METHOD OF TREATING ARTERIOVENOUS MALFORMATIONS BY TARGETING THE EPHRIN PATHWAY
20220347322 · 2022-11-03
Inventors
Cpc classification
G01N33/5008
PHYSICS
A61K49/0008
HUMAN NECESSITIES
International classification
Abstract
The disclosure provides a mouse model of arteriovenous malformation, such as found in Hereditary Hemorrhagic Telangiectasia, that accurately and persistently models the disease progression in various organisms, including humans. The disclosure further provides a mouse comprising a mutant Ephrin pathway gene, such as Alk1, in brain endothelial cells only, and methods of screening for therapeutically useful modulators of Ephrin pathway gene expression or gene product activity useful in treating or ameliorating a symptom of arteriovenous malformation, such as Hereditary Hemorrhagic Telangiectasia or hemorrhagic stroke.
Claims
1. A method of treating arteriovenous malformation in a subject comprising administering a therapeutically effective amount of a modulator of Eph receptor/ephrinB2 signaling to the subject.
2. The method of claim 1 wherein the modulator inhibits Eph receptor/ephrin B2 signaling.
3. The method of claim 1 wherein the modulator stimulates Eph receptor/ephrin B2 signaling.
4. The method of claim 1 wherein the subject has hereditary hemorrhagic telangiectasia.
5. The method of claim 1 wherein the subject is at risk of, or has had, a hemorrhagic stroke.
6. The method of claim 1 wherein the arteriovenous malformation is in the brain.
7. A method of treating hereditary hemorrhagic telangiectasia in a subject comprising administering a therapeutically effective amount of a modulator of an Eph receptor polypeptide to the subject.
8. The method of claim 7 wherein the modulator is an inhibitor of the Eph receptor.
9. The method of claim 8 wherein the Eph receptor is an Eph type-B receptor.
10. The method of claim 9 wherein the Eph type-B receptor is Eph type-B receptor 4 (EphB4).
11. The method of claim 10 wherein the inhibitor is soluble Eph type-B receptor 4.
12. A method of treating hereditary hemorrhagic telangiectasia in a subject comprising administering a therapeutically effective amount of a modulator of an ephrin polypeptide to the subject.
13. The method of claim 12 wherein the modulator is a stimulator of the ephrin polypeptide.
14. The method of claim 13 wherein the ephrin polypeptide is an ephrin type-B polypeptide.
15. The method of claim 14 wherein the ephrin type-B polypeptide is ephrin B2.
16. A method of screening for a therapeutic to treat hereditary hemorrhagic telangiectasia comprising (a) administering a candidate compound to an organism with a brain-specific arteriovenous malformation (BAVM organism); (b) maintaining the organism for a time suitable for AVM symptoms to arise; (c) measuring a property associated with brain arteriovenous malformation (BAVM) in a BAVM organism; and (d) comparing the level of the property in the BAVM organism receiving the candidate compound to the level of the property in a BAVM organism not receiving the candidate compound, wherein a candidate compound is identified as a therapeutic to treat hereditary hemorrhagic telangiectasia if the level of the property differs in the BAVM organism receiving the candidate compound relative to the level of the property in the BAVM organism not receiving the candidate compound.
17. The method of claim 16 wherein the BAVM organism is a mouse.
18. The method of claim 16 wherein the property is a symptom of BAVM.
19. The method of claim 18 wherein the symptom is intracranial bleeding.
20. The method of claim 18 wherein the symptom is hemorrhagic stroke.
21. The method of claim 16 wherein the property is AVM onset, AVM size, extent of hemorrhage, or time to moribundity.
22. An in vitro method of screening for a therapeutic to treat hereditary hemorrhagic telangiectasia comprising (a) administering a candidate compound to an alk1.sup.−/− endothelial cell derived from a brain; (b) measuring the level of an arteriovenous programming protein; and (c) comparing the level of the arteriovenous programming protein in the presence of the candidate compound to the level of the arteriovenous programming protein in the absence of the compound, wherein a candidate compound is identified as a therapeutic to treat hereditary hemorrhagic telangiectasia if the level of an arteriovenous programming protein is differs in the presence of the candidate compound compared to the level in the absence of the candidate compound.
