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(Circulation. 1997;96:1624-1630.)
© 1997 American Heart Association, Inc.

Articles

Estrogen Restores Endothelial Cell Function in an Experimental Model of Vascular Injury

C. Roger White, PhD; Jonathan Shelton, BS; Shi-Juan Chen, MD; Victor Darley-Usmar, PhD; Leslie Allen, BS; Cheryl Nabors, BS; Paul W. Sanders, MD; Yiu-Fai Chen, PhD; ; Suzanne Oparil, MD

From the University of Alabama at Birmingham, Departments of Medicine, Vascular Biology, and Hypertension Program (C.R.W., J.S., S.-J.C., L.A., P.W.S., Y.-F.C., S.O.), Pathology (V.D.-U.), Physiology (S.O.), and Nephrology (C.N., P.W.S.); the Department of Veterans Affairs Medical Center (P.W.S.); and the Center for Free Radical Biology (C.R.W., V.D.-U.), Birmingham, Ala.

Correspondence to C. Roger White, PhD, Departments of Medicine, Vascular Biology, and Hypertension Program, 1046 Zeigler Research Bldg, 703 S 19th St, Birmingham, AL 35294-0007. E-mail card029{at}uabdpo.dpo.uab.edu

	Abstract

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Background It has been suggested that reendothelialization of damaged blood vessels protects against the vascular injury response. The goal of the present study was to determine whether estrogen restores endothelial cell function in balloon-injured rat carotid arteries.

Methods and Results Ten-week-old male and female Sprague-Dawley rats with intact gonads underwent balloon injury to the right common carotid artery. Female rats were randomized to receive either daily subcutaneous injections of 17ß-estradiol (17ß-E₂; 20 µg · kg^-1 · d^-1) or vehicle over the course of the study. Vessel morphology was assessed 2 weeks after injury. Significant neointima formation was observed in vehicle-treated males. This response was blunted in vehicle-treated and 17ß-E₂–supplemented females. Intima-to-media ratios were 1.28±0.23 (males), 0.72±0.07 (vehicle-treated females), and 0.49±0.07 (17ß-E₂–supplemented females). To test whether reductions in neointimal lesion formation were related to the functional reendothelialization of the damaged vessel, endothelium-dependent relaxation was tested in isolated ring segments from the three experimental groups. Vessels were precontracted with phenylephrine followed by cumulative administration of acetylcholine, an endothelium-dependent vasodilator. Maximum relaxation to acetylcholine was 8.13±1.70% (males), 22.06±4.36% (vehicle-treated females), and 46.47±3.48% (17ß-E₂–supplemented females). The enhanced endothelium-dependent relaxation of rings from 17ß-E₂–supplemented females correlated with reduced neointimal proliferation in this group. The concentration of nitric oxide metabolites in plasma correlated positively with plasma 17ß-E₂ concentration.

Conclusions These results suggest that estrogen protects against neointimal injury in the balloon-injured rat, at least in part, by facilitating the reendothelialization of the damaged vessel.

Key Words: hormones • balloon • vasculature

	Introduction

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Much interest is currently focused on the therapeutic benefits of estrogen. Death from cardiovascular disease is relatively rare in premenopausal women compared with age-matched men. After menopause, however, the incidence of hypertension, atherosclerosis, and stroke increases dramatically, and cardiovascular disease becomes the leading cause of death in women.¹ Emerging data from ongoing clinical studies, including the Women's Health Initiative and the Heart Estrogen-Progestin Replacement Study, show that hormone replacement therapy reduces cardiovascular disease in postmenopausal women.^{1 2 3 4} Increased interest, therefore, has focused on the relationship between sex hormones, especially estrogen, and cardiovascular risk and events. The cardioprotective effects of estrogen include decreases in plasma LDL cholesterol and increases in HDL cholesterol.^{2 3 4} The benefits of estrogen cannot be attributed solely to effects on lipids and lipoproteins, however. Accumulating evidence supports a direct vasoprotective effect of the hormone.⁵

