Background
The rupture of cerebral aneurysms elicits devastating subarachnoid hemorrhage (SAH). Surgical and endovascular treatments can be preventive but are associated with inherent risks and complications [
1]. Therefore, medical therapy with lower risks for complications is needed to prevent SAH.
The incidence of cerebral aneurysms and SAH is higher in post- than premenopausal women [
2,
3]. Post-menopausal estrogen deficiency is thought to play a key role in the pathophysiology of cerebral aneurysms. Our group established a cerebral aneurysm model in ovariectomized rats subjected to hemodynamic stress and hypertension [
4]. The model mimics human postmenopausal hormonal conditions. Estrogen-deficient model rats had a higher incidence of cerebral aneurysms than male rats, and estrogen deficiency promoted endothelial damage and vascular inflammation [
5‐
8]. Estrogen (17β-estradiol) and an estrogen receptor β (ERβ)-, but not an ERα agonist, reduced the incidence of cerebral aneurysm rupture in ovariectomized mice [
9]. These findings suggest that ER-related drugs may help to prevent SAH. In humans, postmenopausal estrogen replacement therapy reduced the risk of SAH [
10‐
12] but increased the risk for breast and endometrial cancer, stroke, and venous thromboembolism [
9,
13‐
15].
Bazedoxifene (BZA), a selective estrogen receptor modulator (SERM), is used to prevent postmenopausal osteoporosis. It has fewer estrogenic side effects than conjugated estrogen. SERM is a ligand of ERα and ERβ; its characteristics are different from pure ER agonists and ER-antagonists because, depending on the target tissue, it exerts agonistic or antagonistic effects [
16]. Its tissue-specific effects are thought to be attributable to its distinct affinity for each ER, a unique conformational change in ERs upon binding to SERM, and the distinct distribution of ERα and ERβ in the tissue [
16,
17]. Therefore, we used our rat model to investigate whether BZA may represent a potential and safer prophylactic agent against cerebral aneurysm rupture in postmenopausal women.
We established a novel rat model of aneurysm rupture by modifying the carotid ligation procedure used in our original model in which ovariectomized rats were subjected to hemodynamic changes and hypertension without pharmacologically elicited degeneration of the arterial wall [
18]. The pathological features of the experimental aneurysms in our ovariectomized hypertensive rats were those of human aneurysms. In the posterior cerebral artery (PCA) where ruptured aneurysms formed most frequently, the mRNA level of interleukin (IL)-1β was higher and the imbalance of matrix metalloproteinase (MMP)-9 and the level of the tissue inhibitor of metalloproteinase-2 (TIMP-2) were greater than at the bifurcation of the anterior cerebral artery-olfactory artery (ACA-OA) where no ruptured aneurysms were observed. We suggested that these molecules associated with inflammation and vascular degradation are at least partly responsible for aneurysmal rupture [
18]. Here, we used our modified rat aneurysm model to test the hypothesis that BZA exerts protective effects against vascular inflammation and aneurysm rupture.
We now demonstrate that BZA reduced the incidence of aneurysmal rupture without affecting the blood pressure in ovariectomized aneurysm-model rats. Its restoration of the decreased expression of both ERs in the aneurysm-prone cerebral artery was associated with the reduction of pro-inflammatory cytokines and vascular degradation molecules. We also show that BZA attenuated the body weight gain seen under conditions of estrogen deficiency and hypertension. Our findings suggest BZA as a potential therapeutic agent for the prevention of SAH in postmenopausal women.
Discussion
We demonstrate that ovariectomy promoted aneurysmal rupture in hypertensive rats subjected to hemodynamic stress. We first document that in our hypertensive, ovariectomized aneurysm-model rats, the downregulation of ERs and the upregulation of pro-inflammatory molecules in the rupture-prone cerebral artery were associated with an increase in the aneurysmal rupture rate and that BZA ameliorated the downregulation of ERs, restored their gene and protein expression, decreased the incidence of aneurysmal rupture, and lowered the level of pro-inflammatory- and vascular degradation molecules. The incidence of ruptured aneurysms and the mRNA level of ERs and pro-inflammatory and vascular degradation molecules were similar to those in HT rats with intact ovaries. These findings suggest that BZA prevented aneurysmal rupture associated with inflammation and vascular degradation via the downregulation of ERs in our ovariectomized aneurysm-model rats.