23. The method of claim 22 wherein the arteriovenous programming protein is a protein in the Ephrin pathway.
24. The method of claim 23 wherein the protein in the Ephrin pathway is an ephrin type-B or an Eph type-B receptor.
25. The method of claim 24 wherein the protein in the Ephrin pathway is Eph type-B receptor 4 or ephrin B2.
26. The method of claim 22 wherein the arteriovenous programming protein is a protein in the Notch pathway or the Transforming Growth Factor-β pathway.
27. A non-human mammal comprising a homozygous Alk1.sup.− inactivating mutation exclusively in brain endothelial cells.
28. The mammal of claim 27 wherein the mammal is a mouse.
29. The mammal of claim 27 wherein the homozygous Mkt inactivating mutation is a deletion of A/k 1.
30. A method of making the non-human mammal of claim 21 comprising the use of Crispr/Cas9 to introduce the mutation.
31. The method of claim 30 further comprising determining that a brain endothelial cell harbors the mutation by single-cell sequencing.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0055] Alk1 is predominantly expressed in arterial endothelial cells (ECs), making it an excellent candidate to investigate the role of AV-specific genes in AVM pathogenesis. Disclosed herein is the deletion of Alk1 specifically from brain ECs, avoiding lethal complications of whole-body Alk1 deletion, using a recently characterized Slco1c1-CreER.sup.T2 allele [Slco1c1-CreER.sup.T2 mice] and another mouse line, i.e., the Alk1.sup.flox/flox mouse line, a temporally inducible Cre under the control of the Slco1c1 promoter (active specifically in the brain endothelium). Deletion of A/kl by Tamoxifen (TAM) in this Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx model (the HHT2 mouse model as referenced herein) led to Alk1 deletions specific to brain ECs, avoiding the lethal complications of whole-body Alk1 deletions. The result was 100% BAVM and intracranial hemorrhages, without detectable defects elsewhere in the body. Using this model, the HHT phenotype was fully characterized within one month, establishing the model disclosed herein as a robust mouse model of HHT.
[0056] The HHT2 mouse model disclosed herein has been characterized using innovative, high-resolution two-photon imaging through a cranial window to access the vasculature in live brains, achieving a 5D perspective (3D vascular structure plus blood velocity over time). The model also provides the tools for screening candidate modulators (e.g., molecular regulators) that promote or hinder HHT2 BAVM formation. In addition, cutting-edge genomic expression profiling is used to elucidate Alk1 target genes.
[0057] The HHT2-BAVM mouse model with brain endothelial cell-specific Alk1 mutations (e.g., deletions) is expected to reveal the hallmarks of BAVM manifestations, including gross pathology, histopathology, hypoxia, perfusion, vessel densities, patterns, and AV shunting. The onset and progression of BAVMs will be apparent from 5D live brain imaging through cranial windows and will allow for neurobehavioral assessment in these mice. The mouse model of HHT2-BAVM will be useful in assessing HHT2 and in identifying modulators of pathways involved in HHT development, including proteins involved in the Ephrin pathway, the Notch pathway and the Transforming Growth Factor-13 pathway. There are sixteen Erythropoietin-Producing Hepatocellular (Eph) receptors, divided into the A- and B-subclasses: EphA (1-10) and EphB (1-6). They share the same structural features, including an N-terminal extracellular domain that binds with their respective ephrin ligands, a short single-pass hydrophobic transmembrane domain, and an intracellular cytoplasmic signaling domain containing a canonical tyrosine kinase catalytic domain, as well as other protein interaction sites. The ligands for the Eph receptors are the ephrins (also known as Eph-receptor-interacting proteins). Ephrins comprise nine different molecules, also divided into A- and B-subclasses. There are six A-subclass ephrins (ephrin-A1 to ephrin-A6) and three B-subclass ephrins (ephrin-B1 to ephrin-B3). Members of the ephrin A-subclass possess a globular extracellular domain that preferentially only binds EphA receptors and is tethered to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol linkage. In contrast, the three B-subclass ephrins have an extracellular structure that preferentially only binds to EphB receptors (except for EphA4, which can interact with both A- and B-subclass ephrins). Like the Eph receptors, these B-subclass ephrins possess a single-pass hydrophobic transmembrane domain. However, unlike the Eph receptors, these ligands (commonly referred to as ephrin Bs) do not have an intracellular catalytic domain. Instead, they have a short, highly conserved cytoplasmic tail. They are capable of bidirectional signaling, eliciting both forward as well as reverse signaling.