Transluminal balloon injury is a commonly used paradigm for the study of mechanisms of response to vascular damage and atherosclerosis.^{6 7 8} Balloon injury models are characterized by the formation of a concentric fibromuscular lesion that encroaches on the arterial lumen.⁶ This injury response is characterized by the proliferation of smooth muscle cells in the intima. These cells also adopt a secretory phenotype, resulting in excessive production of extracellular matrix. In addition, platelets and macrophages adhere to the vessel wall and release cytokines and growth factors. These stimulate further smooth muscle cell proliferation and act as chemoattractants for other cell types, which in turn become incorporated into the neointimal lesion.^{6 9}

Recent reports indicate that estrogen protects against neointimal hyperplasia resulting from balloon injury in the rat carotid artery¹⁰ and rabbit iliac artery.¹¹ In the balloon-injured, nongonadectomized male rabbit, estrogen treatment resulted in reduced neointimal thickening, [³H]thymidine incorporation, and DNA content of injured vessels compared with rabbits not receiving estrogen. These results suggest that estrogen inhibited cell proliferation in this model of vascular injury. The protective effects of estrogen were similar to those exerted by angiopeptin, a somatostatin analogue, which has previously been shown to inhibit neointimal hyperplasia.¹² Estrogen may also protect against the development of atherosclerosis. In animal models of hypercholesterolemia, estrogen replacement therapy prevents the development of atheromatous lesions and defects in endothelium-dependent relaxation via mechanisms unrelated to changes in plasma lipoprotein concentration.^{13 14}

Pharmacological interventions can prevent or inhibit the neointimal response after vascular injury. Angiopeptin, a somatostatin analogue, administered by a Wolinsky porous balloon inhibits myointimal proliferation in the balloon-injured rabbit aorta.¹² Interestingly, hyperplasia was not inhibited at the site of local delivery but rather at regions of the aorta downstream from the site of application. The authors suggested that the protective downstream effect of angiopeptin may be due to healing of the damaged endothelium.¹² Endothelial cell seeding techniques have also been used to test whether the reendothelialization of the damaged vessel limits the neointimal response. In a model of bilateral iliac artery balloon injury, vessels seeded with venous endothelial cells demonstrated a higher degree of reendothelialization than nonseeded control vessels.¹⁵ However, both treated and untreated vessels demonstrated similar extents of neointimal hyperplasia.¹⁵

The goal of the present study was to determine whether estrogen protects against the vascular injury response by stimulating the regrowth and functional recovery of the endothelium. Our results indicate that estrogen inhibits neointima formation and partially restores the endothelium-dependent vasodilator response of the balloon-injured rat carotid artery. It is hypothesized that these protective effects of estrogen are mediated by reendothelialization of the damaged region of the artery and that enhanced release of NO plays an important role in this process.

	Methods

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Animals
Ten-week-old male and female Sprague-Dawley rats were obtained from Charles River Breeding Laboratories (Wilmington, Mass). All rats were maintained at constant humidity (60±5%), temperature (24±1°C), and light cycle (6 AM to 6 PM) and were fed a standard rat pellet diet (Ralston Purina Diet) ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985). Body weights were determined before and 2 weeks after balloon injury of the carotid artery.

Balloon Injury Procedure
Male and female rats underwent balloon injury of the right common carotid artery as described previously.¹⁰ Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg), and the right carotid artery was isolated by a middle cervical incision. The distal right common carotid artery and the region of the bifurcation were exposed. A Fogarty 2F balloon catheter (Baxter V. Mueller) was inserted into the external carotid artery and advanced into the thoracic aorta. The balloon was inflated with saline to distend the common carotid artery and was pulled back to the external carotid artery. The catheter was advanced and then withdrawn for five additional passes to denude the vessel wall of endothelium. The catheter was then removed, and the external carotid artery was tied off before closure of the wound. The left carotid artery was not damaged and served as a control.

Estrogen Supplementation
Female rats with intact gonads were randomly assigned to one of two treatment groups. The first group (n=6) received daily injections of 17ß-E₂ (20 µg/kg in 100 µL cottonseed oil SC daily), and the second group (n=9) received vehicle (100 µL cottonseed oil SC daily). 17ß-E₂ was obtained from Sigma Chemical Co. Male rats (n=7) also received daily vehicle treatment.

