De Novo Cerebrovascular Malformation in the Adult Mouse After Endothelial Alk1 Deletion and Angiogenic Stimulation
Background and Purpose—In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse.
Methods—Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter >15 μm per 200 vessels) were quantified 10 days after tamoxifen administration.
Results—Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk12f/2f mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs.
Conclusions—Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.
- AVM (arteriovenous malformation) intracranial
- models, animal
- telangiectasia, hereditary hemorrhagic, type 2
Brain arteriovenous malformations (AVMs) cause life-threatening intracranial hemorrhage, but brain AVM (bAVM) pathobiology is poorly understood. Previously, we induced a bAVM phenotype in adult mice by focal pan-cellular Alk1 gene deletion and vascular endothelial growth factor (VEGF) stimulation.1 Alk1 is predominantly expressed in the arterial endothelial cell (EC).2 We tested the hypotheses that deletion of Alk1 in EC is sufficient to cause bAVM after angiogenic stimulation, and that increased growth of Alk1-deficient EC is a key mechanism underlying bAVM development.
Detailed methods are described in the online-only Data Supplement. Experimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco and conformed to the National Institutes of Health Guidelines for the care and use of animals in research.
Alk12f/2f mice (Alk1 exons 4–6 flanked by loxP sites)3 were bred with Pdgfb-iCreER mice that express tamoxifen-inducible cre recombinase (iCreER) in EC.4 EC-Alk1 deletion was induced5 by intraperitoneal injection of tamoxifen 14 days after intrabrain injection of adeno-associated viral serotype 1–packaged adeno-associated viral-VEGF with cytomegalovirus promoter driving VEGF expression. Mice with NG2 promoter (NG2-iCreER) driving cre expression were used to induce pericyte-Alk1 deletion. Vascular morphology was analyzed using latex casting and immunostaining 10 days after tamoxifen administration. EC proliferation and dysplasia index were quantified. Experimental design and groups are shown in Figure 1. Data are mean±SD.
EC-Alk1 Deletion Resulted in AVM in the Brain Angiogenic Region and Other Organs
Alk1iECKO+VEGF mice developed tangles of irregular vessels in the brain angiogenic region, whereas Alk1iECKO+LacZ or wildtype+VEGF did not (Figure 2A and 2B). The dysplasia index was higher in the Alk1iECKO+VEGF group (11.5±4.1) than in the wildtype+VEGF (1.2±0.4; P<0.001) and Alk1iECKO+LacZ (1.8±1.2; P<0.001) groups (Figure 2C). Macrophage infiltration and microhemorrhage were observed around the malformed vessels (Figure I in the online-only Data Supplement). EC specificity of PDGFb-iCreER was confirmed in the brain of Alk1iECKO+VEGF mice (Figure 1B) using Ai14 cre reporter (Figure II in the online-only Data Supplement).
AVMs also developed in the intestine, lung, and around ear-tag wounds of Alk1iECKO+VEGF and Alk1iECKO+LacZ mice. Alk1iECKO mice had pale paws and darkened feces, indicating anemia and gastrointestinal bleeding, and died 6 to 13 days after tamoxifen administration (Figures III and IV in the online-only Data Supplement).
EC Proliferation Increased in bAVM
Compared with wildtype+VEGF mice, Alk1iECKO+VEGF mice had more newly proliferated ECs (5-bromo-2'-deoxyuridine/ETS family transcription factor [BRDU+/ERG+], 285±48 versus 91±25 cells/mm2; P<0.001; Figure 3) and a higher vessel density in the brain angiogenic region (P<0.001; Figure V in the online-only Data Supplement). The newly proliferated (BrdU+) and proliferating (ki67+) ECs were clustered around dysplastic vessels (Figure 3).
Pericyte-Alk1 Deletion Did Not Cause AVM
NG2-iCreER was used to delete pericyte-Alk1. The cre activity in pericytes was confirmed using Ai14 reporter (Figure VIA in the online-only Data Supplement). No AVM was detected in adult NG2-iCreER;Alk12f/2f mice (Figure VIB in the online-only Data Supplement).
Previously, we reported that injection of adeno-associated viral-VEGF into the brain of adult Alk1+/- mice induces a capillary level of dysplasia,6 which served as a surrogate model for bAVM until we developed a macroscopic level of a bAVM model through focal homozygous Alk1 deletion plus VEGF stimulation.1 In that model, an adenoviral vector that has cytomegalovirus promoter driving cre expression (Ad-Cre) was used to induce focal pan-cellular Alk1 deletion.1 Here, we show that deletion of Alk1 in adult EC results in a bAVM phenotype similar to that in the pan-cellular Alk1 deletion model. Both have macrophage infiltration and microhemorrhage.7 EC-specific Alk1 deletion in the adult mouse has also resulted in AVM in the small intestine and lung and around the skin wound, which recapitulated the phenotype of global Alk1 deletion in adult mice.5 Thus, deletion of Alk1 in adult EC is sufficient to induce AVMs.
SM22α-Cre–driven Alk1 deletion has been reported to result in brain and spinal cord AVMs,8 suggesting that loss of Alk1 from pericytes/vascular smooth muscle cells can cause AVMs. In contrast, we find that conditional deletion of Alk1 in adult pericytes did not trigger AVMs in any organ, around the ear-tag wound, or in the VEGF-stimulated brain. As revealed by Rosa-LacZ cre reporters, SM22α promoter driving cre expression results in nonspecific recombination in EC lineage.8 Therefore, we propose that Alk1 deletion in pericytes alone is not sufficient and that gene deletion in EC is required to initiate AVM development.
We found that deletion of Alk1 in ECs in the adult brain increased EC proliferation in response to VEGF stimulation. Although the expression of Ai14 reporter indicated that cre was activated in ECs of all vessels in the angiogenic region, only a few dysplasia vessels were formed. The dysplastic vessels have more proliferating ECs than the surrounding normal capillaries. As it is statistically unlikely that the dominance of the proliferating cells in dysplastic vessels is the result of multiple independent cre recombination events, we propose that ECs with homozygous Alk1 deletion undergo clonal expansion and have a higher proliferation rate than wildtype EC or Alk1+/– EC, which results in unevenly enlarged abnormal vessels. This hypothesis needs to be further validated.
In summary, deletion of Alk1 in EC leads to increased focal EC proliferation during brain angiogenesis and de novo AVM development. Knowledge of the importance of EC in AVM development will help in understanding AVM pathogenesis and in designing specific therapies.
The authors thank Voltaire Gungab for assistance with article preparation, S. Paul Oh at the University of Florida for providing Alk12f/2f mice, and members of the University of California, San Francisco Brain Arteriovenous Malformation Study Project (http://avm.ucsf.edu) for their support.
Sources of Funding
Support was provided by grants to Dr Su from the National Institutes of Health (R01NS027713 and P01NS044155) and from the Leslie Munzer Foundation. Additional support was provided by a grant to Dr Young from the Michael Ryan Zodda Foundation.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.003655/-/DC1.
- Received September 24, 2013.
- Revision received November 27, 2013.
- Accepted December 16, 2013.
- © 2014 American Heart Association, Inc.
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