[0058] Also contemplated is a mouse model of HHT2 with one Alk1 germline knockout allele and one floxed Alk1 allele, to provide an alternative genetic basis for assessing HHT2 and for identifying modulators of the aforementioned pathways involved in HHT development, including proteins involved in the Ephrin pathway, the Notch pathway and the Transforming Growth Factor-β pathway.
[0059] More particularly, the HHT2-BAVM mouse model (homozygous Alk1 mutation) is expected to identify triggers that lead to BAVM formation, including AV programming, endothelial barrier, inflammation, endothelial-to-mesenchymal transition (EndMT), and superoxide production in mice with Alk1 deletion in the brain endothelium. In addition, ribosomal profiling is contemplated to identify molecular candidates downstream of Alk1 in a genome-wide expression analysis and to obtain global gene expression patterns after Alk1 deletion in the brain endothelium. Using bioinformatic techniques, identified genes are categorized based on their functional characteristics, especially as they relate to processes that may contribute to BAVM formation in the HHT2-BAVM mouse model. Findings are validated by immunofluorescence, qPCR, and/or in situ hybridization by RNA-scope. Identified genes are expected to be molecular regulators and mediators crucial for HHT2 BAVM development.
[0060] The HHT2-BAVM mouse model has been used to develop a method to treat HHT, showing that inhibiting EphB4 can attenuate disease progression. Further, it is shown that stimulation of ephrin B2 can have an analogous effect on HHT2. It is expected that inhibition of an Eph type-B receptor, such as EphB1, EphB2, EphB3 or EphB6 in addition to EphB4 will have a beneficial attenuating effect on the progression of Hereditary Hemorrhagic Telangiectasia, including HHT2. If is further expected that stimulating an ephrin type-B ligand polypeptide, including ephrin B1 and ephrin B3 as well as ephrin B2 will have a beneficial effect on the progression of HHT, such as HHT2.
[0061] The following examples are presented by way of illustration and are not intended to limit the scope of the subject matter disclosed herein.
EXAMPLES
Example 1
[0062] Engineering a Mouse Model of HHT2
[0063] Alk1 was specifically from brain ECs, avoiding the lethal complications of whole-body Alk1 deletion, using the Slco1c1-CreER.sup.T2 allele, a temporally inducible Cre under the control of the Slco1c1 promoter, which is a promoter active specifically in brain endothelium. Deletion of Alk1 from postnatal day (P) 13 with tamoxifen (TAM) in this Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx model led to 100% BAVM and intracranial hemorrhages, without detectable defects elsewhere in the body. Although this mouse model has 100% penetrance for BAVM formation, the model is readily adaptable for use with particular chosen time points to compare AVM onset and size, extent of hemorrhage, moribundity time (
[0064] The mouse model of HHT2 is a valuable preclinical tool for understanding pathogenesis, identifying new therapeutic targets, and informing new treatment strategies for HHT2. The model is also useful in testing the hypothesis that abnormal AV programming underlies HHT2 AVM development. We previously characterized AV molecular programming in AVMs, but in a Notch-based model of AVM, rather than an HHT model. The Notch work was predicated on the established premise that Notch regulates AV fate, and we showed that Notch arterializes veins in AVMs. It was our expectation that HHT genes would also affect AV programming in AVM formation, but there was no empirical data supporting this position. The mouse model disclosed herein allows for the testing of this hypothesis by examining AVMs in mice lacking brain endothelial Alk1. The versatile mouse model of HHT2 disclosed herein is also useful in testing whether superoxide production underlies HHT2 AVM formation. These efforts are aided by the in vivo 2-photon (2P) microscopy protocol (
[0065] The mouse model of HHT is also beneficial in using a lineage tracing approach to track whether EndMT contributes to HHT2 AVM pathogenesis. It is also contemplated that the approach taken in engineering the mouse model of HHT, i.e., the use of the Slco1c1-CreER.sup.T2 allele to target site-specific mutations in brain ECs, to further our understanding of BAVM. The experimental data disclosed herein also establishes the value of ribosomal profiling to generate an unbiased genome-wide expression profile to identify Alk1 transcriptional targets in brain ECs. The experiments disclosed herein overcome a barrier in the field, establishing a mouse model of HHT2 that reveals molecular triggers in AVM pathogenesis, thus identifying new therapeutic targets.