Vessel Reactivity Studies
Two weeks after balloon injury of the right carotid artery, heparinized rats were euthanized with an overdose of sodium pentobarbital (75 mg/kg IP). Endothelium-dependent relaxation of isolated carotid arteries was assessed 2 weeks after injury. Right and left carotid arteries were excised and placed in prewarmed and oxygenated Krebs-Henseleit solution and cleansed of fat and adhering tissue. The undamaged left carotid artery served as a control in functional studies of the balloon-injured vessel. Individual ring segments (2 to 3 mm in width) were cut from injured and control vessels. Each vascular ring was mounted on two stainless steel hooks and suspended from a force-displacement transducer in a water-jacketed tissue bath. The ring was anchored to a hook at the base of the chamber, and a passive load of 1.5 g was applied to the vessel. Ring segments were bathed in a Krebs-Henseleit solution of the following composition (mmol/L): NaCl 118, KCl 4.6, NaHCO₃ 27.2, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.75, Na₂EDTA 0.03, and glucose 11.1 (pH 7.4). The solution was continuously aerated with a 95%O₂/5% CO₂ gas mixture and maintained at 37°C. Changes in isometric tension were measured with capacitive force transducers (Radnoti Glass Technology, Inc). Real-time data were acquired and digitized with an IBM-compatible computer and stored for later analysis with commercially available software (Experimenter's Workbench). Dose-response profiles for different experimental conditions were analyzed and tested to determine differences in contraction and relaxation responses.

Isometric tension was measured in isolated ring segments of control and balloon-injured carotid arteries. After an equilibration period of 45 minutes, ring segments were depolarized with KCl (70 mmol/L) to determine the contractile capacity of the vessel. Rings were then thoroughly washed and allowed to equilibrate for an additional 45 minutes. All subsequent experiments were performed in the presence of indomethacin (5 µmol/L) to eliminate the effects of cyclooxygenase-derived vasoactive molecules. In initial studies, contractile responses of balloon-injured vessels were tested by cumulative administration of PE (10^-9 to 3x10^-6 mol/L) or 5-HT (10^-8 to 10^-4 mol/L). NO-dependent relaxation was tested in PE-contracted ring segments by exposure to ACh (10^-9 to 3x10^-6 mol/L), an endothelium-dependent vasodilator. In other studies, PE-contracted ring segments were exposed to SNP (10^-9 to 3x10^-5 mol/L) to elicit endothelium-independent relaxation.

Vessel Morphometry
After completion of the in vitro blood vessel bioassay, each ring was placed in 10% buffered formalin solution. Vessels were embedded in paraffin and serially sectioned (5 µm) for morphometric analysis. Tissue was stained with hematoxylin-eosin. Neointimal proliferation was assessed in thin sections of balloon-injured and control carotid arteries 2 weeks after injury. The undamaged left carotid artery served as a control in these studies. Morphometric analysis of each arterial ring segment was performed with a computer-based Bioquant II Morphometric system. At least three sections of each vessel were examined, and measurements were averaged for statistical analysis. The cross-sectional areas of the media and neointima were measured. The degree of neointima formation of the injured carotid artery was expressed as the absolute area of neointima and the ratio of the neointimal area to the medial area. All morphometric measurements were performed by the same individual, who was blinded to the treatment groups.

Plasma Estradiol Assay
Blood samples were collected when the animals were killed to monitor the effect of 17ß-E₂ treatment on circulating levels of the hormone. Plasma 17ß-E₂ levels were determined by radioimmunoassay with a commercially available kit (Diagnostic Products Corp). Assay sensitivity was 8.0 pg/mL, and intra-assay and interassay coefficients of variation for estradiol were 5.3% and 6.4%, respectively.

Plasma NO₃^- and NO₂^- Measurements
To determine whether the protective effects of estrogen were related to a stimulatory effect of the hormone on NO formation, the plasma concentrations of NO₃^- and NO₂^-, the primary metabolites of NO, were determined as described previously.¹⁶ NO has a relatively short half-life in plasma, but NO₂^- and NO₃^- are stable products. The Griess reagent (1% sulfanilamide/0.1% N-[1-naphthyl]ethylenediamine dihydrochloride) was used in the measurement of NO₃^- and NO₂^-. Because this reagent reacts only with nitrite, plasma NO₃^- was first reduced to NO₂^- by incubation of plasma samples with Escherichia coli rich in nitrate reductase. After a 2-hour incubation period with nitrate reductase, the Griess reagent was added to plasma samples and incubated at room temperature for 10 minutes. After this incubation period, the absorbance of samples was read at 540 nm. Plasma NO₂^- concentrations were then estimated by comparing absorbance values with those obtained from a standard curve for NaNO₂ (0 to 200 µmol/L).

