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(Circulation Research. 2001;0:hh1401.094281.)
© 2001 American Heart Association, Inc.

Report

Abnormal Balance in the Angiopoietin-Tie2 System in Human Brain Arteriovenous Malformations

Presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, Calif, October 17, 2000, and published in abstract form (Anesthesiology. 2000;93:A360).

Tomoki Hashimoto, Tiffany Lam, Nancy J. Boudreau, Andrew W. Bollen, Michael T. Lawton William L. Young

From the Departments of Anesthesia and Perioperative Care (T.H., T.L., W.L.Y.), Surgery (N.J.B.), Pathology (A.W.B.), Neurological Surgery (M.T.L., W.L.Y.), and Neurology (W.L.Y.), University of California, San Francisco, Calif.

Correspondence to William L. Young, MD, Center for Cerebrovascular Research, University of California, San Francisco, 1001 Potrero Ave, 3C-38, San Francisco, CA 94110. E-mail CCR{at}anesthesia.ucsf.edu

Abstract

Brain arteriovenous malformations (BAVMs) are congenital vascular lesions that often present as cerebral hemorrhage in young adults. The variable nature of the clinical course, especially with respect to spontaneous hemorrhage, recurrence, growth, and regression, suggests that BAVMs are lesions with active angiogenesis and vascular remodeling. We examined mRNA and protein expression of angiopoietin 1 (Ang1) and Ang2 by semiquantitative reverse transcriptase–polymerase chain reaction, in situ hybridization, and Western blot in BAVMs and control brains obtained from temporal lobectomy for medically intractable seizures. Although Ang1 mRNA levels were similar in BAVMs and controls, Ang1 protein levels were 30% lower in BAVMs than in controls. Ang2 mRNA levels were 40% higher and Ang2 protein levels were 8-fold higher in BAVMs than in controls. In situ hybridization showed that the Ang2 mRNA was localized to the perivascular area in BAVMs. This abnormal balance in the Ang-Tie2 system may, in part, explain the aberrant vascular phenotype in BAVMs.

Key Words: KEY WORDS: cerebral arteriovenous malformations • vascular malformations • endothelium • angiopoietin • Tie2

Brain arteriovenous malformations (BAVMs) are presumed to be sporadic congenital lesions resulting from abnormal vascular development.^{1 2} The variable nature of the clinical course, especially with respect to spontaneous hemorrhage,³ recurrence,¹ growth, and regression,⁴ strongly suggests active vascular changes in most lesions. Such changes have been speculated to involve angiogenesis and vascular remodeling.^{1 2}

Angiopoietins (Ang) and their receptor, Tie2, play a critical role in angiogenesis and vascular stability.^{5 6 7 8 9} Ang1, an agonist for the Tie2 receptor, promotes interaction between endothelial cells (ECs) and peri-EC support cells to stabilize vessels.^{5 6} Ang2 is an antagonist for the Tie2 receptor that acts to destabilize these attachments by preventing Ang1 stimulation of Tie2.⁷ The vascular phenotypes observed by overexpression of Ang2⁷ or homozygous disruption of the Tie2 gene^{5 8 9} are strikingly similar to BAVM vessels. They display abnormally dilated vessels that lack mature peri-EC support structure. Similarly, loss-of-function mutations in the Tie2 gene are found in patients with familial mucocutaneous venous malformations,¹⁰ vascular lesions resembling BAVMs. Previously, we demonstrated decreased Tie2 expression in BAVMs. In this study, we describe an abnormal balance between Ang1 and Ang2 expression at both protein and mRNA levels.

Materials and Methods

Materials
We collected BAVM specimens from 7 women and 9 men (32±14 years of age), as previously described.² All patients received embolization therapy before BAVM resection. Control subjects consisted of 2 women and 2 men (30±11 years of age). The protocol of this study complies with the guidelines for the conduct of research involving human subjects by the National Institutes of Health and the Committee on Human Research at the University of California San Francisco and Columbia University College of Physicians and Surgeons.