[0066] To investigate the function of cerebral endothelial Alk1 in regulating brain vascular structure and function postnatally, Alk1 was specifically deleted in the brain endothelium using a novel mouse genetic tool, i.e., the Slco1c1-CreER.sup.T2 allele. In the Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx mouse strain, CreER.sup.T2 is driven by the brain endothelial specific promoter Slco1c1, which allows deletion of both floxed Alk1 alleles in the brain endothelium. The data disclosed herein shows that deleting both floxed Alk1 alleles from P13 led to HHT-like symptoms by P22 in 100% of mice (
[0067] Mutant and littermate control mice (Table 1) are generated by breeding Slco1c1-CreER.sup.T2 Alk1.sup.fx/+ mice with Alk1.sup.fxf/x mice. Both parental lines were established prior to breeding. Tamoxifen (TAM; Sigma) (0.5 mg) is injected intraperitoneally (IP) at P13 to delete the floxed Alk1 alleles from the brain endothelium. If needed, the dose and time of TAM injection is optimized to most closely model HHT phenotypes. To verify Alk1 gene deletion, immunostaining is performed using a well-established commercial antibody against mouse Alk1.sup.24,30 at 2 and 4 days after TAM injection. Deletion of Alk1 in ECs systemically led to defects two days after TAM injection. The following analyses are performed on the mice. A moribundity curve is generated. Moribundity is assessed in an unbiased manner by a daily routine health check by trained veterinary nurses who are unaware of the experimental status of the mice, and through researchers recording animal weight, activity, posture, and appearance. We will monitor neurological behavior is monitored as part of routine observations and video recordings are made of the onset and occurrence of any neurodysfunction, Once moribund, mice are harvested for analysis.
TABLE-US-00001 TABLE 1 Brain Endothelial Cell (EC) Alk1 Deletion Slco1c1-CreER.sup.T2; Alk1.sup.fxfx+ Slco1c1-CreER.sup.T2; Alk1.sup.fx/+ Slco1c1-CreER.sup.T2; Alk1.sup.+/+
[0068] Moribund mutant and control mice will be dissected to assess the most severe phenotype, gross pathology, heart/body weight ratio, brain microbleeds, vascular defects, and brain abnormalities. These analyses are also performed at P14 (1 day after TAM, no expected detectable abnormalities), P15 (2 days after TAM, expected detectable abnormalities), and P17 (4 days after TAM, expected intermediate phenotype), to characterize initial defects. Histopathological evaluation and a hypoxia assay with Hypoxyprobe™ immunostaining (HPi-100, HPI, Inc.) is performed using published protocols.sup.2,33 as AV shunting results in hypoxia.
[0069] At P15, P17, and moribund (i.e., the time moribundity occurs), brain vascular structure is evaluated by perfusion with fluorophore-labeled lectin (Vector Labs), alone or with immunostaining for ECs using an anti-CD31 antibody (BD Pharmingen). Mouse parietal cortex is sectioned to 2-3 mm, stained, and flat-mounted to image cortical surface vessels. Densities and patterns of all vessels (CD31+) and perfused vessels (lectin+) are quantified by Image J. EC proliferation is evaluated by Ki67 staining and apoptosis is evaluated by cleaved caspase-3 staining, along with Erg co-staining to label EC nuclei in frozen sections. Five sections per mouse brain are quantified by Image J.
[0070] AV shunting is assessed by a microsphere passage assay at P17 and moribund. In this assay, 15 μm FITC-labeled beads, too large to pass through normal brain capillaries, are injected into carotid arteries. If abnormal brain capillaries are present, beads pass through the brain and lodge in the lungs, functionally defining brain AV shunts. Vascular topology is assessed in whole brains by casting with MICROFIL® compound (FlowTech, Inc.) at moribund. We will determine if bleeding occurs before and independently of AVM. Lectin perfusion and gross pathologic analysis will be performed as described herein to detect hemorrhage. Subsequently, half of the brain is stained with H&E, and the other half is used for vascular imaging to detect AVMs.