Statistical Analysis
All results are expressed as mean±SEM. Two to three observations for a given treatment were obtained from each animal. These values were averaged so that a single mean value is reported for each animal. Dose-response profiles for the different experimental treatments were analyzed and tested to determine differences in contraction and relaxation responses by use of the StatView statistical analysis program. Unpaired observations were assessed by one-way ANOVA and multiple-range tests. Pearson's correlation analysis was used to compare treatment responses in the experimental groups.

	Results

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Two weeks after balloon injury of the right carotid artery, body weights of male rats were significantly greater than in vehicle-treated and 17ß-E₂–supplemented female rats (Table). Radioimmunoassay of plasma from each experimental group showed that circulating 17ß-E₂ was elevated in both vehicle- and estrogen-supplemented female rats compared with males. The increase in plasma 17ß-E₂ in female rats receiving daily injections of the hormone confirms previous observations showing that this dosage (20 µg · kg^-1 · d^-1) of 17ß-E₂ induces a twofold increase in plasma estrogen concentration over the 2-week course of the study.¹⁰ Morphometric analysis of thin sections of balloon-injured carotid arteries was performed to determine the magnitude of the vascular injury response in male and female rats (Table). Although medial areas were not different among males, vehicle-treated females, and 17ß-E₂–supplemented females, neointimal area and intima-to-media ratios were greatest in the male. These responses to balloon injury were blunted in both vehicle-treated and 17ß-E₂–supplemented females (Table). Plasma 17ß-E₂ concentration correlated inversely with neointimal area (r=-.48, P<.05). Analysis of undamaged left carotid arteries showed that the medial areas of left and right carotid arteries were similar both within the same animal and between treatment groups (data not shown). The intimal area of undamaged carotid arteries was negligible (a single cell layer thick).

View this table: [in this window] [in a new window]   Table 1. Effects of Sex and Estrogen Administration on Body Weight and Vessel Morphology After Balloon Injury of Right Carotid Arteries of Male and Female Sprague-Dawley Rats

In vitro studies were designed to determine whether estrogen treatment influenced vascular reactivity in balloon-injured and control carotid artery ring segments of males, vehicle-treated females, and 17ß-E₂–supplemented females. Results of cumulative PE dose-response experiments showed that the force-generating capacity of balloon-injured ring segments was similar in all groups despite differences in vessel morphology (Fig 1). Furthermore, the EC₅₀ for maximum contraction of balloon-injured ring segments was similar in all treatment groups (Fig 2). Similar responses were observed when ring segments were exposed to cumulative concentrations of the vasoconstrictor 5-HT (Fig 3). Although tension development of balloon-injured vessels was less than that of undamaged carotid ring segments for each treatment group (data not shown), control and injured vessels displayed similar sensitivities to PE (Fig 2). These results indicate that vasoconstrictor responses of balloon-injured carotid ring segments are similar in the three treatment groups.

View larger version (20K): [in this window] [in a new window]   Figure 1. Force development in balloon-injured rat carotid arteries. Carotid ring segments from male (, n=7), vehicle-treated female (, n=8), and 17ß-E2–supplemented female rats (, n=6) were exposed to cumulative concentrations of PE. Force development (in grams tension) was similar in all experimental groups. Data are mean±SEM.

View larger version (16K): [in this window] [in a new window]   Figure 2. Cumulative dose-response profiles for PE in carotid ring segments 2 weeks after injury. Ring segments excised from (A) undamaged (open symbols) and (B) balloon-injured (solid symbols) carotid arteries of males (, n=6; , n=7), vehicle-treated females (, n=8; , n=8), and 17ß-E2–supplemented females (, n=6; , n=6) were exposed to cumulative concentrations of PE. Dose-response profiles for balloon-injured and control vessels from all treatment groups were similar. Data are mean±SEM.

