarly for measuring water consumption, which did not differ significantly between groups. In this feeding paradigm, it was assumed that the loss of liquids due to the dripping would be similar between ethanol- and/or water-containing bottles. Mean ethanol consumption by each mouse over the course of each week was averaged and adjusted to the body weight to derive mean ethanol consumption per week in grams per kilogram of body weight for each mouse. Data obtained from individual mouse for the course of 6 wk were pooled to calculate ethanol consumption as a group. Ethanol- or water-containing bottles were frequently switched to avoid side preference by mice. Circulating blood ethanol levels were monitored weekly in each animal in the blood obtained from tail vein, using commercially available kit (Sigma, St. Louis, MO).

Mouse carotid injury and reendothelialization
Six weeks after E2 or placebo pellet implantation and ethanol feeding, carotid artery injury (denudation of endothelial layer) surgery was performed in 10 mice in each of the four groups mentioned above. Carotid injury was performed essentially as described previously from our laboratory (4). Surgery was performed using a dissecting microscope. Briefly, mice were anesthetized with an ip injection of ketamine (45 mg/kg body weight). The bifurcation of the left external carotid artery was surgically exposed via a midline incision on the ventral aspect of the neck. Size 6–0 surgical silk sutures are placed around the common carotid, internal and external carotid arteries to temporarily restrict the flow of blood to the area of surgical manipulation. The artery was injured using a .014-in.-diameter flexible angioplasty wire (ACS, Temecula, CA) modified to create a barotraumatic/stretch injury, which was introduced into the external carotid artery and advanced to the common carotid artery. The wire was advanced and withdrawn three times to ensure a reliable effect. The wire was removed from the artery, the external carotid artery permanently ligated, and the temporary ligatures released to allow blood flow to be restored through the internal carotid artery. The connective tissue of the subcutis was closed with interrupted 6–0 absorbable sutures and skin closed with interrupted 6–0 silk sutures. Antibiotics (0.1 ml sc; Di-trim, Orion, Finland) were given immediately postoperatively via sc injection. Seven days after injury and before mice were killed, arteries were perfused with 0.5% Evans Blue dye. Area of arteries stained with Evans Blue was considered to be reendothelialized. Excised arteries were cut open to expose endothelial layer and subjected to microscopy to evaluate and quantify the reendothelialized area, essentially as previously described (4). The ratio between the area stained in blue and the total carotid artery area was calculated. The surface of the area that remained deendothelialized was indexed to the total carotid artery area to take into account the changes in vessel area due to both the elasticity of the carotid artery and the flattening of the vessel between slides.

Hind limb ischemia, laser Doppler perfusion imaging, and histology
Hind limb ischemia was established at the end of 6 wk of ethanol feeding in the remaining 10 mice in each group, as described before by the excision of left femoral artery (16). Briefly, mice were anesthetized with an ip injection of ketamine. Under aseptic conditions, a 5-mm incision was made on the left thigh region. A ligation was made around the femoral artery and all arterial branches were removed. A small segment of the artery was then dissected free. The connective tissues of the subcutis were closed with interrupted 6-0 absorbable sutures and the skin closed with wound clips. Using well-established laser Doppler perfusion imaging, the blood flow recovery in the ischemic limb was measured and ratio of perfusion in the ischemic limb to that of control noninjured limb was plotted, before and immediately after surgery (before and after surgery, d 0) and at d 7 after surgery (red color denotes high blood flow and purple no blood flow). On d 7, mice were killed; tissues were harvested, fixed, and sectioned. Sections were subjected to immunochemical staining with anti-CD31 antibodies to identify and quantify capillary density. Similar hind limb surgical procedures were carried out in ER-{alpha} and -ß null mice and genotype-matched C57BL/6J WT mice.

Cell culture
Human umbilical vein endothelial cells (HUVECs) were cultured as described (16). For synchronization, cells were serum starved for 48–72 h. For ethanol exposure, cells were cultured in the presence of indicated concentrations of ethanol for 48 h. Cells were treated with indicated doses of E2.

