Cerebral aneurysm progression suppressed by blockage of endothelin B receptor

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Object

Cerebral aneurysm is a major cause of subarachnoid hemorrhage, but the mechanisms of its development remain unclear. Mechanical stretch has been reported to induce vascular smooth-muscle cell apoptosis via endothelin B receptors (ETBRs). The objectives of this study were to clarify the expression and localization of ETBR in cerebral aneurysms and to examine the effect of ETBR blockage on the development of experimental cerebral aneurysms.

Methods

Seventy-two rats underwent a cerebral aneurysm induction procedure and were divided into four groups according to the duration of postoperative study periods. Expression of ETBR was confirmed by reverse transcription–polymerase chain reaction and immunohistochemical analysis. The authors also studied the effect of K-8794, an oral selective antagonist of ETBR, to see whether it would influence the formation of cerebral aneurysms.

Two weeks after the aneurysm induction procedure, ETBR was rarely detected in anterior cerebral artery–olfactory artery bifurcations, but it was weakly expressed in experimental cerebral aneurysms at 1 month after the procedure, and markedly expressed at 3 months. The administration of K-8794 for 1 month after the procedure significantly reduced the number of advanced aneurysms and the number of apoptotic smooth-muscle cells.

Conclusions

These results suggest that ETBR might play a significant role in the progression of cerebral aneurysms and have the potential to improve prevention and treatment of cerebral aneurysms.

Abbreviations used in this paper:ACA = anterior cerebral artery; α-SMA = α–smooth muscle actin; BP = blood pressure; ETAR = endothelin A receptor; ETBR = endothelin B receptor; IEL = internal elastic lamina; JNK = c-jun N-terminal protein kinase; MCA = middle cerebral artery; NO = nitric oxide; OA = olfactory artery; PCR = polymerase chain reaction; RT-PCR = reverse transcriptase PCR; ssDNA = single-stranded DNA; VSMC = vascular smooth-muscle cell.

Article Information

Address reprint requests to: Kazuhiko Nozaki, M.D., Ph.D., Department of Neurosurgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan. email: noz@kuhp.kyoto-u.ac.jp.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Photomicrographs of right ACA–OA bifurcations. A: Control group. B: 2W group. There is no apparent aneurysmal invagination. C: 1M group. A small aneurysm (arrow) is seen at the bifurcation. D: 3M group. Bar = 100 μm.

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    Photomicrographs of ETBR-immunostained sections of experimental cerebral aneurysms. Panels A and C depict images of adjacent sections from a single animal as do panels B and D. A: An orcein-stained specimen from the 2W group. The IEL is disconnected near the apex of the ACA–OA bifurcation, and there is no apparent aneurysmal dilation; this is classified as an early aneurysmal change. B: An orcein-stained specimen from the 1M group. The IEL has completely disappeared, and a bulge has appeared; this is classified as an advanced aneurysm. C: There was no apparent ETBR immunoreactivity in the 2W group. D: Some spotty immunostaining (arrows) is visible in the medial layer of this section from a rat from the 1M group. E: Marked expression of ETBR is evident in and around the advanced aneurysm from an animal from the 3M group. F: In this higher magnification of the image in panel E, it is apparent that ETBR is localized in the medial layer as well as in the intimal layer. Bar = 100 μm.

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    A and B: Photomicrographs showing expression of ETBR in a human cerebral aneurysm. Expression of ETBR is mainly apparent in the medial layer of the arterial wall. C: Photomicrograph of a section of a normal MCA from an autopsy sample revealing ETBR immunoreactivity only in the intimal layer. Bars = 500 μm (A), 100 μm (B), and 50 μm (C). Lu = lumen.

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    Photomicrographs of stained sections of experimental cerebral aneurysms. Double staining reveals the colocalization of ssDNA, α-SMA (aSMA), cleaved caspase-3, and ETBR in experimental cerebral aneurysms (arrows) from the 3M group (A, B, C, G, H, I) and the 1M group (D, E, F). Panels C, F, and I show the merged images of A and B, D and E, and G and H, respectively. The ETBR immunoreactivity was partly colocalized with α-SMA (C) and ssDNA (F, arrowheads). Note that the ETBR immunoreactivity is strong and limited around the cerebral aneurysm of the 3M group (A). The number of apoptotic VSMCs was assessed by double staining for ssDNA and α-SMA (I, arrowheads). The distribution of ssDNA (J) is almost the same as that of cleaved caspase-3 (K, specimen adjacent to that shown in J). Bar = 100 μm.

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    A: Results of semiquantitative RT-PCR of ACA–OA bifurcation samples. B: Graph showing the relative expression ratios of ETBR and β-actin. *p < 0.0001, †p < 0.05.

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    Photomicrographs showing the results of the K-8794 study. A and B: An orcein-stained section from an animal in the K-8794 group (A) and an animal in the 1M group (B). In the K-8794 group, an early aneurysmal change (A) was observed in five of eight rats. In the 1M group, no early aneurysmal change was observed and advanced aneurysms (B) were found in all rats. C and D: Sections stained for ssDNA. There were significantly fewer ssDNA-positive cells in sections from animals in the K-8794 group (C) than in those from animals in the 1M group (D). Bar = 100 μm.

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