View larger version (17K): [in this window] [in a new window]   Figure 3. Cumulative dose-response profiles for 5-HT in carotid ring segments 2 weeks after injury. Ring segments excised from (A) undamaged (open symbols) and (B) balloon-injured (solid symbols) carotid arteries of males (, n=4; , n=5), vehicle-treated females (, n=3; , n=3), and 17ß-E2–supplemented females (, n=3; , n=3) were exposed to cumulative concentrations of 5-HT. Dose-response profiles for balloon-injured and control vessels from all treatment groups were similar. Data are mean±SEM.

Additional experiments were designed to determine whether estrogen treatment resulted in the functional reendothelialization of the damaged vessel. Endothelium-dependent relaxation was assessed in isolated ring segments from the three experimental groups. Vessels were first contracted with PE followed by administration of ACh, an endothelium-dependent vasodilator. Relaxation responses were calculated as percentage change in tension from the predose response level. Dose-response profiles to ACh were similar in undamaged left carotid arteries in all treatment groups (Fig 4A). The ACh-induced relaxation of balloon-injured carotid arteries was diminished in all treatment groups, with ring segments of male rats displaying the most severe impairment (Fig 4B). Maximum relaxation in response to ACh was 8.13±1.70% (males), 22.06±4.36% (vehicle-treated females), and 46.47±3.48% (E₂-supplemented females). Although E_max was significantly increased in both groups of female rats compared with males, the recovery of ACh-induced relaxation was greatest in E₂-supplemented females (Fig 4B). The E_max value correlated positively with plasma 17ß-E₂ (r=.50, P<.05) (Fig 5). The enhanced endothelium-dependent relaxation of rings from 17ß-E₂–supplemented females also correlated with reduced neointima formation in this group (r=-.59, P<.05). In related experiments, PE-contracted ring segments taken from balloon-injured arteries were exposed to the endothelium-independent vasodilator SNP. Dose-response profiles to SNP were similar in all groups (Fig 6), suggesting that the differential relaxation response of vessels from the three treatment groups was not due to an altered responsiveness of the VSMC. Rather, these data suggest that the enhanced relaxation of carotid artery rings from 17ß-E₂–supplemented rats is due to increased production of endothelium-derived NO in these vessels relative to those isolated from vehicle-treated male and female rats.

View larger version (13K): [in this window] [in a new window]   Figure 4. Endothelium-dependent relaxation of rat carotid artery ring segments. Ring segments excised from (A) undamaged (open symbols) and (B) balloon-injured (solid symbols) carotid arteries were contracted with PE followed by cumulative administration of ACh. Endothelium-dependent relaxation of control vessels from male (, n=7), vehicle-treated female (, n=9), and 17ß-E2–supplemented female (, n=6) rats were similar. Relaxation of balloon-injured vessels from all groups was impaired with respect to control ring segments. Female rats supplemented with 17ß-E2 (, n=5) for 2 weeks after injury displayed a significant improvement in endothelium-dependent relaxation compared with males (, n=6) and vehicle-treated females (, n=6). Data are mean±SEM. *P<.05 and **P<.01 vs male group; P<.01 vs vehicle-treated female group.

View larger version (13K): [in this window] [in a new window]   Figure 5. Positive correlation between plasma 17ß-E2 and Emax, maximum relaxation response to acetylcholine. Calculated correlation coefficient (r) was .50 (P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 6. Endothelium-independent relaxation of rat carotid artery ring segments. Ring segments excised from balloon-injured carotid arteries were contracted with PE followed by cumulative administration of SNP. Endothelium-independent relaxation of damaged vessels from males (, n=3), vehicle-treated females (, n=3), and 17ß-E2–supplemented females (, n=3) was similar 2 weeks after injury. Data are mean±SEM.