Thymidine uptake assays
Cell proliferation was assayed by tritiated-thymidine uptake assays as described previously (16, 17). Briefly, cells were rendered quiescent by culturing in low serum (0.2%) for 48–72 h and were further exposed or not to different concentrations of ethanol (1–25 mM) for 48 h in complete medium supplemented with E2 (10–8 M). Eighteen hours before harvest, cells were pulsed with 1µCi of 3H-thymidine/well.

Tube formation assay
HUVECs were preexposed to 25 mM ethanol with or without 10–8 M E2 for 48 h. Cytokine-reduced matrigel matrix (300 µl; Becton Dickinson, Lincoln Park, NJ) was used to coat each well of a four-well glass slide. Control and treated HUVECs (50,000 cells/slide) were plated on matrigel chambers and tube formation was assessed after 5 h using light microscope. Two investigators in a blinded fashion observed tubes in 10 different 1-mm2 areas. Morphological changes in tube structures were recorded and photographed.

Apoptosis
Immunoflurorescence staining was performed to determine HUVEC apoptosis [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining kit; Roche Diagnostics, Indianapolis, IN], essentially as described earlier (18) and following the manufacturer’s instructions.

Statistical analyses
Results are presented as means ± SEM. Ethanol consumption comparisons were done by two-way ANOVA (factor groups and weeks) (GB-STAT, Dynamic Microsystem Inc., Silver Spring, MD), followed by Scheffé’s post hoc analysis. Statistical differences in blood ethanol concentrations (BEC), body weight, percent reendothelialization, and hind-limb perfusion were calculated by Student’s t test. All tests were two sided and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 supplementation increases ethanol consumption in OVX mice
Mice receiving E2 pellets consumed significantly more ethanol consistently over the 6-wk period than those receiving placebo (Fig. 1AGo). Analysis of data for E2 vs. placebo factor revealed the following; F (1, 49) = 12.8, P < 0.01). Mice receiving E2 pellet consumed increasing amount of ethanol at each of the following weeks (average 1.1 g/kg at wk1 to an average of 2.8 g/kg at wk 5), whereas the consumption of ethanol by mice receiving placebo remained the same at each week [average 1.03 g/kg; F (1, 24) = 9.33, P < 0.01]. The increased consumption of ethanol in mice implanted with E2 was also reflected in higher BEC, compared with those mice that were implanted with placebo pellets (an average of 67 mg/dl in E2 group vs. 26 mg/dl in placebo group; P < 0.01 E2 vs. placebo). The BEC values showed a similar trend as was seen with the ethanol consumption data, i.e. the BEC values increased over the course of each week. For example, the BEC values after 1 and 5 wk ethanol consumption were an average of 31 and 135 mg/dl (P < 0.001) in the E2 group and 21 and 26.5 mg/dl, respectively, in the placebo group. This trend in higher blood ethanol concentrations thus correlated with the grams per kilogram consumption data (r = 0.92, n = 20, P < 0.01), suggesting that increased consumption of ethanol by E2 group of mice was not due to extra spillage or evaporation of ethanol from the feeding bottles. Figure 1BGo shows the average weekly weight of mice in the two groups. The E2 group of mice displayed a lower body weight only in the first 2 wk (P < 0.05 placebo vs. E2) after which the difference in body weights was nonsignificant. These data corroborated the observations from other studies indicating a stimulatory role for E2 in inducing increased voluntary alcohol consumption.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 1. OVX mice supplemented with estradiol consume more ethanol. Female BALB/c mice 8–10 wk of age were OVX and implanted with either E2 pellets or a placebo pellet and were given access to both water and 10% ethanol in two bottle drinking paradigm. A, Consumption of ethanol (expressed as grams per kilogram of body weight) by each mouse was recorded each day and was averaged over each week. Mice supplanted with E2 consumed more ethanol, compared with those receiving placebo (placebo vs. E2, P < 0.05 for wk 1 and 2, P < 0.01 for wk 3–6). B, Body weight was recorded twice a week and averaged over each week. Mice receiving E2 weighed significantly less than those receiving placebo only for first 2 wk (P < 0.05, E2 vs. placebo).

