Reduction of post injury neointima formation due to 17beta-estradiol and phytoestrogen treatment is not influenced by the pure synthetic estrogen receptor antagonist ICI 182,780 in vitro.

BACKGROUND
Animal and organ culture experiments have shown beneficial inhibitory estrogen effects on post injury neointima development. The purpose of this study was to investigate whether such estrogen effects are influenced by the estrogen receptor antagonist ICI 182,780. Different concentrations of 17beta-estradiol and the phytoestrogens genistein and daidzein were tested.


METHODS
Female New Zealand White rabbits were benumbed. In situ vascular injury of the thoracic and abdominal aorta was performed by a 3F Fogarty catheter. Segments of 5 mm were randomised and held in culture for 21 days. Three test series were performed: 1) control group--20 microM ICI--30 microM ICI--40 microM ICI. 2) control group--20 microM ICI--40 microM 17beta-estradiol--40 microM 17beta-estradiol + 20 microM ICI. 3) control group--20 microM ICI--40 microM daidzein--40 microM daidzein + 20 microM ICI--20 microM genistein--20 microM genistein + 20 microM ICI. After 21 days the neointima-media-ratio was evaluated.


RESULTS
1) Treatment with ICI 182,780 did not reduce neointima formation significantly (p = 0.05). 2) 40 microM 17beta-estradiol alone (p < 0.0001) and in combination with 20 microM ICI (p < 0.0001) reduced neointima formation significantly. 3) 20 microM genistein alone (p = 0.0083) and combined with 20 microM ICI (p = 0.0053) reduced neointima formation significantly. 40 microM daidzein did not have a significant (p = 0.0637) effect.


CONCLUSIONS
The estrogen receptor antagonist ICI 182,780 did not modulate the inhibitory estrogen effects on post injury neointima formation. These results do not support the idea that such effects are mediated by vascular estrogen receptors.


Background
Beneficial estrogen effects in the cardiovascular system are still under investigation and have been discussed contro-versially [1][2][3]. Clinical benefits such as the reduction of cardiovascular mortality, as suggested by several retrospective studies, [4,5] have not been supported by ran-domised [6] and interventional [7,8] trials. However, there are plenty of experimental data demonstrating beneficial estrogen effects on distinct aspects of the cardiovascular system, i.e., lipid metabolism [9] and lipid peroxidation [10], post injury smooth muscle cell and neointimal proliferation [11][12][13][14], and the vascular tone [15][16][17][18]. Some of these effects are also described for phytoestrogens like genistein and daidzein which therefore may be suggested as a possible therapeutic option in postmenopausal women [19].
In previous organ culture experiments we have demonstrated that 17β-estradiol and the phytoestrogens genistein (with) [20] and daidzein (without protein tyrosine kinase activity) [21] were able to inhibit neointima formation after vascular injury in a dose dependent manner [22,23]. These effects have been described in aortic rings not only from female but from male rabbits as well [24].
In further experiments we titrated the lowest concentrations of these three estrogens which were able to reduce neointima formation significantly and we moreover demonstrated that this effect was unlikely caused by toxicity [25].

In vitro model
A total of 7 mature female New Zealand White (NZW) rabbits (Tierforschungszentrum, University of Ulm, Germany) were benumbed by shooting a bolt into the back of their heads and exsanguinated by cutting their carotid arteries. The abdomen was opened with a scalpel and the aortic vessel prepared by removal of the connective tissue. Endothelium denudation of the abdominal and thoracic aorta was then performed in all animals in situ with a 3F Fogarty catheter (Baxter Inc., Unterschleissheim, Germany) which was pushed into the vessel through an incision at the iliac bifurcation. After inflation with natrium chloride 0.9 % the balloon was pulled through the whole vessel one time. The now denuded aortas were excised by saving the adventitial tissue. Each aorta was cut into sec-tions of 5 mm and these aortic rings were randomised into 14 groups. Three experimental test series were performed ( Table 1): 1) Effect of the pure synthetic estrogen receptor antagonist ICI 182,780 (Schering, Berlin, Germany) at concentrations of 20, 30 and 40 µM (n = 8 each). 2) Effect of 40 µM 17β-estradiol (Sigma, Deisenhofen, Germany) alone and in combination with 20 µM ICI 182,780, the lowest concentration having been found to have no effect on neointimal proliferation in test series 1 (n = 12 each).

