Effect of elevation of vascular endothelial growth factor level on exacerbation of hemorrhage in mouse brain arteriovenous malformation.

OBJECTIVE: A high level of vascular endothelial growth factor (VEGF) has been implicated in brain arteriovenous malformation (bAVM) bleeding and rupture. However, direct evidence is missing. In this study the authors used a mouse bAVM model to test the hypothesis that elevation of focal VEGF levels in bAVMs exacerbates the severity of bAVM hemorrhage. METHODS: Brain AVMs were induced in adult mice in which activin receptor-like kinase 1 (Alk1, a gene that causes AVM) gene exons 4-6 were floxed by intrabasal ganglia injection of an adenoviral vector expressing Cre recombinase to induce Alk1 mutation and an adeno-associated viral vector expressing human VEGF (AAV-VEGF) to induce angiogenesis. Two doses of AAV-VEGF (5 × 109 [high] or 2 × 109 [low]) viral genomes were used. In addition, the common carotid artery and external jugular vein were anastomosed in a group of mice treated with low-dose AAV-VEGF 6 weeks after the model induction to induce cerebral venous hypertension (VH), because VH increases the VEGF level in the brain. Brain samples were collected 8 weeks after the model induction. Hemorrhages in the bAVM lesions were quantified on brain sections stained with Prussian blue, which detects iron deposition. VEGF levels were quantified in bAVM tissue by enzyme-linked immunosorbent assay. RESULTS: Compared to mice injected with a low dose of AAV-VEGF, the mice injected with a high dose had higher levels of VEGF (p = 0.003) and larger Prussian blue-positive areas in the bAVM lesion at 8 or 9 weeks after model induction (p = 0.002). VH increased bAVM hemorrhage in the low-dose AAV-VEGF group. The overall mortality in the high-dose AAV-VEGF group was 26.7%, whereas no mouse died in the low-dose AAV-VEGF group without VH. In contrast, VH caused a mortality of 50% in the low-dose AAV-VEGF group. CONCLUSIONS: Using mouse bAVM models, the authors provided direct evidence that elevation of the VEGF level increases bAVM hemorrhage and mouse mortality.

Adult mouse venous hypertension model: common carotid artery to external jugular vein anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases. We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed. This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

Endoglin deficiency in bone marrow is sufficient to cause cerebrovascular dysplasia in the adult mouse after vascular endothelial growth factor stimulation.

BACKGROUND AND PURPOSE: Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng(+/-)) mice. We hypothesized that Eng(+/-) BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. METHODS: BM transplantation was performed using adult wild-type (WT) and Eng(+/-) mice as donors/recipients. An adeno-associated viral vector expressing VEGF was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 µm/100 vessels), and BMDCs in the angiogenic foci were analyzed. RESULTS: The dysplasia index of WT/Eng(+/-) BM mice was higher than WT/WT BM mice (P<0.001) and was similar to Eng(+/-)/Eng(+/-) BM mice (P=0.2). Dysplasia in Eng(+/-) mice was partially rescued by WT BM (P<0.001). WT/WT BM and WT/Eng(+/-) BM mice had similar numbers of BMDCs in the angiogenic foci (P=0.4), most of which were CD68(+). Eng(+/-) monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. CONCLUSIONS: Endoglin-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.

Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain.

OBJECTIVE: Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell coverage. METHODS AND RESULTS: Adult Alk1(1f/2f) mice (loxP sites flanking exons 4-6) and wild-type mice were injected with 2×10(7) PFU adenovious-cre recombinase and 2×10(9) genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1(1f/2f) mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×10(3) pixels/mm(2) versus 40±13×10(3); P=0.001), iron deposition (508±506 pixels/mm(2) versus 6±49; P=0.04), and Iba1(+) microglia/macrophage infiltration (888±420 Iba1(+) cells/mm(2) versus 240±104 Iba1(+); P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P<0.001), fewer vascular-associated pericytes (503±179/mm(2) versus 931±115, P<0.001), and reduced platelet-derived growth factor receptor-ß expression. CONCLUSIONS: Reduction of mural cell coverage in response to vascular endothelial growth factor stimulation is a potential mechanism for the impairment of vessel wall integrity in hereditary hemorrhagic telangiectasia type 2-associated brain arteriovenous malformation.