 
Increased ethanol consumption inhibits E2-mediated enhanced reendothelialization in injured carotid arteries and neovascularization in the ischemic hind limbs in OVX mice
Ten mice from each group (placebo/no ethanol; E2/no ethanol; placebo/ethanol; E2/ethanol) were subjected to carotid artery denudation and reendothelialization (ReEndo) was assessed on d 7 by Evans Blue staining. As shown in representative Fig. 2AGo and quantified in Fig. 2BGo, ReEndo was significantly reduced in animals receiving placebo and ethanol than those receiving placebo but no ethanol (percent ReEndo = 23 vs. 56%, respectively; P < 0.05). ReEndo was almost complete in mice receiving E2 without ethanol (90%); however, ReEndo was significantly inhibited in mice implanted with E2 and having access to ethanol (percent ReEndo = 78% in the E2 group vs. 21% in the E2+EtOH group; P < 0.001). These data suggest that higher consumption of ethanol by E2-implanted mice negates the enhanced endothelial recovery observed in mice without access to ethanol.


Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		FIG. 2. Increased ethanol consumption impairs E2-induced endothelial recovery in denuded carotid arteries. A, Mice from each group were subjected to carotid artery denudation and reendothelialization was examined 7 d after injury by Evans Blue staining. Representative images of Evans Blue-stained carotid arteries from each group of mice are shown. B, Quantification of percent endothelial recovery in each group of mice. ReEndo was significantly reduced in animals receiving E2 and consuming ethanol than those receiving E2 but no ethanol (P < 0.001). Animals receiving placebo and having access to 6 wk alcohol drinking also showed delayed ReEndo, compared with those with placebo but no access to ethanol (P < 0.05).

 
Enhanced ethanol consumption blunts E2-mediated blood flow recovery and neovascularization in the ischemic hind limbs
Next we examined the physiological effect of ethanol consumption on neovascularization in a critical hind limb ischemia model. As shown in representative perfusion imaging pictures in Fig. 3AGo and quantified in Fig. 3BGo, compared with mice implanted with placebo, perfusion on d 7 after surgery in the ischemic hind limb was significantly higher in mice implanted with E2 pellets (P < 0.01 placebo vs. E2 groups). However, perfusion in the ischemic hind limbs of mice receiving both E2 and ethanol was significantly lower, compared with the E2-alone group (P < 0.05). The reduction in perfusion by ethanol, despite E2 supplementation, was further corroborated by the reduction in capillary density in the ischemic hind limbs of mice in E2+ethanol group, compared with the E2 group alone (Fig. 3CGo, P < 0.01). These studies indicate that E2-mediated enhanced ethanol consumption in mice negate the beneficial effects of E2 on vascular repair.


Figure 3
View larger version (53K):
[in this window]
[in a new window]

  		FIG. 3. Enhanced ethanol consumption blunts E2-mediated blood flow recovery and neovascularization in the ischemic hind limbs. A, Hind limb ischemia was established in each group of mice by the excision of femoral artery. Perfusion was recorded in ischemic and control limbs by laser Doppler perfusion imaging. Representative images showing blood flow recovery on d 7 after surgery are shown (red color indicates higher perfusion and purple color denotes no perfusion). B, Quantification of blood flow recovery in ischemic limb on d 7 after surgery. Data are plotted as the ratio of perfusion in ischemic limb to nonischemic limb (P < 0.01, placebo vs. E2; P < 0.05, E2 vs. E2/ethanol). C, Capillary density was significantly reduced in the ischemic hind limbs of mice implanted with E2 and consuming ethanol, compared with those receiving E2 alone (P < 0.01, E2 vs. E2/EtOH).