Reverse Transcriptase–Polymerase Chain Reaction, Northern Blot, In Situ Hybridization, and Western Blot
Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), in situ hybridization, Western blot, and Northern blot were performed as previously described.^{2 11 12} Primer sets for Ang1, Ang2, and Tie2 were from R&D Systems. Primary antibodies against Ang1 and Ang2 were provided by Regeneron Pharmaceutical (Tarrytown, NY).

Statistical Analysis
mRNA levels are normalized by 18S rRNA. Data (mean±SD) are presented as a relative expression, with mean control as 100%, and compared by the Mann-Whitney test.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Results

Using semiquantitative RT-PCR, we observed that Ang1 mRNA levels were similar in the BAVMs (n=5) and controls (n=3) (105±6% versus 100±4%, P=0.46) (Figure 1A). Western blot analysis, however, revealed that Ang1 protein levels were lower in the BAVMs (n=10) compared with controls (n=3) (66±15% versus 100±14%, P=0.01) (Figure 1B). Using semiquantitative RT-PCR, we found that Ang2 mRNA levels were higher in the BAVMs (n=5) compared with the controls (n=3) (139±17% versus 100±12%, P=0.03) (Figure 1C). Northern blot analysis confirmed high levels of Ang2 mRNA in the BAVMs, whereas Ang2 mRNA was not detectable in the two controls (see the online data supplement, available at http://www.circresaha.org). Western blot analysis showed that Ang2 protein levels were markedly higher in the BAVMs (n=10) compared with controls (n=3) (754±147% versus 100±40%, P=0.01) (Figure 1D). Analysis using semiquantitative RT-PCR revealed that Tie2 mRNA was lower in the BAVMs (n=6) than in controls (n=3) (65±15% versus 100±16%, P=0.02).

View larger version (45K): [in this window] [in a new window]   Figure 1. Expression of Ang1 mRNA, Ang1 protein, Ang2 mRNA, and Ang2 protein in BAVMs and the control brain. In each panel (A, Ang1 mRNA; B, Ang1 protein; C, Ang2 mRNA; and D, Ang2 protein), top shows densitometric intensity of mRNA or protein levels expressed as a percentage of values obtained in control brain samples (mean±SD). (Ang mRNA levels are normalized by 18S rRNA.) Bottom shows representative gel from semiquantitative RT-PCR or Western blot. , Different sets of primer for 18S rRNA yielding different product sizes were used. Ang1 protein appeared at 150 kDa under nonreduced conditions (B). Ang2 protein appeared at 75 kDa under reduced conditions (D). CB indicates control brain; PC, positive control. *P<0.05.

H&E staining of the BAVMs demonstrated a preponderance of vessels that were free of intraluminal inflammation or organized thrombus (Figures 2A and 2B). CD31 immunohistochemistry showed that the EC layer in the BAVMs was intact (Figures 2E and 2F). In situ hybridization showed Ang1 mRNA in the perivascular area of both BAVM and control vessels (Figures 2I through 2K). Ang2 mRNA was abundantly expressed in the perivascular area of the BAVM vessels (Figures 2L and 2M). Ang2 mRNA was not detectable in the controls (Figure 2N). No signal was observed using sense probes (see the online data supplement).

View larger version (97K): [in this window] [in a new window]   Figure 2. Localization of Ang1 and Ang2 in BAVMs and the control brain. Photomicrographs of serial sections from BAVMs (top and middle) and the control brain (bottom) in H&E staining (A through D), CD31 immunostaining (E through H), in situ hybridization for Ang1 (I through K), and in situ hybridization for Ang2 (L through N). Middle panels show higher magnification of the insets in the top panels. Arrows indicate endothelial cell layer (top and middle) and cortical vessel (bottom). Endothelial layer was generally intact in both BAVMs (A, B, E, and F) and the CB (C, D, G, and H). BAVM vessels expressed both Ang1 (I and J) and Ang2 (L and M) in the perivascular areas. Cortical vessel in control brain expressed Ang1 (K) in the perivascular area but not Ang2 (N). Bar=100 µm.