Because reports suggest that increased production of NO protects against vascular injury^{17 18} and that estrogen stimulates constitutive NOS activity,^{19 20 21} studies were designed to test whether NO production was increased in 17ß-E₂–supplemented rats. We used the Griess assay to measure plasma concentrations of NO₂^- and NO₃^-, the primary metabolites of NO. Increases in plasma NO₂^- and NO₃^- were observed in vehicle-treated and 17ß-E₂–supplemented rats compared with males (Fig 7). The enhanced NO₂^- and NO₃^- levels correlated positively with increased plasma 17ß-E₂ levels (r=.46, P<.05) and maximum vessel relaxation responses (r=.84, P<.01) in these animals and inversely with neointimal area (r=-.49, P<.05) (Fig 8) and intima-to-media ratios (r=-.47, P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 7. Fig 7. NO2-+NO3- levels in plasma of males (solid bar, n=7), vehicle-treated females (crosshatched bar, n=7), and 17ß-E2–supplemented females (open bar, n=6). Plasma NO2-+NO3- concentration was significantly elevated in vehicle-treated and 17ß-E2–supplemented females compared with males. Data are mean±SEM. *P<.05 and **P<.01 vs male group.

View larger version (15K): [in this window] [in a new window]   Figure 8. Inverse correlation between plasma NO2-+NO3- concentrations and neointimal area of balloon-injured vessels. Calculated correlation coefficient (r) was -.49 (P<.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The focus of these studies was to determine whether 17ß-E₂ supplementation prevents structural and functional alterations in vessels exposed to balloon injury and endothelial cell denudation. Balloon-injury studies are commonly performed in rodents, particularly rats, to investigate mechanisms of vascular injury. This model has been used extensively to study VSMC migration and proliferation.^{10 22} In the present experiments, we used the endothelium-dependent relaxation of isolated carotid ring segments as an index of reendothelialization after vascular injury. Results of these studies indicate that supplementation of normal female, balloon-injured rats with 17ß-E₂ partially restores endothelium-dependent vasodilator function in mechanically injured blood vessels and also prevents the neointimal response to vascular injury. These results suggest that 17ß-E₂ protects against the vascular injury response by stimulating the reendothelialization of damaged blood vessels.

Recently, treatment of balloon-injured rats with 17ß-E₂ was shown to inhibit neointimal lesion formation in gonadectomized rats of both sexes.¹⁰ Similar results have been described in a rabbit model of balloon injury in which estrogen supplementation reduced [³H]thymidine incorporation in injured vessels and inhibited neointimal cell proliferation.¹¹ Estrogen treatment inhibits neointima formation in injured carotid arteries of gonadectomized rats.^{10 23} The inhibition of cell proliferation correlated with a decrease in the expression of the proto-oncogene c-myc, whose induction has been linked to the hyperplastic response of blood vessels.¹⁰

Increased production of NO attenuates vascular damage in balloon injury models.^{17 18} In the rat, intravenous infusion of the NO donor CAS-1609 inhibits neointimal hyperplasia and restores the vasodilator response to ACh. CAS-1609 has a stimulatory effect on endothelial cell proliferation in culture while inhibiting platelet-derived growth factor–stimulated VSMC growth. These results suggest that NO minimizes vessel damage and promotes the functional recovery of endothelial cells in this model of carotid vessel injury.¹⁷

Estrogen stimulates the synthesis of NO in numerous tissues, including the uterine artery, heart, uterus, and skeletal muscle.¹⁹ Both pregnancy and estrogen supplementation enhance NOS-I and NOS-III expression, whereas NOS-II is unaffected.²⁰ Furthermore, the 5'-flanking region of the gene for NOS-III contains an estrogen response element.²⁴ Estrogen-mediated stimulation of NOS activity occurs by a receptor-dependent mechanism, because NOS-III expression is inhibited by the estrogen receptor antagonists tamoxifen and ICI182780.²⁵ In the present study, plasma concentrations of NO metabolites correlated positively with plasma 17ß-E₂ levels and negatively with the extent of neointima formation. Thus, increases in NO production may be a fundamental mechanism by which estrogen blunts vascular injury.

Indirect evidence suggests that the basal release of NO is elevated in female rabbits compared with males.^{26 27} Endothelium-dependent relaxation of aortic rings was impaired in ovariectomized rabbits compared with intact females, and dose-response profiles were similar in magnitude to the responses seen in male rabbits. Furthermore, the impaired response of ring segments from ovariectomized females correlated with the reduction in plasma E₂ concentration.²⁶ In another study, progesterone was shown to antagonize the facilitative effects of estrogen on endothelium-dependent relaxation of coronary arteries of ovariectomized dogs.²⁸ It is clear, therefore, that steroid hormones differentially regulate vascular function.

Results of animal and human studies suggest that acute exposure to 17ß-E₂ induces rapid changes in vessel tone and that treatment with NOS inhibitors abolishes estrogen-mediated vascular relaxation.^{29 30} Plasma concentrations of NO₂^- and NO₃^-, metabolites of NO, are elevated in postmenopausal women receiving hormone replacement therapy.³¹ In postmenopausal women, short-term estrogen replacement therapy results in increased coronary blood flow^{30 32} and peripheral vasodilator responses.^{33 34} These data suggest that protective effects of estrogen on the vasculature may be related to the enhanced production of NO. Results of the present study show that plasma 17ß-E₂ levels correlate positively with plasma concentrations of NO metabolites. The ability of estrogen to attenuate neointima formation may, therefore, be related to NO-mediated inhibition of VSMC proliferation and platelet adhesion.

Several reports suggest that the regrowth of the endothelium may attenuate vascular damage in balloon-injury models.^{17 18} Endothelial cell growth factors may play an important role in this protective response. Intravenous treatment of balloon-injured rabbits with bFGF, an endothelial cell mitogen, resulted in a significant reendothelialization of damaged iliac arteries compared with controls not receiving bFGF. The extent of neointimal thickening was not different, however, between the two groups. Functional responses of bFGF-exposed and control vessels were tested by in vitro bioassay of endothelium-dependent relaxation. Iliac arteries of rabbits receiving bFGF treatment demonstrated enhanced ACh-mediated relaxation compared with controls. Those authors suggested that angiogenic growth factors such as bFGF may facilitate the recovery of endothelial cell function in injured vessels.³⁵

Other mitogens, including VEGF, promote endothelial cell growth and proliferation^{36 37} and angiogenesis,^{38 39} increase endothelial barrier permeability,^{40 41} and modulate blood vessel tone.⁴² Local delivery of VEGF to balloon-injured rat carotid arteries results in diminished neointimal hyperplasia and enhanced reendothelialization of damaged vessels at periods up to 4 weeks after injury.²² VEGF treatment was associated with reductions in proliferating cell nuclear antigen immunostaining and neointimal thickening. The authors suggest that the protective effect of VEGF in balloon-injured vessels was mediated by the reendothelialization of the vessel wall.²² VEGF is secreted by a variety of cell types in the vessel wall, including VSMCs and macrophages,⁴³ and 17ß-E₂ has been shown to regulate VEGF mRNA expression in uterus and endometrial carcinoma cells.^{44 45} The extent of mitogen-induced endothelial cell growth and angiogenesis in vivo can be regulated by other factors. For instance, progesterone stimulates the production of thrombospondin-1, an extracellular matrix glycoprotein, which suppresses angiogenesis in vivo in human endometrium.⁴⁶ Thus, the stimulatory effects of estrogen on endothelial cell growth may be regulated by other cellular mediators.

Results of the present studies clearly show that estrogen supplementation inhibits the vascular injury response and promotes reendothelialization, as demonstrated by enhanced endothelium-dependent relaxation in carotid arteries of normal female rats subjected to balloon injury. The subsequent localized increase in NO production in damaged carotid arteries of estrogen-supplemented rats may play an important role in limiting neointima formation, because NO is known to inhibit smooth muscle cell proliferation and vascular adhesion processes.⁴⁷ Further studies are required to define the cellular and molecular mechanisms underlying the estrogen-mediated reendothelialization of the balloon-injured vessel.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine bFGF = basic fibroblast growth factor 17ß-E2 = 17ß-estradiol 5-HT = serotonin NO2- = nitrite NO3- = nitrate NOS = NO synthase NOS-I = neuronal NO synthase isoform NOS-II = inducible NO synthase isoform NOS-III = endothelial NO synthase isoform PE = phenylephrine SNP = sodium nitroprusside VEGF = vascular endothelial growth factor VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported in part by grants HL-44195, HL-47081, HL-07457, and HL-50147 (Dr Oparil); PO1-HL-48676 (Drs White and Darley-Usmar); and DK-46199 (Dr Sanders) from the National Institutes of Health and a Grant-in-Aid from the American Heart Association, Alabama Affiliate (Dr White).

Received January 22, 1997; revision received March 5, 1997; accepted March 7, 1997.

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