 
E2-induced increase in alcohol consumption is not affected by the loss of either ER{alpha} or ERß
Although previous studies have shown that E2 modulates alcohol consumption, whether ERs play a role in this stimulatory action of E2 is not known. To clarify these issues, ethanol consumption was studied in ER-{alpha} and ER-ß null mice and in age-, gender-, and genotype-matched WT mice (C57BL/6J). Absence of either ER-{alpha} or ER-ß did not affect increase in ethanol consumption in E2 supplemented mice when compared with placebo groups in any genotype (Fig. 4AGo). Over the course of 5 wk, compared with placebo, E2 supplementation led to similar increase in average ethanol consumption (grams per kilogram of body weight) in WT, ER-{alpha}, and ER-ß null mice. The body weights of mice taken twice a week did not significantly differ in any of the genotypes (data not shown). Adjusted for body weight, ethanol consumption was significantly higher in E2-supplemented WT, ER{alpha}-knockout and ERß-knockout mice, compared with those receiving placebo [E2 vs. placebo, F (2, 24) = 8.99, P < 0.01]. Another interesting finding was irrespective of the genotype, each group of mice supplemented with E2 consumed significantly higher amount of ethanol at wk 5, compared with the amount consumed at wk 1 (P < 0.001). The difference between ethanol consumption between placebo and E2-supplemented mice (of C57BL/6J background) was even more striking than that seen in BALB/c mice (Fig. 1Go), suggesting that the background of mice may have a differential preference of ethanol consumption.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 4. E2-induced increment in ethanol consumption in mice occurs despite the loss of either ER-{alpha} or ER-ß. A, WT, ER-{alpha} knockout, and ER-ß knockout mice (n = 20 each) were OVX and implanted with either E2 or placebo pellets (n = 10/group). E2 supplementation-mediated enhancement in ethanol consumption was similar in mice of all genotypes at all time points up to 5 wk (P < 0.01 placebo vs. E2 for each genotype). B, After 5 wk of ethanol feeding, hind limb ischemia was established in each group of mice. Blood flow recovery was determined by laser Doppler imaging and is shown as the ratio of perfusion between ischemic limb to nonischemic limbs at d 7 after surgery (ER-{alpha} null placebo vs. ER-{alpha} null E2, P < 0.01).

 
Mice of all three genotypes, receiving either placebo or E2 (n = 10 for each treatment group) were subjected to hind limb surgery to examine the effect of E2, either alone or in combination with ethanol, on blood flow recovery. E2-induced blood flow recovery in WT mice (P < 0.001 placebo vs. E2) was significantly lower in ER-{alpha} null mice (Fig. 4BGo; P < 0.01; E2/WT vs. E2/ER-{alpha} null mice). Interestingly, E2-mediated blood flow recovery in ER-{alpha} null mice was further blunted when these mice were exposed to ethanol drinking (Fig. 4BGo, P < 0.01). Blood flow recovery responses in ER-ß mice were comparable with those observed in WT mice (P = 0.08).

