Specific repression of the preproendothelin-1 gene in intracranial arteriovenous malformations

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✓ Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction—specifically, deregulation of endothelin-1 (ET-1) secretion—contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge—Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.

Article Information

Address reprint requests to: Youssef G. Comair, M.D., F.R.C.S.(C), Department of Neurosurgery, Cerebrovascular Biology Laboratory, Cleveland Clinic Foundation, 9500 Euclid Avenue, Desk S80, Cleveland, Ohio 44195.

© AANS, except where prohibited by US copyright law.

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    Photomicrographs showing that AVM vascular lesions fail to secrete detectable levels of ET-l peptide. Frozen sections of normal brain (A and B) or AVMs (C-F) were selected to localize ET-l secretion using immunohistochemistry. The ET-l secretion is visualized an as orange-brown stained immunoreactivity found in the endothelium (arrows) of normal brain (A), but not in AVMs with thin-walled (C) or thick-walled (E) vessels. Control specimens tested with nonimmune serum in normal (B) and AVM (D and F) endothelium demonstrated that immunoreactivity was specific for ET-1. Identical results were observed in all 17 patients with AVMs. Original magnification × 100.

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    Photomicrographs demonstrating that ET-l secretion is specifically inhibited in AVM vasculature. The ET- 1 secretion was analyzed using immunohistochemistry in blood vessels immediately proximal to or distal from the AVM vasculature. The ET-I secretion by the endothelium (arrows) was normal in smaller (A) and larger (C) vessels that were not directly involved with the AVM lesions. Testing of negative controls (B and D) was performed using nonimmune serum as described. Results are representative of six independent specimens from different AVM patients. A and B: original magnification × 200; C and D: original magnification × 100.

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    Photomicrographs showing that vascular expression of ET-I was elevated in Sturge-Weber vascular malformations. An immunohistochemical analysis of specific (A) and nonspecific (B) ET- 1 staining in frozen sections from Sturge-Weber vascular malformations was performed. As shown in A, ET- 1 secretion was elevated in Sturge-Weber malformations compared to normal controls. Identical results were observed in all seven sections tested from different patients. Original magnification × 100.

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    Results of the RT-PCR analysis of ppET-1 mRNA levels. A: Schematic diagram of the human ppET-1 gene indicating the exon/intron structure and positions of upstream and downstream primers spanning exons 1 and 3 (El and E3). The coding sequences are shaded. B: Autoradiogram. Increasing amounts of total RNA were analyzed for ppET-1 mRNA levels using RT-PCR and the 372.bp amplicon was separated by agarose gel electrophoresis. C: Graph of the results of scanning densitometry of the gel shown in B demonstrating that the amount of amplicon produced was directly proportional to the amount of input DNA, verifying that the assay was linear under these conditions.

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    Repression of preproET-1 (ppET-1) mRNA in AVM lesions. A: Autoradiogram. Total RNA was extracted from a microdissected AVM, normal brain (NB), and Sturge—Weber vessels and analyzed by RT-PCR for ppET-1 mRNA levels. Normal brain cerebrovasculature expresses abundant amounts of the ppET-1 mRNA, whereas levels in AVMs are essentially undetectable. The mRNA levels in Sturge—Weber lesions are elevated. NB-RT = RT-PCR in NB in which the RT reaction was omitted; ET-1 and GAPDH are PCR control reactions using cDNA inserts as templates. B: Graph displaying the results of a densitometric analysis of ppET-1 mRNA from six patients with AVMs, four patients with Sturge—Weber lesions, and four normal controls. Data are expressed as the mean ± SEM.

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    Bar graph demonstrating that ET-1 peptide secretion is repressed in endothelial cell strains cultured from AVM vessels. Endothelial cell strains from normal cerebrovasculature and AVMs were incubated in serum-free medium for 4 hours. The medium was extracted and measured for immunoreactive ET-1 as described in Materials and Methods. Data are corrected for cellular protein in each well. Human umbilical vein endothelial cells (HUVEC) were used as a positive control. Data are expressed as the mean ± SEM from two independent experiments in cell strains obtained from two subjects.

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    Repression of preproET-1 (ppET-1) mRNA in cultured AVM endothelial cells. prot. = protein. A: Autogradiogram showing the results of an RT-PCR analysis of ppET-1 mRNA levels in total RNA extracted from AVM (lane 1) and normal brain (NB) cerebrovascular (lane 2) endothelial cells. B: Bar graph displaying the results of a densitometric analysis of RT-PCR measurements of ppET-1 mRNA levels in two independent endothelial cell strains derived from patients with normal or AVM vessels. Data are expressed as the mean ± SEM.

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    Autoradiograms showing the results of an immunoprecipitation of ETA and ETB receptors from AVM and normal cerebrovasculature in vivo. The AVM and normal cerebrovasculature were microdissected and homogenized in RIPA buffer. Polyclonal anti-peptide antisera raised against specific epitopes in rat ETA and ETB were used for immunoprecipitation. The immunoprecipitates were resolved by electrophoresis, transferred to nitrocellulose, and the same antibodies were used to localize ETA and ETB proteins using chemiluminescent detection as described in Materials and Methods. Reactions with nonimmune serum confirmed the specificity of the immunoprecipitation; the band at 55 kD is the heavy chain of sheep IgG. Identical results were observed in two independent experiments using different patient samples.

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