[0071] Experiments are conducted and data is obtained blindly to test group genotypes. Inclusion of mice with different biological variables, including sex, ensures a rigorous comparison in all experiments. Statistical analyses are used to determine the sample size and outcome of all experiments. Differences between two groups are analyzed by Student's t-test. Differences between multiple groups are analyzed by ANOVA, followed by Tukey's post-test for pairwise comparisons. If a normal distribution cannot be assumed, the non-parametric Mann-Whitney U test and Kruskal-Wallis test are used in place of the t-test and ANOVA, respectively. Statistical significance is assumed when p<0.05. For example, to detect a difference of 20 μm in AV connection diameter between groups, assuming a standard deviation of 2.5 μm in controls and 20 μm in mutants, 10 mice are needed per treatment group, based upon a 2-tailed power calculation with power >0.80. Sample sizes for proposed experiments are assessed by power analysis with appropriate parameters.
[0072] We expect that the experiments described in this Example will provide a comprehensive characterization of the HHT2 mouse model disclosed herein, documenting the core pathologies and kinetics of their development, including AV shunting, bleeding, behavior changes, and illness.
Example 2
[0073] Second HHT2 Mouse Model that Simulates Human Disease
[0074] An HHT2 mouse model is also developed using Slco1c1-CreER.sup.T2; Alk1.sup.−/fxx, which more closely reflects the dominant genetic lesion of human HHT2. In this mouse model, the null allele represents the germline ALK1 mutation seen in HHT patients. The floxed Alk1 allele is excised in brain ECs, causing loss of heterozygosity (LOH) in these cells. We will characterize the phenotypes in this model are characterized and compared to those in the Slco1c1-CreER.sup.T2 Alk1.sup.fx/fx model.
[0075] Mutant and littermate control mice (Table 2) are generated by breeding Slco1c1-CreER.sup.T2 Alk1.sup.fx/+ with Alk1.sup.−/fx mice. Mice are injected IP with 0.5 mg TAM at P13 to delete the floxed Alk1 allele from brain ECs. Mouse weight, activity, and moribundity are analyzed as described in Example 1. Results are compared to existing data on Slco1c1-CreER.sup.T2 Alk1.sup.fxf/x mice. The TAM regimen is optimized to establish a model with a longer healthy period to better resemble the human disease. With the optimized TAM regimen, gross pathology, histology, and vascular structure are analyzed, as described in Example 1.
TABLE-US-00002 TABLE 2 Brain EC Alk1 deletion Slco1c1-CreER.sup.T2; Alk1.sup.−/fx Slco1c1-CreER.sup.T2; Alk1.sup.−/+ Slco1c1-CreER.sup.T2; Alk1.sup.+/+
[0076] The Slco1c1-CreER.sup.T2, Alk1.sup.−/fx model is expected to be ideal for modeling human HHT2 and will develop BAVM like the Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx mice, but BAVM will occur more quickly with the same TAM regimen, as there is only one floxed allele to excise. We expect to identify the optimal TAM regimen to achieve a mouse model most similar to human disease progression.
