Upregulation of estrogen receptor α and mediation of 17β-estradiol vasoprotective effects via estrogen receptor α in basilar arteries in rats after experimental subarachnoid hemorrhage

Laboratory investigation

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Object

The authors previously demonstrated that 17β-estradiol benzoate (E2) treatment prevents subarachnoid hemorrhage (SAH)–induced cerebral vasospasm and preserves endothelial nitric oxide synthase (eNOS) in male rats. Changes in the expression of estrogen receptor (ER) subtypes ERα and -β and their roles in the E2-mediated preservation of eNOS in SAH remain unknown. In the present study the effects of SAH on the expression of ERα and -β in the cerebral arteries were clarified, and the receptor roles in the E2-mediated preservation of eNOS expression in SAH were differentiated.

Methods

A 2-hemorrhage SAH model was induced by 2 autologous blood injections into the cisterna magna of adult male rats. The effect of SAH on ERα and -β expression was evaluated. Other rats subcutaneously received implanted Silastic tubes containing corn oil with E2 and daily injections of various doses of an ERα- (methyl-piperidinopyrazole [MPP]) or ERβ-selective antagonist (R,R-tetrahydrochrysene) after the first hemorrhage. The protein levels of ERα, ERβ, eNOS, and inducible nitric oxide synthase (iNOS) from basilar arteries were examined using Western blot analysis, and their mRNAs were evaluated by reverse transcription–polymerase chain reaction.

Results

The ERα but not the ERβ was upregulated in the basilar artery after SAH. Treatment with MPP eliminated E2-mediated effects in SAH, relieved cerebral vasospasm, preserved eNOS expression, and suppressed iNOS expression.

Conclusions

Estrogen receptor α is upregulated in the basilar artery after SAH. Note that E2 exerts its protective effects through ERα-dependent pathways to relieve cerebral vasospasm and preserve eNOS expression. A selective ERα agonist may be the drug of choice for the treatment of patients with SAH.

Abbreviations used in this paper: BA = basilar artery; E2 = 17β-estradiol benzoate; eNOS = endothelial nitric oxide synthase; ER = estrogen receptor; FoxM1 = Forkhead box; GAPDH = glycer-aldehyde-3-phosphate dehydrogenase; IgG = immunoglobulin G; iNOS = inducible NOS; MPP = methyl-piperidino-pyrazole; NADPH = nicotinamide adenine dinucleotide phosphate; PCR = polymerase chain reaction; ROS = reactive oxygen species; RT = reverse transcription; R,R-THC = R,R-tetrahydrochrysene; SAH = subarachnoid hemorrhage; SD = standard deviation; STAT5 = signal transducer and activator of transcription 5.

Article Information

Address correspondence to: Shen-Long Howng, M.D., Ph.D., Department of Neurosurgery, Kaohsiung Medical University Hospital, Taiwan, Republic of China. email: slhowng@cc.kmu.edu.tw, or to Yi-Ren Hong, Ph.D., Graduate Institute of Biochemistry, Kaohsiung Medical University, Taiwan, Republic of China. email: m835016@cc.kmu.edu.tw.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Gel blots and bar graphs demonstrating SAH-induced changes in the levels of ERα (a) and -β (b) mRNA in the BAs. The ERα mRNA was upregulated after SAH (a), whereas ERβ mRNA remained unchanged after SAH (b). Graphs represent quantitative results of 3 pooled samples (9 rats) per group (18 rats total). Values represent the means ± SD. The RT-PCR products of β-actin were used as internal controls. **p < 0.01.

  • View in gallery

    Gel blots and bar graphs revealing SAH-induced changes in the levels of ERα and -β protein levels in BAs. The ERα protein was upregulated after SAH (a), whereas ERβ protein remained unchanged after SAH (b). Graphs show the quantitative results of 3 pooled samples per group (9 rats per group, 18 rats total). Membrane was probed with anti–β-actin antibody to verify equivalent loading. **p < 0.01.

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    Photomicrographs of BA cross-sections and bar graphs showing the effect of E2 on SAH-induced vasospasm. a: Note that E2 inhibited SAH-induced vasospasm in male rats. Pretreatment with MPP (2.0 mg/kg body weight) blocked the E2 effects on SAH-induced vasospasm. b: Note the MPP dose–dependent inhibition of E2-induced protective effects. Graphs represent quantitative results of 27 rats (3 in each group). Five cross-sectional areas from each BA were averaged. **p < 0.01, *p < 0.05. Bar = 100 μm. The SAH+, E2+, and MPP-groups and the SAH+, E2+, and MPP (2 mg/kg) groups in panel a are the same as in panel b.

  • View in gallery

    Photomicrographs of BA cross-sections and bar graph revealing the effect of R,R-THC on E2-mediated relief of SAH-induced vasospasm. Pretreatment of rats with 3 escalating doses of R,R-THC (0.05, 0.1, 0.2 mg/kg body weight) failed to block the E2 effects on SAH-induced vasospasm. Graph represents quantitative results for 18 rats (3 in each group). **p < 0.01. Bar = 100 μm.

  • View in gallery

    Gel blots and bar graphs demonstrating ERα-dependent inhibition of E2 in the SAH-induced suppression of eNOS mRNA and induction of iNOS mRNA in rat BAs. Pretreatment of rats with E2 and MPP (2.0 mg/kg body weight) inhibited E2-mediated preservation of eNOS mRNA in SAH (a) and E2-mediated suppression of iNOS mRNA in SAH (b). Note that R,R-THC (0.1 mg/kg body weight) failed to inhibit the E2-mediated effects on SAH-induced eNOS mRNA decreases or iNOS mRNA increases. Graphs represent quantitative results of 3 pooled samples per group (9 rats per group, 54 rats total). The RT-PCR products of GAPDH were used as internal controls. **p < 0.01.

  • View in gallery

    Gel blots and bar graphs showing the ERα-dependent inhibition of E2 in the SAH-induced suppression of eNOS protein and induction of iNOS protein in rat BAs. Pretreatment of rats with E2 and MPP (2.0 mg/kg body weight) inhibited E2-mediated preservation of eNOS protein in SAH (a) and E2-mediated suppression of iNOS protein in SAH (b). Note that R,R-THC (0.1 mg/kg body weight) failed to reverse the E2-mediated effects on SAH-induced eNOS protein decreases or iNOS protein increases. Graphs represent quantitative results of 3 pooled samples per group (9 rats per group, 54 rats total). Membrane was probed with anti-β–tubulin antibody to verify equivalent loading. **p < 0.01.

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