Immunohistochemistry, morphometry, statistical evaluation
The sections were fixed in 4 % formaline, embedded in paraffin, and serially cut (4 µm slices) until the maximal thickness of the neointima was reached. Haemalaun and Eosin staining was performed for a first morphological analysis. To identify smooth muscle cells and myofibroblasts among medial and neointimal cells immunohistochemical staining (biotin avidin peroxidase method) was performed with a monoclonal antibody against α-actin (mouse-anti-human; Renner Inc, Darmstadt, Germany). Elastica-van-Gieson's staining was performed for the morphometry of the neointima and media (software package from Bilaney Consulting Inc, Düsseldorf, Germany). The neointimal area was defined the area between lamina elastica interna and lumen. The media area was defined the area between lamina elastica interna and externa. Histomorphometry was done in a blinded fashion. The effect on neointima formation is expressed as the neointima/ media ratio (median and 1 st /3 rd quartile). The Wilcoxon two-sample test was used to determine statistical significance at a level of p = 0.05.

Morphoplogical aspects
Aortic rings were morphologically intact after 21 days of cultivation and treatment as seen by microscopy. The lamina elastica interna and externa was intact as well. The media area (area between lamina elastica interna and externa) contained vascular smooth muscle cells which were made visible by α-actin staining. Sections had different amounts of neointima formation that contained vasculars mooth muscle cells surrounded by connective tissue (Figure 2).

Effect of ICI 182,780 on post injury neointima formation
Administration of ICI 182,780 in concentrations of 20, 30 and 40 µM did not cause a significant (p = 0.05) effect on post injury neointima formation (neointima/media-ratio) over 21 days (Figure 3). Regarding the highest concentration of 40 µM ICI 182,780 treatment the p-value did only reach 0.0875 which was in part due to the wide range of the measured data.

Effect of the phytoestrogens genistein and daidzein on post injury neointima formation
Treatment with 20 µM Genistein resulted in a significant (p = 0.0083) reduction of post injury neointima formation (neointima/media-ratio) after 21 days ( Figure 5). This effect was not modulated by additional treatment with 20 µM ICI 182,780. However, treatment with 40 µM daidzein did not result in a statistically significant neointima formation (p = 0.0637) in this experiment. Additional treatment with 20 µM ICI 182,780 also did not cause a significant effect.

Discussion
The purpose of this in vitro experiment was to investigate whether inhibitory estrogen and phytoestrogen effects on post injury neointima formation, as demonstrated previously [22][23][24][25], can be influenced by the pure synthetic estrogen receptor antagonist ICI 182,780. Because aortic rings from the whole aortic tree were used as target organs and because the lumen diameter varies between different aortas and parts of the aortic tree (thoracic and abdominal aorta), the neointima-/media-ratio was calculated as the basis for statistical analysis. In a first step the effect of different ICI 182,780 dosages from 20 µM to 40 µM was tested. These dosages corresponded to estrogen and phytoestrogen dosages that had been established as effective in this organ culture model before [25]. ICI 182,780 treatment in the three dosages did not cause a significant effect on post injury neointima formation (neointima-/ media ratio). Focussing on neointima formation independent from the media area (data not shown) there was a statistically significant (p = 0,0392) inhibitory effect at a concentration of 40 µM ICI 182,780. Because it has been shown previously that ICI 182,780 at concentrations >50 µM directly affects the metabolism of 17β-estradiol it did not make sense to use higher concentrations in this experiment [34].