Example 3
[0077] Initiation and Progression of BAVMs in Slco1c1-CreER.sup.T2; Alk1.sup.fxf/x Mice Using 5D Two Photon Live Imaging
[0078] To reveal the development of Alk1-mediated BAVM formation longitudinally, live 5D imaging (
[0079] The Notch model of AVM has shown cellular changes leading to AVMs using the Cdh5 (PAC)-CreER.sup.T2; R26R-Confetti line.sup.2, where Cre positive cells have GFP+ nuclei and YFP+ cytoplasm (
[0080] Experimental and control Slco1c1-CreER.sup.T2, Alk1.sup.fxf/x mice with the arterial nuclear reporter (ephrin-B2H2B-eGFP) and the R26R-RG reporter are generated as in Table 3, by breeding Slco1c1-CreER.sup.T2; Alk1.sup.fx/+; R26R-RG mice with Alk1.sup.fxf/x; ephrin-B2H2BeGFP mice. We have produced the R26R-RG reporter has been constructed. To image the live brain, a cranial window is created over the parietal cortex at P12.sup.2,34. Images are taken from P13, prior to TAM injection to induce Alk1 deletion, followed by imaging at P14, P16, P18, and P20 to document the onset and progression of the phenotype.sup.2,34. For each imaging session, blood is perfusion-labeled with Cascade Blue-dextran to visualize vessels, allowing for lumen diameter and red blood cell velocity measurements. Through the window, artery, arteriole, capillary, venule, and vein branches are identified by hierarchical structure and blood flow, allowing for determination of the location of marked ECs and the path of migration relative to their vessel compartments.sup.2,34,36. The following quantitative data is acquired: AV connection diameter; red blood cell (RBC) velocity in AV connections; and the number, position, and area (“footprint”) of ephrin-B2 positive (arterial) and ephrin-B2 negative (non-arterial) ECs. Changes in: minimal AV connection diameter; RBC velocity; cell number; cell position (rate and direction of migration, rate of directional migration); and cell area are then determined by extrapolation.
TABLE-US-00003 TABLE 3 Alk1 deletion with reporters for imaging Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; ephrin-B2.sub.H2B-eGFP; R26R-RG Slco1c1-CreER.sup.T2; Alk1.sup.fx/+; ephrin-B2.sub.H2B-eGFP; R26R-RG Slco1c1-CreER.sup.T2; Alk1.sup.+/+; ephrin-B2.sub.H2B-eGFP; R26R-RG
[0081] It is expected that data on the onset and progression of AVMs in mice lacking Alk1 specifically in brain endothelium, is acquired, providing the first longitudinal, high resolution imaging of HHT2 BAVM development. The time of AVM initiation also provides crucial information as to whether bleeding occurs prior to AVM formation.
Example 4
[0082] Mechanisms of BAVM Development in Mice with Brain-Endothelial-Specific Alk1 Deletion
[0083] The mouse model disclosed herein is used to investigate candidate events leading to BAVM progression in HHT2 including AV programming, inflammation, endothelial barrier, EndMT, and superoxide production. Although the mouse model has 100% penetrance of BAVMs, carefully chosen time points are used to compare AVM onset and size, extent of hemorrhage, moribundity time, and other readouts to ascertain effects of a candidate trigger.
[0084] An avenue of inquiry relevant to BAVM development in mice with brain-specific Alk1 deletions is the role of AV molecular programming, which is investigated using Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx mice. We have characterized AV molecular programming in AVMs in a Notch-based model of AVM. The Notch work was predicated on the established premise that Notch regulates AV fate, and we showed that Notch arterializes veins in AVMs. Inspired by the Notch work, in which we showed that AV specification/programming is a key molecular mechanism in AVM formation, we expected that HHT genes would also affect AV programming in AVM formation. There is evidence that Alk1 knockout animals have reduced ephrinB2 expression in embryos.sup.37. Without wishing to be bound by theory, we expect Alk1 loss of function to venulize arteries. The experiments disclosed herein will assess this expectation in the HHT2 AVM setting. Data from experiments performed to date show that EphB4 expression was not changed in arteries lacking Alk1, rather EphB4 expression expanded into capillaries in this background (
Example 5
[0085] LacZ Reporter Assays Identify the Molecular Identities of Alk1 Mutant Vessels
[0086] AVMs are a nidus of enlarged vessels connected by abnormal AV shunting. AVMs have historically been investigated as abnormal vessel growth, which may be a consequence of other primary lesions. We have proposed that abnormal AV programming underlies AVM development in a Notch AVM mouse model, where we showed that Notch arterialized veins.sup.2,34. Based on the data that Alk1 is primarily expressed in arteries and not in veins.sup.29 (