Ethanol blunts E2-induced EC proliferation, tube formation, and survival
Because significant reduction in postinjury arterial ReEndo and postsurgery recovery from hind limb ischemia in animals supplemented with E2 and consuming higher volumes of ethanol suggested impairment in EC proliferation and survival, we determined the dose effect of ethanol exposure on E2-induced proliferation and survival of HUVECs, in vitro. As shown in Fig. 5AGo, exposure to ethanol dose-dependently inhibited E2-induced HUVEC proliferation. Significant inhibition of proliferation was observed at doses greater than 5 mM. Our previous studies in ECs (17) have shown that 48 h exposure of 25 mM ethanol (a concentration equivalent to the in vivo blood ethanol concentration achieved with 4–6 wk ethanol feeding models) mimics cellular response to long-term alcohol abuse in humans. All further in vitro studies were therefore conducted using 25 mM ethanol exposure for 48 h. We also determined the effect of alcohol exposure on the functional ability of ECs to form tube-like structures, in vitro. HUVECs were exposed or not to 25 mM ethanol with or without 10–8 M E2 for 48 h and were plated on cytokine reduced matrigel matrix (300 µl) four-well glass slide. As shown in Fig. 5BGo, exposure to ethanol significantly altered the morphology and inhibited the tubulogenesis by HUVECs. The number of well-formed tube (square millimeter area) was also significantly reduced (43 ± 2.2 tubes/mm2 in E2-treated vs. 13 ± 1.6 in E2+EtOH treated cells, P < 0.001). Inclusion of E2 in ethanol-treated HUVECs provided some support for tubulogenic activity over ethanol-alone treatment; however, the organization and morphology of tube-like structures was severely impaired in cells exposed to E2+ethanol, compared with E2 alone. Because ethanol exposure to cultured cells is known to induce cell apoptosis, whereas E2 has been shown to protect ECs from apoptosis (4), we also determined whether exposure of HUVECs to ethanol might neutralize antiapoptotic effects of E2. HUVECs were exposed or not to 25 mM ethanol for 48 h with or without 10–8 M E2. TUNEL staining shown in Fig. 5CGo revealed that treatment of HUVECs with 25 mM ethanol for 48 h induced significant apoptosis, and simultaneous treatment of cells with E2 did not rescue the proapoptotic effect of ethanol. Together these data suggest that ethanol negates the beneficial effect of E2 on EC proliferation, survival, and function.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 5. Ethanol impairs E2-mediated EC proliferation, function, and survival. A, Exposure of HUVECs to ethanol dose-dependently inhibited E2-induced proliferation. Significant inhibition of proliferation was observed at doses greater than 5 mM ethanol (E2 vs. E2+ >5 mm EtOH, P < 0.01). B, HUVECs exposed to 25 mM ethanol significantly lose their ability to form tubular structure in matrigel-coated wells, despite E2 treatment. Representative photomicrographs are shown for each treatment group. C, E2 fails to inhibit ethanol-mediated apoptosis in HUVECs. Representative pictures of TUNEL staining are shown. All experiments were conducted at least three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Much of our knowledge of alcoholism and its effect on organ damage has been gathered from studies conducted in males. Recent evidence has revealed that drinking differs between men and women. Moreover, studies of women alcoholics in treatment suggest that they often experience greater physiological impairment including cardiac impairments earlier in their drinking careers, despite having consumed less alcohol than men (19), possibly due to: 1) lower total body water content than men; 2) diminished activity of alcohol dehydrogenase, the primary enzyme involved in the metabolism of alcohol; 3) faster metabolism of alcohol into toxic acetaldehyde; and 4) alcohol’s interactions with hormones that contribute to the pathological drive to consume alcohol. The latter explanation has just begun to emerge, and further research on estrogen-alcohol interactions may provide valuable insight into the mechanisms by which alcohol-seeking behavior and ensuing health problems can be controlled in women. Because the beneficial effects of estrogen on the endothelium generally mirror the effects on cardiovascular events overall, improvement in endothelial function is an important mechanism by which estrogen replacement therapy could provide cardioprotection. At present, little information exists regarding the cross-action of alcohol and estrogen on endothelial function. The data obtained in this study thus provide a critical framework for clinically relevant understanding on the impact of alcohol consumption on E2-mediated enhancement in ischemic neovascularization and endothelial functions.

Several studies in pre- and postmenopausal women reported a correlation between circulating E2 levels to the increased alcohol consumption (8, 9, 10, 20, 21). Several recent epidemiological studies also reported higher alcohol consumption in postmenopausal women using supplemental estrogen/hormone therapy, compared with nonusers (22, 23, 24). These studies, although suggestive of a hormonal role in alcohol consumption, were mostly based on self-reported alcohol consumption and thus subject to bias and essentially not designed to study the estradiol-alcohol connection. On the other hand, well-controlled animal studies seeking a connection between estradiol modulations of alcohol consumption are more striking and conclusive, although molecular mechanisms mediating these changes are yet to be defined. Using various models of controlled, voluntary alcohol intake, a series of studies have shown that in OVX and intact female rodents, estradiol supplementation enhances voluntary alcohol consumption (8, 11, 12, 13, 14, 15). Few studies, however, have not confirmed E2’s stimulatory role in alcohol consumption to a similar extent (14, 15, 25). The discrepancy between these observations may reflect species (rats vs. mice), gender (males vs. females), and variations in estrogen supplementation protocols (single injection, oil, dose of E2, E2 pellets with constant release). Our data indicating that given equal opportunity to drink water and ethanol, OVX mice on E2 supplementation consistently drink more alcohol than those without E2 is in concert with the majority of reported observations. Despite this compelling evidence on E2’s stimulatory role in alcohol consumption, no information regarding pathophysiological effects of E2-enhanced alcohol consumption on cardiovascular disease and endothelial dysfunction is currently available. To the best of our knowledge, our study is the first study to show that enhanced alcohol consumption in mice after estrogen supplementation neutralizes the beneficial effects of estrogen on endothelial and ischemic injury in physiologically relevant models.