Neointima-/ Media -Ratio
periments [25]. While a weak tendency of an inhibitory effect was visible in this present experiment, speculations may be allowed on the effect of higher concentrations of daidzein. Additional treatment with 20 µM ICI 182,780 did not influence the daidzein effect.
Genistein is well investigated as an inhibitor of protein tyrosine kinases [20,21]. This may account for estrogen receptor independent antiproliferative properties. Genistein's analogue daidzein lacks tyrosine kinase activity which could explain the different effects in our experiment [21,35,36].
On the base of this presented data the inhibitory estrogen effect of both, 17β-estradiol and the phytoestrogens, is unlikely mediated by estrogen receptor pathways. Our data support observations from experiments with female mice which were deficient of one of the two known estrogen receptor (ER) subtypes α or β. Physiologic levels of 17β-estradiol, when compared with ovariectomized animals, were able to reduce neointimal proliferation after vascular injury in both, ER-a [37] and ER-β [38] deficient mice. Moreover, our data support findings from Dubey et al. who demonstrated that phytoestrogens like genistein and daidzein were able to inhibit human aortic smooth muscle cell proliferation and migration by estrogen receptor independent, i. e., mitogen-activated protein (MAP-) kinase modulated pathways [39]. The authors, too, used the synthetic ICI 182,780 for their control experiments. ICI 182,780 was first introduced by Wakeling et al. [33] and has been characterized as a competitive antagonists to estrogens with high affinity to the estrogen receptor and without a significant estrogenic activity itself.
But our data is in contrast to previous in vivo findings from Bakir et al. [40]. The authors demonstrated an inhibitory ICI 182,780 effect on 17β-estradiol induced reduction of post injury neointima formation in rat carotid arteries. However, another organ culture experiment (pulmonary arteries from hypoxic rats) by Karamsetty et al. demonstrated that 10 µM 17β-estradiol, 30 µM genistein, and 30 µM daidzein enhanced the relaxation response to carbachol. This was found to be a result of increased nitric oxide synthesis and release. But this effect could not be mediated by additional treatment with 10 µM ICI 182,780 [41]. The authors used estrogen and ICI 182,780 at concentrations comparable to our experiment. New findings on slow genomic (transcriptional) and rapid nongenomic (i.e., MAP kinase) estrogen effects [39,42,43] and on different estrogen receptor subtypes (estrogen receptor α and β) [1-3] may lead to deeper insights into the pharmacology of this system. Quite subtle ICI 182,780 effects on estrogen receptors could be demonstrated in specific cells of the sheep uterus [44] and in yeast genetic systems [45]. The acute and rapid estrogen induced calcium-dependent release of NO could be blocked by ICI 182,780 in bovine aortic endothelial cells [46] and in a human vascular endothelial cell (HUVEC) system [47].
The question remains which estrogen receptor independent mechanism(s) may have led to the demonstrated in-  Neointima-/ Media -Ratio hibitory estrogen/phytoestrogen effect on post injury neointima formation in our present experiment. Dubey et al. were able to demonstrate that not 17β-estradiol but endogenous metabolites like methoxyestradiols, with no affinity to estrogen receptors, could be responsible for ERindependent antimitogenic effects on vascular smooth muscle cells [48]. Metabolites like 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was 17βestradiol in inhibiting DNA synthesis, collagen synthesis, cell proliferation and migration [34]. Cytochrome-P450 played a key role in this local estrogen metabolism and ICI 182,780 at concentrations >50 µM inhibited this metabolism. In addition, other nongenomic, i.e., antioxidant mechanisms have to be discussed. Yoon at al. demonstrated inhibitory 17β-estradiol effects on vascular smooth muscle cell proliferation (VSMC from rats) induced by lysophosphatidylcholine (component of oxidized LDL) or reactive oxygen species (ROS) [49]. The inhibitory 17βestradiol effect was not antagonized by ICI 182,780. Furthermore, calcium antagonistic gender independent properties have been described for 17β-estradiol [15,16] and for several phytoestrogens [50].