[0087] To test the AV molecular identities of Alk1 mutant vessels, LacZ reporter assays.sup.33,34 are used, where ephrinB2.sup.LacZ/+ and EphB4.sup.LacZ4/+ mark arterial and venous vessels, respectively. First, the mice identified in Table 4 are generated by breeding Slco1c1-CreER.sup.T2; Alk1.sup.fx/+ mice with Alk1.sup.fx/fx; ephrinB2.sup.LacZ/+ or Alk1.sup.fx/fx; EphB4.sup.LacZ/+ mice. The mice identified by asterisks in Table 4 are analyzed by LacZ staining at P16, following TAM injection at P13 (
TABLE-US-00004 TABLE 4 Alk1 deletion with AV reporters *Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; ephrinB2.sup.LacZ/+ *Slco1c1-CreER.sup.T2; Alk1.sup.fx/+; ephrinB2.sup.LacZ/+ Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; ephrinB2.sup.LacZ/+ Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx *Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; EphB4.sup.LacZ/+ *Slco1c1-CreER.sup.T2; Alk1.sup.fx/+; EphB4.sup.LacZ/+ Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; EphB4.sup.LacZ/+ Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx
[0088] We originally expected reduced arterial markers and increased venous markers in arteries in Alk1-deficient mice. However, the data show no change in these markers in arteries, but upregulation of EphB4 in veins and capillaries as revealed by LacZ staining in Slco1c1-CreER.sup.T2 Alk1.sup.fx/fx; EphB4.sup.LacZ/+ mice following TAM treatment at P13 (
Example 6
[0089] Determining Whether ephrinB2 or EphB4 is Required for BAVM Formation in Alk1 Mutant Mice
[0090] The LacZ reporters described herein are knockins (i.e., one copy of ephrinB2 or EphB4 is knocked out by the LacZgene). Experiments will reveal whether having heterozygous ephrinB2 or EphB4 affects the BAVM phenotype in mice lacking Alk1. The data show that Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx; EphB.sup.LacZ/+ mice treated with TAM from P13 show reduced BAVM formation and hemorrhage and delayed moribundity compared to Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx mice (
[0091] To determine if having heterozygous ephrinB2 or EphB4 affects the BAVM phenotype in mice lacking Alk1, the mice identified in Table 4 in black letters are analyzed using methods described herein. For the EphB4 study, BAVM formation has been examined at the single time point of P20 (
[0092] A pharmacological approach to inhibition of EphB4 is also undertaken. The soluble extracellular domain of EphB4 (sEphB4) completely inhibits EphB4 signaling in mice.sup.38. sEphB4 is injected intraorbitally.sup.34 into Slco1c1-CreER.sup.T2; Alk1.sup.fx/fx mice to test its ability to prevent and treat BAVMs. Imaging studies through a cranial window offers unprecedented insight into the efficacy of a candidate drug such as sEphB4 in AVM prevention and regression in real time. Such a study without a cranial window would require a great number of experimental mice and would lack definitive proof that an established AVM had regressed. To determine if sEphB4 prevents BAVM formation, at P12 a cranial window is implanted and also begin daily injections of sEphB4 (about 4 mg/kg) into Slco1c1-CreERT2; Alk1fx/fx mice are begun. Imaging begins at P13, followed by immediate TAM injection and imaging at P14, P16, P18, and P20. To determine if sEphB4 causes regression of BAVMs, a second cohort of mice are treated with TAM at P13, followed by cranial window implantation at P17. Mice are imaged at P18, after BAVM formation, followed by sEphB4 treatment daily, and imaging at P19, 20, and 22. Recombinant human fibronectin is injected as a negative control. We expect the experiment to reveal that pharmacological repression of EphB4, like genetic reduction of EphB4, leads to prevention or reduction of BAVMs in Alk1 mutant mice. More generally, we expect ephrinB2 and EphB4 to be important for Alk1-mediated AVM formation, and we expect that heterozygosity of these genes (i.e., mice harboring heterozygous mutant Alk1 in brain ECs) will affect AVM formation, as revealed in our Slco1c1-CreER.sup.T2 Alk1″ model. Data from completed experiments show that heterozygous deletion of EphB4 inhibits cerebral hemorrhage and delays moribundity (
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[0152] All publications and patents mentioned in the application are herein incorporated by reference in their entireties or in relevant part, as would be apparent from context. Various modifications and variations of the disclosed subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the described modes for making or using the disclosed subject matter that are obvious to those skilled in the relevant field(s) are intended to be within the scope of the following claims.