Cerebral Cavernous Malformations Arise Independent of the Heart of Glass Receptor
Background and Purpose—The Heart of Glass (HEG) receptor binds KRIT1 and functions with KRIT1, CCM2, and PDCD10 in a common signaling pathway required for heart and vascular development. Mutations in KRIT1, CCM2, and PDCD10 also underlie human cerebral cavernous malformation (CCM) and postnatal loss of these genes in the mouse endothelium results in rapid CCM formation. Here, we test the role of HEG in CCM formation in mice and in humans.
Methods—We constitutively or conditionally deleted Heg and Ccm2 genes in genetically modified mice. Mouse embryos, brain, and retina tissues were analyzed to assess CCM lesion formation.
Results—In postnatal mice, CCMs form with Ccm2−/− but not with Heg−/− or Heg−/−;Ccm2+/- endothelial cells. Consistent with these findings, human patients with CCM who lack exonic mutations in KRIT1, CCM2, or PDCD10 do not have mutations in HEG.
Conclusions—These findings suggest that the HEG-CCM signaling functions during cardiovascular development and growth, whereas CCMs arise because of loss of HEG-independent CCM signaling in the endothelium of the central nervous system after birth.
Cerebral cavernous malformations (CCMs) are common vascular malformations that arise primarily in the central nervous system.1 CCMs are typically diagnosed in middle age and constitute an important cause of stroke and neurological deficit in younger individuals. One third of CCMs are familiar, and positional cloning studies have identified loss of function mutations in 3 genes, KRIT1, CCM2 and PDCD10, as causal for this disease.1 KRIT1, CCM2, and PDCD10 encode intracellular adaptor proteins that have been shown to form a single biochemical complex that is bound by the transmembrane receptor Heart of Glass (HEG),2 but the role of HEG in CCM disease has not been defined.
Fish and mouse genetic studies have demonstrated that HEG, KRIT1, CCM2, and PDCD10 function together in endothelial cells during formation of the heart and vasculature.2–5 In addition, inducible endothelial deletion of Krit1, Ccm2, or Pdcd10 in neonatal mice results in the formation of retinal and hindbrain CCMs that accurately reproduce the human disease.6,7 In the present study, we use genetically modified mice and studies of human patients with familial CCM to test the role of HEG in CCM formation rigorously.
Materials and Methods
Mutant Heg and Ccm2 mouse alleles and Cre transgenic mice have been described previously.2,8–10 The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal protocols.
Endothelial Cell Isolation and Quantitative Polymerase Chain Reaction
Lung endothelial cells were isolated using anti-PECAM beads, and quantitative polymerase chain reaction was performed after cDNA synthesis using SYBR Green (Applied Biosystems).
Evans Blue Dye Extravasation Assay
A concentration of 3 mg/g Evans blue dye was administrated via tail vein injection 16 hours before euthanization and pulmonary vascular perfusion with saline.
Embryos at desired developmental stages were dissected and analyzed as previously described. Rat anti-Pecam (1:500, BD PharMingen) were used to stain blood vessels. To induce postnatal gene inactivation, pups were given 100 μg tamoxifen via intragastric injection at P1 followed by oral administration of 50 μg at P3 and P5.
Twenty-one unrelated patients and 6 healthy controls were used. The study was approved by the local ethics committee.
Sequencing and Quantitative Multiplex Polymerase Chain Reaction of Short Fluorescent Fragment Analysis
HEG1 sequencing was performed after coding exon amplification using primers indicated in Table I in the online-only Data Supplement. HEG genomic rearrangements were assessed using the Quantitative Multiplex Polymerase Chain Reaction of Short Fluorescent fragments method as described.
We have previously found that Heg−/−;Ccm2+/- die at E10 with vascular defects identical to those detected in Krit1−/− and Ccm2−/− embryos.2 To determine whether HEG and CCM proteins function together within endothelial cells, we generated mice lacking both Heg alleles and a single Ccm2 allele exclusively in the endothelium. At E9.5 Tie2-Cre;Hegfl/− and Heg−/−;Ccm2fl/+, embryos exhibited normal blood circulation and patent branchial arch arteries but Tie2-Cre;Hegfl/-;Ccm2fl/+ littermates lacked patent branchial arch arteries, a phenotype identical to that of Heg−/−;Ccm2+/- embryos (Figure 1A–1D). Thus Heg and Ccm2 interact within endothelial cells during early cardiovascular development.