Despite the reported cardioprotective effects of low to moderate alcohol intake, clinical, experimental, and epidemiological data have pointed to the relationship between heart disease and higher alcohol consumption (26, 27). Part of the complexity regarding the effect of alcohol on the cardiovascular system may be the result of contrasting influences of light vs. heavy alcohol consumption on the vascular endothelium, with animal studies indicative of favorable effects on endothelial function with low-dose alcohol exposure but the induction of endothelial dysfunction with higher doses (reviewed in Ref. 28). At least part of these aberrations in endothelial function results from modulations in nitric oxide production by ECs (28). Because beneficial effects of E2 on endothelium mainly involve nitric oxide-mediated relaxation and vasodilatation, effects that may be compromised by chronic alcohol exposure result in endothelial dysfunction. Moreover, a model of ischemic injury, in which endothelial proliferation and survival is essential for neovascularization, the combined influence of estrogen and alcohol on endothelial function has yet not been studied. We have earlier shown that exposure to chronic ethanol dose-dependently reduces EC proliferation in vitro (17). Our data thus provide new evidence regarding the effect of ethanol consumption on relevant vascular repair models, in vivo.

Another interesting observation in our study reveals that enhanced ethanol consumption in E2-supplemented mice is not affected by the absence of either ER-{alpha} or ER-ß. These data, however, should be interpreted with caution, given the small sample size in our study. It should be noted that receptor-independent actions of E2 have been also described (29, 30). It is also possible that E2 does change the expression of relevant genes mediating the urge to consume more alcohol by ER-{alpha}/ß, but estrogen receptors other than ER-{alpha}/ß may be involved. This, however, remains speculative and will require systematic experimental evidence.

The beneficial effects of E2 on the endothelium have repeatedly been shown to mirror the effects on cardiovascular events overall. Accordingly, it is apparent that improvement in endothelial function is an important mechanism by which estrogen replacement therapy could provide cardioprotection (3, 4, 5). Recently we have also shown E2 mediated enhancement of the reendothelialization in OVX mice receiving estradiol via mechanisms involving endothelial nitric oxide synthase -mediated endothelial progenitor cell mobilization and inhibition of apoptosis (4). Our data showing that enhanced ethanol intake in OVX mice receiving E2 suppresses the beneficial effect of E2 on endothelial recovery would argue that control on ethanol consumption would be a desirable step to fully realize the beneficial effects of estrogen on endothelial functions. Our data may argue that ethanol’s putative negative effects on endothelial function may persist, even after ethanol consumption is withdrawn, as was the case in our studies in which at the onset of vascular injuries, the ethanol feeding was stopped. It should be noted that end points of in vivo studies were completed on d 7 after injury and evaluation of vascular repair at longer time points may not show significant differences. It is also noteworthy that resident endothelial cells are not the sole participants in vascular repair but endothelial progenitor cells (EPCs) also actively participate in this process. E2 has been shown to mobilize these EPCs from the bone marrow to the circulation and impacts their homing to the area of injury. It may be hypothesized that long-term presence of ethanol in the system compromises the ability of E2 as an EPC-mobilizing agent. The exact functional impact and molecular mechanisms of ethanol actions on EC function after withdrawal are, however, not known, and further experimental evidence to determine the exact mechanisms by which ethanol competes with beneficial effects of E2 on endothelial cells would be required.

In summary, our data provide novel physiologically relevant insights regarding estrogen-alcohol interactions. We hope that these data will provide a critical framework for understanding the role of enhanced alcohol consumption and its impact on E2-mediated benefits on EC function. This body of information could lead in the future to a better understanding of how alcohol’s putative stimulatory actions integrate with those of estrogen and regulate EC gene expression, growth, and function.


   Footnotes
 
This work was supported in part by American Heart Association Grant 0530350N and National Institutes of Health Grant AA14575 (to R.K.).

Current address for J.R., G.Q., D.W.L., and R.K.: Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, Illinois 60611.

Disclosure Summary: All authors have nothing to disclose.

First Published Online May 3, 2007

Abbreviations: BEC, Blood ethanol concentration; E2, 17ß-estradiol; EC, endothelial cell; EPC, endothelial progenitor cell; ER, estrogen receptor; HUVEC, human umbilical vein endothelial cell; OVX, ovariectomized; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; WT, wild type.