Bevacizumab attenuates VEGF-induced angiogenesis and vascular malformations in the adult mouse brain.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) expression is elevated in human brain arteriovenous malformations (bAVM). We have developed a bAVM model in the adult mouse by focal Alk1 gene deletion and human VEGF stimulation. We hypothesized that once the abnormal vasculature has been established, tonic VEGF stimulation is necessary to maintain the abnormal phenotype, and VEGF antagonism by bevacizumab (Avastin) would reduce vessel density and attenuate the dysplastic vascular phenotype. METHODS: Angiogenesis and bAVM were induced by injection of adeno-associated viral vector expressing human VEGF alone into the brain of wild-type mice or with adenoviral vector expressing Cre recombinase (Ad-Cre) into Alk1(2f/2f) mice. Six weeks later, bevacizumab or trastuzumab (Herceptin, bevacizumab control) was administered. Vessel density, dysplasia index, vascular cell proliferation and apoptosis, and human IgG were assessed (n=6/group). RESULTS: Compared with trastuzumab (15 mg/kg), administration of 5, 10, and 15 mg/kg of bevacizumab to adeno-associated viral vector expressing human VEGF treated wild-type mice reduced focal vessel density (P<0.05); administration of 5 mg/kg bevacizumab decreased proliferating vascular cells (P=0.04) and increased TUNEL-positive vascular cells (P=0.03). More importantly, bevacizumab (5 mg/kg) treatment reduced both vessel density (P=0.01) and dysplasia index (P=0.02) in our bAVM model. Human IgG was detected in the vessel wall and in the parenchyma in the angiogenic foci of bevacizumab-treated mice. CONCLUSIONS: We provide proof-of-principle that, once abnormal AVM vessels have formed, VEGF antagonism may reduce the number of dysplastic vessels and should be evaluated further as a therapeutic strategy for the human disease.

Minimal homozygous endothelial deletion of Eng with VEGF stimulation is sufficient to cause cerebrovascular dysplasia in the adult mouse.

BACKGROUND: Brain arteriovenous malformations (bAVMs) represent a high risk for hemorrhagic stroke, leading to significant neurological morbidity and mortality in young adults. The etiopathogenesis of bAVM remains unclear. Research progress has been hampered by the lack of animal models. Hereditary Hemorrhagic Telangiectasia (HHT) patients with haploinsufficiency of endoglin (ENG, HHT1) or activin receptor-like kinase 1 (ALK1, HHT2) have a higher incidence of bAVM than the general population. We previously induced cerebrovascular dysplasia in the adult mouse that resembles human bAVM through Alk1 deletion plus vascular endothelial growth factor (VEGF) stimulation. We hypothesized that Eng deletion plus VEGF stimulation would induce a similar degree of cerebrovascular dysplasia as the Alk1-deleted brain. METHODS: Ad-Cre (an adenoviral vector expressing Cre recombinase) and AAV-VEGF (an adeno-associated viral vector expressing VEGF) were co-injected into the basal ganglia of 8- to 10-week-old Eng(2f/2f) (exons 5 and 6 flanked by loxP sequences), Alk1(2f/2f) (exons 4-6 flanked by loxP sequences) and wild-type (WT) mice. Vascular density, dysplasia index, and gene deletion efficiency were analyzed 8 weeks later. RESULTS: AAV-VEGF induced a similar degree of angiogenesis in the brain with or without Alk1- or Eng-deletion. Abnormally patterned and dilated dysplastic vessels were found in the viral vector-injected region of Alk1(2f/2f) and Eng(2f/2f) brain sections, but not in WT. Alk1(2f/2f) mice had about 1.8-fold higher dysplasia index than Eng(2f/2f) mice (4.6 ± 1.9 vs. 2.5 ± 1.1, p < 0.05). However, after normalization of the dysplasia index with the gene deletion efficiency (Alk1(2f/2f): 16% and Eng(2f/2f): 1%), we found that about 8-fold higher dysplasia was induced per copy of Eng deletion (2.5) than that of Alk1 deletion (0.3). ENG-negative endothelial cells were detected in the Ad-Cre-treated brain of Eng(2f/2f) mice, suggesting homozygous deletion of Eng in the cells. VEGF induced more severe vascular dysplasia in the Ad-Cre-treated brain of Eng(2f/2f) mice than that of Eng(+/-) mice. CONCLUSIONS: (1) Deletion of Eng induces more severe cerebrovascular dysplasia per copy than that of Alk1 upon VEGF stimulation. (2) Homozygous deletion of Eng with angiogenic stimulation may be a promising strategy for development of a bAVM mouse model. (3) The endothelial cells that have homozygous causal gene deletion in AVM could be crucial for lesion development.