There was no correlation between expression of Ang1 or Ang2 and the interval between embolization treatment and surgery (median, 1 day; range, 1 to 49 days) or the number of embolization treatments (median, 2; range, 1 to 4 days) (see the online data supplement).

Discussion

In this study, we found increased Ang2 mRNA and protein levels and decreased Ang1 protein levels in BAVMs. Previously, we described markedly decreased Tie2 protein in BAVMs,² consistent with the decreased Tie2 mRNA observed in this study. The decreased level of Tie2 receptor expression along with the reduction in the Ang1 would be expected to reduce Tie2 signaling, thus leading to vascular instability. Furthermore, a marked increase in the expression of the Tie2 antagonist Ang2 might be expected to additionally impair vascular stability in BAVMs. This may, in part, explain the aberrant vascular phenotype in BAVMs (ie, dilated vessels with a relative lack of mature peri-EC support).

The underlying causal mechanisms of our observed changes remain to be determined. We observed decreased Ang1 protein levels despite normal Ang1 mRNA levels, which may suggest increased proteolytic activity in BAVMs.¹³ Stimuli such as shear stress or ischemia may also affect the expression of Ang and Tie2. However, BAVM vessels are exposed to high rates of fully oxygenated blood flow² and are therefore unlikely to be ischemic. Normal eNOS expression in BAVMs² suggests that, at least at the time of microsurgical resection, shear stress may not be the sole cause of vascular remodeling in BAVMs. Because BAVM resection is associated with brain swelling,¹⁴ the decreased Ang1 may have some mechanistic involvement in vasogenic edema.¹⁵

Although embolization treatment may provoke angiogenesis through the formation of organized thrombi or recanalization processes, the majority of vessels in our study were free of intraluminal organizing thrombus. Moreover, Ang mRNAs were expressed in both embolized and nonembolized vessels in BAVMs, suggesting that embolization treatment is not the sole cause of the abnormal Ang expression.

The precise roles of the Ang-Tie2 system in healthy^{5 6 7 8 9} and diseased states^{10 16 17} are not completely understood. For example, vessels with different sizes may have different patterns of Ang expression. At high concentrations, Ang2 may act as an agonist for Tie2 receptor.¹⁸

Because of the practical difficulty in obtaining normal human brain specimens, we used structurally normal brain tissue obtained from temporal lobectomy for epilepsy treatment as our controls. Our previous report⁴ showed that, like normal ECs, the ECs in the control samples exhibited normal morphology and were quiescent. We also showed that BAVM and control specimens have a similar EC mass per unit weight using the method described by others.^{2 5 9}

In summary, there is an abnormal balance in the Ang-Tie2 system in BAVMs that may, in part, explain the pathological vascular phenotype of BAVMs.

Acknowledgments

This work was supported in part by National Institutes of Health Grants RO1-27713 and K24-NS02091 (to W.L.Y.). Regeneron, Inc, Tarrytown, NY, provided angiopoietin antibodies. The authors thank Gabriele Bergers, PhD, Dhanesh Gupta, MD, and Christopher Quick, PhD, for insightful suggestions; the neurosurgical services at Columbia University, New York and at the University of California, San Francisco (UCSF) for kindly providing tissue specimens; the Brain Tumor Research Center at UCSF (Dolores Dougherty and King Chiu); Nancy Quinnine, RN; and the members of the Center for Cerebrovascular Research and the UCSF BAVM Study Project for their continued support.

Footnotes

Original received December 28, 2000; resubmission received April 9, 2001; revised resubmission received June 7, 2001; accepted June 7, 2001.

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 89/2/111    most recent hh1401.094281v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hashimoto, T. Articles by Young, W. L. Search for Related Content PubMed Articles by Hashimoto, T. Articles by Young, W. L. Related Collections Angiogenesis Developmental biology Cerebral Aneurysm, AVM, & Subarachnoid hemorrhage Other Vascular biology