Unlike Ccm2−/− animals, Heg−/− mice survive past birth and approximately half live to advanced age.2 To test the role of HEG in CCM formation, we compared the brains of Heg−/− animals at 10 and 17 months with those of animals in which Ccm2 had been deleted postnatally in endothelial cells. All Tie2-CreERT2;Ccm2fl/fl mice exhibited CCM formation in the meninges, cerebellum, and retina (Figure 2A). In contrast, Heg−/− animals failed to exhibit CCM formation, even at 17 months (Figure 2B and 2C; n=34; Table II in the online-only Data Supplement). Thus, the loss of HEG, in contrast to loss of CCM2, does not result in CCM formation in mice.
To address the role of HEG in CCM formation further, we conditionally deleted 1 Ccm2 allele in Tie2-CreERT2;Heg−/−;Ccm2fl/+ animals, creating postnatal animals with the same genotype as embryos that exhibit vascular defects identical to those of endothelial Ccm2−/− animals. Although endothelial deletion of Ccm2 immediately after birth conferred rapid CCM formation by P17 (Figure 3A and 3B; Table II in the online-only Data Supplement; n=4), deletion of 1 allele of Ccm2 in Heg−/− animals failed to confer CCM formation in either the retina or the hindbrain (Figure 3C; Table II in the online-only Data Supplement; n=16). The number of visible hindbrain CCM lesions in endothelial Ccm2−/− animals did not differ from that in endothelial Heg−/−;Ccm2−/− animals (mean lesion number of 17 and 16, respectively; n=3 for both groups; Figure 3D).
To determine whether HEG1 might be a human CCM disease gene, we analyzed this gene in 21 unrelated patients with CCMs identified by cerebral MRI and pathological examination, and in whom no point mutation or copy number anomaly was detected in KRIT1, CCM2, or PDCD10. Eighteen cases were sporadic with multiple lesions and 3 were familial (≥1 relative with CCM lesions; Table III in the online-only Data Supplement). None of these patients with CCM exhibited germline mutations or large deletions in HEG1. Sixteen exonic polymorphisms were detected. All were present in single-nucleotide polymorphism databases (Table IV in the online-only Data Supplement), and 14 have a frequency >5%.
A role for HEG-CCM signaling in endothelial barrier function has been demonstrated in vitro and in vivo and proposed to participate in CCM disease pathogenesis.11,12 Unlike CCM2-deficient lung endothelial cells, lung endothelial cells harvested from Heg−/− mice expressed normal levels of the endothelial cell junction genes Claudin5 and VE-cadherin by quantitative polymerase chain reaction (Figure I in the online-only Data Supplement). In addition, although endothelial loss of Ccm2 conferred a 60% increase in Evans blue extravasation in the lungs of Cdh5-CreERT2;Ccm2fl/fl mice, no difference was observed in Heg−/− mice (Figure I in the online-only Data Supplement). The role of endothelial barrier function in CCM pathogenesis remains speculative, but these studies suggest that HEG is not required in the CCM signaling pathway that supports vascular integrity.
How loss of CCM signaling causes CCM formation and why CCMs form so specifically in the central nervous system remain unanswered questions. Our studies reveal roles for HEG during embryonic CCM signaling but not in the postnatal pathway that underlies CCM pathogenesis.
One interpretation of these studies is that there exist multiple upstream inputs to the CCM signaling pathway in endothelial cells (eg, HEG during cardiovascular growth and another to prevent CCM formation and perhaps maintain vascular barrier function elsewhere). Definitive proof of distinct upstream activators of CCM signaling will require the molecular identification of such proteins and genetic studies linking their function to CCM formation. Alternatively, it remains possible that HEG couples to CCM signaling in the central nervous system endothelium but that its loss does not disable the pathway to the extent required for lesion formation. The lack of CCMs in postnatal Tie2-CreERT2;Heg−/−;Ccm2fl/+ animals that carry an endothelial deficiency state equivalent to that which causes embryonic phenotypes identical to those conferred by complete KRIT1 or CCM2 deficiency suggests that these studies have a reasonable sensitivity to detect a role for HEG in CCM formation. In either case, our studies indicate that HEG cannot be the sole upstream activator of CCM signaling in the central nervous system endothelium; thus the remarkable specificity of this disease for that organ is likely to reflect the function of other activator(s) that remain to be identified.
Sources of Funding
These studies were supported by National Institutes of Health grants R01HL094326 and R01HL102128 (Dr Kahn) and American Heart Association grant 11SDG7430025 and T32HL007971 (Dr Zheng).
Dr Kleaveland is now affiliated with the Massachusetts General Hospital, Boston, MA.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.004809/-/DC1.
- Received January 13, 2014.
- Revision received February 7, 2014.
- Accepted February 10, 2014.
- © 2014 American Heart Association, Inc.
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