Received October 5, 2006.

Accepted for publication April 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grodstein F, Manson JE., Colditz GA, Willett WC, Speizer FE, Stampfer MJ 2000 A prospective, observational study of postmenopausal hormone therapy and primary prevention of cardiovascular disease. Ann Intern Med 133:933–941[Abstract/Free Full Text]
  2. Lindenfeld J, Ghali JK, Krause-Steinrauf HJ, Khan S, Adams K, Goldman S, Peberdy MA, Yancy C, Thaneemit-Chen S, Larsen RL, Young J, Lowes B, Rosenberg YD 2003 Hormone replacement therapy is associated with improved survival in women with advanced heart failure. J Am Coll Cardiol 42:1238–1245[Abstract/Free Full Text]
  3. Mendelsohn ME, Karas RH 1999 The protective effects of estrogen on the cardiovascular system. N Engl J Med 340:1801–1811[Free Full Text]
  4. Iwakura, A, Luedemann C, Shastry S, Hanley A, Kearney M, Aikawa R, Isner JM, Asahara T, Losordo DW 2003 Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury. Circulation 108:3115–3121[Abstract/Free Full Text]
  5. Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U, Priller J, Laufs K, Ghaeni L, Milosevic M, Bohm M, Nickenig G 2003 Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation 107:3059–3065[Abstract/Free Full Text]
  6. Writing Group for the Women’s Health Initiative Investigators 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the women’s health initiative randomized controlled trial. JAMA 288:321–333[Abstract/Free Full Text]
  7. Grady D, Herrington D, Bittner V, Blumenthal R, Davidson M, Hlatky M, Hsia J, Hulley S, Herd A, Khan S, Newby LK, Waters D, Vittinghoff E, Wenger N 2002 HERS Research Group. Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA [Erratum (2002) 288:1064] 288:49–57[CrossRef]
  8. Hilakivi-Clarke L 1996 Role of estradiol in alcohol intake and alcohol-related behaviors. J Stud Alcohol 57:162–170[Medline]
  9. Purohit V 1998 Moderate alcohol consumption and estrogen levels in postmenopausal women: a review. Alcohol Clin Exp Res 22:994–997[CrossRef][Medline]
  10. Sarkola T, Makisalo H, Fukunaga T, Eriksson CJ 1999 Acute effect of alcohol on estradiol, estrone, progesterone, prolactin, cortisol, and luteinizing hormone in premenopausal women. Alcohol Clin Exp Res 23:976–982[CrossRef][Medline]
  11. Ford MM, Eldridge JC, Samson HH 2004 Determination of an estradiol dose-response relationship in the modulation of ethanol intake. Alcohol Clin Exp Res 8:20–28
  12. Reid ML, Hubbell CL, Reid LD 2003 A pharmacological dose of estradiol can enhance appetites for alcoholic beverages. Pharmacol Biochem Behav 74:381–388[CrossRef][Medline]
  13. Reid LD, Marinelli PW, Bennett SM, Fiscale LT, Narciso SP, Oparowski CJ, Reid ML, Merrigan BA, Moricone J, Hubbell CL, Gianoulakis C 2002 One injection of estradiol valerate induces dramatic changes in rats’ intake of alcoholic beverages. Pharmacol Biochem Behav 72:601–616[CrossRef][Medline]
  14. Juarez J, Vazquez-Cortes C, Barrios-De Tomasi E 2005 Different stages in the temporal course of estrogen treatment produce opposite effects on voluntary alcohol consumption in male rats. Alcohol 36:55–61[CrossRef][Medline]
  15. Marinelli PW, Quirion R, Gianoulakis C 2003 Estradiol valerate and alcohol intake: a comparison between Wistar and Lewis rats and the putative role of endorphins. Behav Brain Res 139:59–67[CrossRef][Medline]
  16. Kishore R, Qin G, Luedemann C, Bord E, Hanley A, Silver M, Gavin M, Goukassian D, Losordo DW 2005 The cytoskeletal protein ezrin regulates endothelial cell proliferation and angiogenesis via TNF induced transcriptional repression of cyclin A. J Clin Invest 115:1785–1796[CrossRef][Medline]