Cerebrovascular casting of the adult mouse for 3D imaging and morphological analysis.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents. In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)(1). In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis. The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level(2), and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

Tissue inhibitor of metalloproteinases-3 mediates the death of immature oligodendrocytes via TNF-α/TACE in focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Oligodendrocyte (OL) death is important in focal cerebral ischemia. TIMP-3 promotes apoptosis in ischemic neurons by inhibiting proteolysis of TNF-α superfamily of death receptors. Since OLs undergo apoptosis during ischemia, we hypothesized that TIMP-3 contributes to OL death. METHODS: Middle cerebral artery occlusion (MCAO) was induced in Timp-3 knockout (KO) and wild type (WT) mice with 24 or 72 h of reperfusion. Cell death in white matter was investigated by stereology and TUNEL. Mature or immature OLs were identified using antibodies against glutathione S-transferase-π (GST-π) and galactocerebroside (GalC), respectively. Expression and level of proteins were examined using immunohistochemistry and immunoblotting. Protein activities were determined using a FRET peptide. RESULTS: Loss of OL-like cells was detected at 72 h only in WT ischemic white matter where TUNEL showed greater cell death. TIMP-3 expression was increased in WT reactive astrocytes. GST-π was reduced in ischemic white matter of WT mice compared with WT shams with no difference between KO and WT at 72 h. GalC level was significantly increased in both KO and WT ischemic white matter at 72 h. However, the increase in GalC in KO mice was significantly higher than WT; most TUNEL-positive cells in ischemic white matter expressed GalC, suggesting TIMP-3 deficiency protects the immature OLs from apoptosis. There were significantly higher levels of cleaved caspase-3 at 72 h in WT white matter than in KO. Greater expression of MMP-3 and -9 was seen in reactive astrocytes and/or microglia/macrophages in WT at 72 h. We found more microglia/macrophages in WT than in KO, which were the predominant source of increased TNF-α detected in the ischemic white matter. TACE activity was significantly increased in ischemic WT white matter, which was expressed in active microglia/macrophages and OLs. CONCLUSIONS: Our results suggested that focal ischemia leads to proliferation of immature OLs in white matter and that TIMP-3 contributes to a caspase-3-dependent immature OL death via TNF-α-mediated neuroinflammation. Future studies will be needed to delineate the role of MMP-3 and MMP-9 that were increased in the Timp-3 wild type.

Coexpression of angiopoietin-1 with VEGF increases the structural integrity of the blood-brain barrier and reduces atrophy volume.

Vascular endothelial growth factor (VEGF)-induced neovasculature is immature and leaky. We tested if coexpression of angiopoietin-1 (ANG1) with VEGF improves blood-brain barrier (BBB) integrity and VEGF neuroprotective and neurorestorative effects using a permanent distal middle cerebral artery occlusion (pMCAO) model. Adult CD-1 mice were injected with 2 × 10(9) virus genomes of adeno-associated viral vectors expressing VEGF (AAV-VEGF) or ANG1 (AAV-ANG1) individually or together in a 1:1 ratio into the ischemic penumbra 1 hour after pMCAO. AAV-LacZ was used as vector control. Samples were collected 3 weeks later. Compared with AAV-LacZ, coinjection of AAV-VEGF and AAV-ANG1 reduced atrophy volume (46%, P=0.004); injection of AAV-VEGF or AAV-ANG1 individually reduced atrophy volume slightly (36%, P=0.08 and 33%, P=0.09, respectively). Overexpression of VEGF reduced tight junction protein expression and increased Evans blue extravasation. Compared with VEGF expression alone, coexpression of ANG1 with VEGF resulted in upregulation of tight junction protein expression and reduction of Evans blue leakage (AAV-ANG1/AAV-VEGF: 1.4 ± 0.3 versus AAV-VEGF: 2.8 ± 0.7, P=0.001). Coinjection of AAV-VEGF and AAV-ANG1 induced a similar degree of angiogenesis as injection of AAV-VEGF alone (P=0.85). Thus, coexpression of ANG1 with VEGF improved BBB integrity and resulted in better neuroprotection compared with VEGF expression alone.

Arteriovenous malformation in the adult mouse brain resembling the human disease.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are an important cause of hemorrhagic stroke. The underlying mechanisms are not clear. No animal model for adult bAVM is available for mechanistic exploration. Patients with hereditary hemorrhagic telangiectasia type 2 (HHT2) with activin receptor-like kinase 1 (ALK1; ACVRL1) mutations have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) stimulation with regional homozygous deletion of Alk1 induces severe dysplasia in the adult mouse brain, akin to human bAVM. METHODS: Alk1(2f/2f) (exons 4-6 flanked by loxP sites) and wild-type (WT) mice (8-10 weeks old) were injected with adenoviral vector expressing Cre recombinase (Ad-Cre; 2 × 10(7) plaque forming units [PFU]) and adeno-associated viral vectors expressing VEGF (AAV-VEGF; 2 × 10(9) genome copies) into the basal ganglia. At 8 weeks, blood vessels were analyzed. RESULTS: Gross vascular irregularities were seen in Alk1(2f/2f) mouse brain injected with Ad-Cre and AAV-VEGF. The vessels were markedly enlarged with abnormal patterning resembling aspects of the human bAVM phenotype, displayed altered expression of the arterial and venous markers (EphB4 and Jagged-1), and showed evidence of arteriovenous shunting. Vascular irregularities were not seen in similarly treated WT mice. INTERPRETATION: Our data indicate that postnatal, adult formation of the human disease, bAVM, is possible, and that both genetic mutation and angiogenic stimulation are necessary for lesion development. Our work not only provides a testable adult mouse bAVM model for the first time, but also suggests that specific medical therapy can be developed to slow bAVM growth and potentially stabilize the rupture-prone abnormal vasculature.

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.

Divergent role for MMP-2 in myelin breakdown and oligodendrocyte death following transient global ischemia.

Transient global ischemia causes delayed white matter injury to the brain with oligodendrocyte (OLG) death and myelin breakdown. There is increasing evidence that hypoxia may be involved in several diseases of the white matter, including multiple sclerosis, vascular dementia, and ischemia. Matrix metalloproteinases (MMPs) are increased in rat and mouse models of hypoxic hypoperfusion and have been associated with OLG death. However, whether the MMPs act on myelin or OLGs remains unresolved. We hypothesized that delayed expression of MMPs caused OLG death and myelin breakdown. To test the hypothesis, adult mice underwent hypoxic hypoperfusion with transient bilateral occlusion of the carotid arteries. After 3 days of reperfusion, ischemic white matter had increased reactivity of astrocytes and microglia, MMP-2 localization in astrocytes, and increased protein expression and activity of MMP-2. In addition, there was a significant loss of myelin basic protein (MBP) by Western blot and caspase-3- mediated OLG death. Treatment with the broad-spectrum MMP inhibitor, BB-94, significantly decreased astrocyte reactivity and MMP-2 activity. More importantly, it reduced MBP breakdown. However, MMP inhibition had no effect on OLG loss. Our results implicate MMPs released by reactive astrocytes in delayed myelin degradation, while OLG death occurs by an MMP-independent mechanism. We propose that MMP-mediated myelin loss is important in hypoxic injury to the white matter.

TIMP-3 and MMP-3 contribute to delayed inflammation and hippocampal neuronal death following global ischemia.

Hippocampal neuronal death following transient global ischemia in the mouse takes days to occur, providing a potential timeframe for therapeutic intervention. Since matrix metalloproteinase-3 (MMP-3) enhances inflammation and tissue inhibitor of metalloproteinases-3 (TIMP-3) promotes apoptosis in ischemia, we hypothesized that they are involved in neuronal death secondary to transient global ischemia. Timp-3 knockout (T3KO) and wild type (T3WT) mice underwent 30 min bilateral carotid artery occlusion (BCAO), which causes hippocampal neuronal death 7 days after reperfusion. Mice lacking the Timp-3 gene have significantly less astrocytosis, microglial reactivity, MMP-3 activity and neuronal cell death. In addition, T3KO mice had decreased tumor necrosis factor (TNF) receptor-1 (TNFR1) expression and increased TNF-alpha converting enzyme (TACE) activity. Mmp-3 KO mice with a similar BCAO showed significantly fewer microglial cells, reduced TNF-alpha expression, and less neuronal death than the Mmp-3 WT. To see if TIMP-3 and MMP-3 cell death pathways were independent, we blocked MMPs with the broad-spectrum MMP inhibitor, BB-94, on days 3 through 6 of reperfusion in T3WT and T3KO mice. BB-94 rescued hippocampal neurons at 7 days in both T3WT and T3KO mice, but significantly fewer neurons died in T3KO mice treated with BB-94. Our results indicate a novel additive role for TIMP-3 and MMP-3 in delayed neuronal death, and show that delayed treatment with MMP inhibitors can be used to reduce hippocampal death.

Increased apparent diffusion coefficients on MRI linked with matrix metalloproteinases and edema in white matter after bilateral carotid artery occlusion in rats.

White matter (WM) injury after bilateral common carotid artery occlusion (BCAO) in rat is associated with disruption of the blood-brain barrier (BBB) by matrix metalloproteinases (MMPs). We hypothesized that WM injury as seen on magnetic resonance imaging (MRI) would correlate with regions of increased MMP activity. MRI was performed 3 days after BCAO surgery in rats. Apparent diffusion coefficients (ADC) were calculated and vascular permeability was quantified by the multiple-time graphical analysis (MTGA) method, using gadolinium-diethylenetriamine pentaacid (Gd-DTPA). After MRI, one group of animals had BBB permeability measured in the WM with (14)C-sucrose, and another had Evans blue (EB) injected for fluorescent microscopy for MMP-2, MMP-9, tight junction proteins (TJPs), and in situ zymography. We found that ADC values were increased in WM in BCAO rats compared with controls (P<0.05). WM with increased ADC had leakage of EB. MMP-2 and MMP-9 activity on in situ zymograms corresponded with leakage of EB. Although increased permeability to EB could be visualized, permeability quantification with (14)C-sucrose and Gd-DTPA failed to show increases and TJPs were intact. We propose that increased ADC, which is a marker of vasogenic edema, is related to activity of MMP-2 and MMP-9. MRI provides unique information that can be used to guide tissue studies of WM injury.