Estrogen exerts diverse vascular effects mediated mainly by ERα and ERβ [
23]. The treatment of ovariectomized rats with estradiol reversed the decrease in ERα and ERβ expression in the cerebral vessel wall [
19]. Using our original aneurysm model, we demonstrated that estrogen deficiency elicited by ovariectomy reduced the expression of ERα while the expression of ERβ was retained at the bifurcation of the ACA-OA where unruptured aneurysms were highly and reproducibly induced [
6]. The present study showed that the expression of ERα and ERβ in the PCA prone to aneurysmal rupture was reduced in our ovariectomized aneurysm rats and that BZA upregulated both ERα and ERβ in the PCA. Elsewhere, our group documented that in ovariectomized aneurysm mice, treatment with estrogen or an ERβ-, but not an ERα agonist, reduced the incidence of cerebral aneurysm rupture [
9]. The downregulation of ERβ is thought to be associated with inflammation [
24]. We suggest now that in our current rat aneurysm model, BZA exerted protective effects against rupture through ERβ stimulation.
IL-1β promotes extracellular matrix degradation by increasing the production of MMPs and decreasing the production of TIMPs [
25,
26]. TNF-α and IL-6 also stimulate the expression of MMP-9 [
27,
28]. In our current and earlier studies [
18,
22], the increase in IL-1β and TNF-α and the imbalance between MMP-9 and TIMP-2 were associated with aneurysm rupture in ovariectomized aneurysm rats. BZA abated the increase in IL-1β and the imbalance between MMP-9 and TIMP-2 and resulted in a moderate inhibition of TNF-α. ERβ stimulation decreased cellular inflammasome activity and IL-1β expression after global cerebral ischemia in ovariectomized rats [
24]. Selective ERβ agonists repress the transcription of pro-inflammatory genes including TNF-α [
29]. The ERβ agonistic effects of BZA may be responsible for the downregulation of pro-inflammatory cytokines, resulting in the inhibition of vascular degradation.
Others reported that TNF-α modulates cerebral aneurysm formation and rupture. The TNF-α inhibitor 3,6’dithiothalidomide resulted in aneurysmal stabilization and a significant decrease in the rupture rate [
30]. In contrast, a meta-analysis reiterated that IL-6 was not associated with the pathogenesis of intracranial aneurysms [
31]. These studies support our present findings.
In women, ERα activation in the brain stimulated physical activity and reduced their food intake and body weight gain [
32,
33]. Therefore, the effect of BZA at 1 mg/kg, but not 0.3 mg/kg, on the body weight of our rats may have involved the upregulation of ERα. On the other hand, since ERβ is thought to be associated with vasoprotection, the effect of BZA on the body weight via the upregulation of ERα may be smaller than the vasoprotective effects elicited by ERβ. Estradiol and a selective ERα agonist increased the uterine weight in mice [
15]. However, BZA had no such effects in the current and an earlier study [
20], indicating that its effects are tissue-specific.
Use of our original rat aneurysm model resulted in a high incidence (more than 80%) of unruptured aneurysms at the ACA-OA bifurcation; at most, 10% of these aneurysms ruptured during the 12-week observation period. For the current study, we modified the hemodynamics of our original model and produced a novel model in which approximately 50% of the experimentally induced aneurysms spontaneously ruptured [
18]. With this new model, we demonstrated the protective effect of BZA, a selective ER modulator, on cerebral aneurysm rupture. The efficacy for prevention of aneurysm rupture may provide a new therapeutic option in the clinical setting.
Our study has some limitations. First, we assessed the changes in the mRNA level and the protein expression of the ERs elicited by ovariectomy and BZA. For a better understanding of the effects of individual ERs, studies using specific ER agonists or inhibitors and knockout animal are needed. Second, we started the administration of BZA 2 weeks after aneurysm induction without confirming the presence of cerebral aneurysms, and we did not confirm the presence of unruptured aneurysm in rupture-prone arteries at 12 weeks. Therefore, it is unclear whether BZA prevented their formation or their rupture. Third, the rodent age may affect their vascular response to estrogen [
34], and menopause simulated by ovariectomy in our rats may be different from natural menopause in aged rats. Further studies are underway to address these questions.