  17. Luedemann C, Bord E, Gangjian Q, Zhu Y, Goukassian D, Losordo DW, Kishore R 2005 Ethanol modulation of TNF-{alpha} biosynthesis and signaling in endothelial cells: synergistic augmentation of TNF-{alpha} mediated endothelial cell dysfunctions by chronic ethanol. Alcohol Clin Exp Res 29:930–938[CrossRef][Medline]
  18. Kusano KF, Pola R, Murayama T, Curry C, Kawamoto A, Iwakura A, Shintani S, Ii M, Asai J, Tkebuchava T, Thorne T, Takenaka H, Aikawa R, Goukassian D, von Samson P, Hamada H, Yoon YS, Silver M, Eaton E, Ma H, Heyd L, Kearney M, Munger W, Porter JA, Kishore R, Losordo DW 2005 Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med 11:1197–1204[CrossRef][Medline]
  19. Hill SY 1984 Vulnerability to the biomedical consequences of alcoholism and alcohol-related problems among women. In: Wilsnack SC, Beckman LJ, eds. Alcohol problems in women. Antecedents, consequences, and intervention. New York: Guilford Press
  20. Muti P, Trevisan M, Micheli A, Krogh V, Bolelli G, Sciajno R, Schunemann HJ, Berrino F 1998 Alcohol consumption and total estradiol in premenopausal women. Cancer Epidemiol Biomarkers Prev 7:189–193[Abstract]
  21. Martin CA, Mainous 3rd AG, Curry T, Martin D 1999 Alcohol use in adolescent females: correlates with estradiol and testosterone. Am J Addict 8:9–14[Medline]
  22. Chilvers, CE, Knibb RC, Armstrong SJ, Woods KL, Logan RF 2003 Postmenopausal hormone replacement therapy and risk of acute myocardial infarction—a case control study of women in the East Midlands, UK. Eur Heart J 24:2197–2205[Abstract/Free Full Text]
  23. Chen WY, Colditz GA, Rosner B, Hankinson SE, Hunter DJ, Manson JE, Stampfer MJ, Willett WC, Speizer FE 2003 Use of postmenopausal hormones, alcohol, and risk for invasive breast cancer. Ann Intern Med 137:798–804
  24. Greenfield JR, Samaras K, Jenkins AB, Kelly PJ, Spector TD, Campbell LV 2003 Moderate alcohol consumption, estrogen replacement therapy, and physical activity are associated with increased insulin sensitivity: is abdominal adiposity the mediator? Diabetes Care 26:2734–2740[Abstract/Free Full Text]
  25. Juarez J, Barrios-De Tomasi E 1999 Sex differences in alcohol drinking patterns during forced and voluntary consumption in rats. Alcohol 19:1522
  26. Liao Y, McGee DL, Cao G, Cooper RS 2000 Alcohol intake and mortality: findings from the National Health Interview Surveys (1988 and 1990). Am J Epidemiol 151:651–659[Abstract/Free Full Text]
  27. Mukamal KJ, Kronmal RA, Mittleman MA, O’Leary DH, Polak JF, Cushman M, Siscovick DS 2003 Alcohol consumption and carotid atherosclerosis in older adults: the Cardiovascular Health Study. Arterioscler Thromb Vasc Biol 23:2252–2259[Abstract/Free Full Text]
  28. Puddy IB, Zilkens RR, Croft KD, Beilin LJ 2001 Alcohol and endothelial function: a brief review. Clin Exp Pharmacol Physiol 28:1020–1024[CrossRef][Medline]
  29. Quinones-Jenab V, Jenab S, Ogawa S, Inturrisi C, Pfaff DW 1997 Estrogen regulation of µ-opioid receptor mRNA in the forebrain of female rats. Brain Res Mol Brain Res 47:134–138[Medline]
  30. Kennedy AM, Shogren KL, Zhang M, Turner RT, Spelsberg TC, Maran A 2005 17ß-Estradiol-dependent activation of signal transducer and activator of transcription 1 (STAT1) in human fetal osteoblasts is dependent on Src kinase activity. Endocrinology 146:201–207[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajasingh, J.
Right arrow Articles by Kishore, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajasingh, J.
Right arrow Articles by Kishore, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals