Estradiol Accelerates Functional Endothelial Recovery After Arterial Injury
Background The mechanisms of the established atheroprotective effects of estrogen have not been entirely clarified. Recent data suggest that agents that hasten the recovery of the endothelium after denuding injury will deter the development of neointimal lesions. Because estrogen has been shown to exert angiogenic effects in vitro and in vivo, we performed a series of experiments to evaluate whether estrogen was capable of accelerating reendothelialization.
Methods and Results Ovariectomized Sprague-Dawley rats received estrogen replacement therapy in the form of subcutaneously implanted pellets designed to release 1.5 or 5.0 mg 17β-estradiol over 30 days. Deendothelializing balloon injury was performed 1 week after pellet implantation, and animals were euthanatized after 1 week for evaluation of reendothelialization (Evans blue staining) or 2 weeks for evaluation of reendothelialization and neointimal formation. At both time points, the use of estradiol caused a dose-dependent increase in reendothelialization, which was measured as absolute area and percentage of area that is reendothelialized. Estradiol accelerated functional endothelial recovery, manifested as an increase in nitric oxide production. Neointimal thickening was also shown to be inhibited in a dose-dependent fashion.
Conclusions Estrogen accelerates functional endothelial recovery after barotraumatic deendothelializing injury. These findings, along with the recent demonstration of estrogen receptor expression by endothelial cells, suggest that the antiatherogenic action of estrogen may be mediated in part through direct effects on endothelial cells.
It has generally been assumed that the antiatherogenic effect of estrogen is due to amelioration of serum lipid patterns.1 Recently, however, the demonstration of estrogen receptor expression by vascular smooth muscle cells2 3 and endothelial cells4 5 has suggested that estrogen may also act directly on vascular tissue. Disruption of the anatomic and functional integrity of the endothelium has long been postulated as a mechanism for the initiation of atherosclerosis.6 Recent data also suggest that acceleration of reendothelialization after arterial injury is associated with attenuation of intimal hyperplasia.7
Estrogen has been shown to promote angiogenic activity in vitro as well as in vivo.8 Furthermore, estrogen has been shown to inhibit neointimal thickening after arterial injury in a rat model.9 Accordingly, we performed a series of experiments to test the hypothesis that estrogen accelerates reendothelialization after arterial injury; this hypothesis was tested in the well-characterized model of arterial denudation in the rat carotid artery.
Ovariectomized Sprague-Dawley rats (Charles River Labs) were divided into two groups. The treatment group consisted of 30 ovariectomized animals that received a subcutaneously implanted pellet designed to release 1.5 or 5.0 mg 17β-estradiol over 30 days (Innovative Research). The control groups consisted of (1) rats (n=15) that received no pellet and (2) rats (n=15) that were treated with a subcutaneously implanted placebo pellet. Pellets were implanted subcutaneously through a small incision made in the skin of the back. Implantation was performed 1 week before balloon arterial injury.
All rats underwent balloon denudation of the carotid artery, as previously described,7 10 through withdrawal of a 2F Fogarty balloon inflated with saline from the proximal edge of the omohyoid muscle to the carotid bifurcation. An additional 3 animals from each treatment group were euthanatized on the day of injury to verify that estradiol treatment did not influence the initial injury (eg, through vasodilation).
Evaluation of Reendothelialization
Reendothelialization was assessed through staining with Evans blue dye (0.5 mL of 0.5% Evans blue dye; Sigma Chemical Co.) as previously described.7 Evans blue identifies areas of nonendothelialized artery with a blue stain.11 After fixation in situ in 100% methanol, the injured segment was photographed with a dissecting microscope (Zeiss). Tissues were then imbedded in paraffin for longitudinal sectioning. Planimetric analysis was performed as previously described.7
Measurement of Serum Estradiol Levels
Serum estradiol levels were measured in samples of tail vein blood. Five animals from each treatment group underwent sampling at baseline (before pellet implantation) and 2 weeks after balloon injury. Estradiol levels were determined with an ELISA by a commercial laboratory (SmithKline).
Evaluation of Intimal Hyperplasia
Neointimal thickening was evaluated by measuring the total area of neointima in longitudinal sections of elastic-trichrome–stained arteries. In addition to planimetry of the neointimal area, the area of the media was measured and the intima/media ratio was calculated.
Measurement of Nitric Oxide From Vessel Segments
To determine whether functional recovery of the endothelium was accelerated by estradiol, the production of nitric oxide by excised arterial segments was measured as previously described.12 Three arteries each from placebo pellet–treated and 1.5 mg estradiol–treated animals were excised 1 week after injury and placed in an organ bath. After 15 minutes, the concentration of nitric oxide was measured according to the Griess reaction.12
Rat Vascular Smooth Muscle Cell Culture
Primary cultures of rat aortic vascular smooth muscle cells were grown according to the explant outgrowth technique as previously described.2 Cells in passages 2 to 4 were grown to confluence and placed in phenol red–free medium supplemented with 0.5% charcoal-stripped serum for 48 hours. Cells were then exposed to one of the following for 24 hours: 0.5% stripped serum, 10% stripped serum supplemented with 10−7 mol/L estradiol, or 10% stripped serum without estradiol.
Northern Blot Analysis
Total RNA isolation, electrophoresis, and capillary transfer were performed as previously described.13 A 675-bp fragment of plasmid pSVI.VEGF.21 (the generous gift of N. Ferrara, Genentech, South San Francisco, Calif) encoding human vascular endothelial growth factor (VEGF) (88% homologous to rat VEGF) was labeled with [32P]dCTP as described to a specific activity of 5 to 9×108 cpm/μg. Prehybridization, hybridization, and washes were performed according to standard techniques.13
Results are expressed as mean±SEM. Differences between groups were evaluated with the use of ANOVA, with the Newman-Keuls method applied to evaluate differences between individual mean values. Differences were considered significant at a level of P<.05. Dose-response relationships were evaluated with linear regression analysis.
Estrogen Replacement Therapy Results in Dose-Dependent Acceleration of Reendothelialization
Evans blue staining indicates areas of nonreendothelialized artery with a blue stain (Fig 1A⇓). By standardizing the area of injury in all animals, a comparison can be made between control and treated animals. Fig 1A⇓ shows examples of arteries after the initial injury and harvested 1 and 2 weeks after injury in control and treated animals. The initial injury was equivalent in all treatment groups, whereas at both time points after injury, the estrogen-treated arteries had a greater area reendothelialized than did the animals receiving placebo pellets. No significant differences were detected between no-pellet and placebo pellet–treated rats for any measurement.
The total area of reendothelialization measured at 1 week (Fig 2A⇓) in placebo-treated animals was 4.7±0.3 versus 6.9±0.5 mm2 (P<.01 versus control) in animals receiving the lower dose of estrogen and 7.8±0.4 mm2 in rats receiving the 5 mg estradiol pellet (P<.001 versus control). Similarly, after 2 weeks, both of the treatment groups showed greater reendothelialization than the untreated controls: placebo-treated rats had a total area of reendothelialization of 5.7±0.5 versus 7.3±0.4 mm2 in the 1.5 mg estradiol group (P=NS) and 7.9±0.4 mm2 in the 5 mg estradiol group (P<.01 versus placebo) (Fig 2A⇓). At both 1 and 2 weeks, the dose-response relationship was significant (P<.001 at 1 week, P<.01 at 2 weeks).
Calculation of reendothelialization as a percentage of the total area of injury revealed similar findings (Fig 2B⇑). At 1 week, the placebo-treated animals showed reendothelialization of 37.1±1.5% of the injured area compared with 58.6±2.1% in the low-dose estradiol group (P<.001 versus control) and 61.9±4.1% in the high-dose estrogen group (P<.001 versus control). After 2 weeks, the arteries of the placebo-treated animals had recovered 59.2±5.0% of endothelial surface area compared with 69.9±2.5% in the low-dose estrogen group (P=NS) and 78.4±0.4 in the high-dose estradiol group (P<.01 versus control). Again, the dose-response relationship was significant at both time points (P<.001 at 1 week, P<.01 at 2 weeks).
Estrogen Results in a Dose-Dependent Reduction in Neointimal Thickening After Barotraumatic Balloon Injury
Evaluation of neointimal thickening confirms prior reports of an inhibitory effect of estrogen on myointimal proliferation in this model9 and also demonstrates a dose-response effect. Fig 1B⇑ shows examples of placebo-treated and high-dose estrogen–treated arteries harvested 2 weeks after balloon injury.
Total neointimal area was measured 2 weeks after the initial balloon injury (Fig 2C⇑). The total intimal area in the placebo-treated animals measured 0.51±0.1 mm2 compared with 0.23±0.03 mm2 in the 1.5 mg estradiol group (P<.01 versus control) and 0.124±0.02 mm2 in the 5 mg estradiol group (P<.01 versus control). The calculated intima/media ratio (Fig 2D⇑) also displayed a similar dose-response relationship. The intima/media ratio in the placebo-treated group was 1.2±0.3 compared with 0.64±0.2 in the low-dose estrogen group (P=NS) and 0.27±0.04 (P<.05 versus control) in the high-dose estrogen group. Both the total intimal area and the intima/media ratio manifested significant dose-response relationships (P<.001 and P<.02, respectively).
Nitric Oxide Production Is Increased in Estradiol-Treated Arteries
Nitric oxide production by the estradiol-treated arteries was significantly greater than that of placebo-treated arteries 1 week after injury (8.55±1.7 versus 2.8±0.18 μmol·L−1·mm−2 per 15 minutes, P<.0001) (Fig 2E⇑). This indicates that the acceleration of anatomic recovery, as identified with Evans blue staining, was also accompanied by estrogen-induced acceleration of functional endothelial recovery.
VEGF Expression by Vascular Smooth Muscle Cells Is Increased by Estradiol
Northern analysis revealed increased expression of VEGF mRNA by rat vascular smooth muscle cells exposed to estradiol (Fig 2F⇑).
Numerous previous studies have documented in various animal models the ability of estrogen to inhibit the formation of a neointima after arterial injury.9 14 15 The mechanism of this effect has not been clarified but has been presumed to be due, at least in part, to direct inhibition of smooth muscle cell proliferation. In the present study, we document for the first time the accelerated reendothelialization of denuded arterial segments as a result of estrogen treatment. At both the 1- and 2-week time points, the estrogen-treated animals showed more rapid recovery of the endothelium and its barrier function as demonstrated by exclusion of Evans blue dye. At both time points, the rate of recovery correlated with the dose of estrogen that had been administered. Similarly, inhibition of neointimal formation corresponded inversely with the speed of reendothelialization, with more rapid reendothelialization being associated with decreased neointimal formation; again, a dose-response relationship was observed. Finally, recovery of endothelial function, manifest as enhanced nitric oxide production, was also shown to be accelerated by estradiol treatment.
The mechanism or mechanisms by which estradiol accelerates functional endothelial recovery remain undefined. Estrogen has previously been shown to exert direct angiogenic effects on endothelial cells,8 raising the possibility that this effect could be mediated through the direct action of estrogen on endothelial cells and perhaps mediated by the estrogen receptor expressed by these cells.4 5 Alternatively, a potential role for VEGF is raised by our finding of increased expression of VEGF by vascular smooth muscle cells exposed to estradiol. Recently, Karas et al16 also reported enhanced VEGF gene expression in human smooth muscle cells by an estrogen receptor–mediated mechanism. Together, these findings suggest that VEGF may contribute to the estrogen-induced acceleration of reendothelialization, although other direct and indirect mechanisms remain to be investigated.
These findings are also consistent with the recent demonstration that VEGF, which directly accelerates endothelial recovery, is also capable of attenuating neointimal proliferation after balloon injury.7 In both the present and this prior study, a correlation between enhanced endothelial recovery and diminished neointimal lesion formation is demonstrated. The potential mechanistic significance of this association regarding a negative regulatory influence of the endothelium of neointimal proliferation remains to be explored in future studies.
- Received December 9, 1996.
- Revision received February 10, 1997.
- Accepted February 14, 1997.
- Copyright © 1997 by American Heart Association
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