Bevacizumab Attenuates VEGF-Induced Angiogenesis and Vascular Malformations in the Adult Mouse Brain

Article · May 2012with28 Reads
DOI: 10.1161/STROKEAHA.111.647982 · Source: PubMed
Abstract
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. 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). 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. 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.
    • Although VEGF-A has been shown to directly alter neuronal properties independent of vascular changes (Sondell et al., 1999), it is possible that the observed attenuation of FXS abnormalities are not due to direct neuronal modulation, but rather due to indirect Bevacizumab induced VEGF-A modulation of vascular properties. Blocking VEGF-A (via Bevacizumab ) decreases VEGF-induced angiogenesis in control brains (Walker et al., 2012); however, its effect on already established vascularization in FXS has not been explored. If decreasing VEGF-A expression decreases vascular density in FXS brain, decreased nutrients and other signaling factors being delivered via the blood stream could mediate some or all of the benefits observed.
    [Show abstract] [Hide abstract] ABSTRACT: Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, ). One of the most prominent regulators of vascular growth is VEGF-A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF-A expression. In addition to its role in vascular regulation, VEGF-A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF-A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF-A in brain. These studies suggest that increased VEGF-A expression in FXS is contributing to non-vascular FXS abnormalities. To explore the role of VEGF-A in mediating non-vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF-A. Bevacizumab treatment was found to decrease FXS Synapsin-1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF-A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF-A is elevated in FXS brain and suggest that its expression promotes non-vascular FXS abnormalities.
    Full-text · Article · Jun 2016
    • Dll4-Notch1 signaling suppresses tip cell formation leading to nonproductive sprouting, whereas Jagged-1 antagonizes the Dll4 ligand, thereby promoting sprouting angiogenesis[46]. We and others have found through analysis of human bAVM specimens and animal model development that a pro-angiogenic signal is needed for bAVM development[18,[47][48][49][50][51]. Without Dll4 signaling, a pro-angiogenic state is favored, for example, proliferation of tip at the expense of stalk cells[46,52].
    [Show abstract] [Hide abstract] ABSTRACT: Brain arteriovenous malformation (bAVM) is an important cause of intracranial hemorrhage (ICH), particularly in the young population. ICH is the first clinical symptom in about 50 % of bAVM patients. The vessels in bAVM are fragile and prone to rupture, causing bleeding into the brain. About 30 % of unruptured and non-hemorrhagic bAVMs demonstrate microscopic evidence of hemosiderin in the vascular wall. In bAVM mouse models, vascular mural cell coverage is reduced in the AVM lesion, accompanied by vascular leakage and microhemorrhage. In this review, we discuss possible signaling pathways involved in abnormal vascular development in bAVM.
    Full-text · Article · Jan 2016
    • In patients with HHT, it was suggested that a ''second hit'', an angiogenic stimulus such as VEGF, might be the trigger for AVM formation in a host with an inherited predisposition for endothelial dysfunction [45]. In this respect, conditional knockout mice with tissue-specific Eng or Alk1 gene deletion [19, 46] are more appropriate to study the effects of anti-VEGF therapy on regression of AVMs. Despite no difference in basal VEGF levels, Eng ?/-and
    [Show abstract] [Hide abstract] ABSTRACT: Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angi-ogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor b (TGF-b) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombo-spondin-1 (TSP-1) in Eng ?/-versus a rise in angiopoietin-2 (Ang-2) in Alk1 ?/-mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng ?/-mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.
    Full-text · Article · Oct 2015
    • Alternatively, if VEGF is neutralized following initial AVM formation then it can block further progression of established AVMs or even cause regression of early AVMs (Han et al., 2014). Similarly, anti-VEGF treatment leads to attenuation of murine brain AVMs that form following the combination of local angiogenic stimulation and Acvrl1 depletion (Walker et al., 2012). These findings bode well for current anti-VEGF therapies in clinical trial for HHT patients (Kanellopoulou and Alexopoulou, 2013).
    [Show abstract] [Hide abstract] ABSTRACT: Hereditary Haemorrhagic Telangiectasia (HHT) is a genetic disorder characterised by a multi-systemic vascular dysplasia and haemorrhage. The precise factors leading to these vascular malformations are not yet understood and robust animal models of HHT are essential to gain a detailed understanding of the molecular and cellular events that lead to clinical symptoms, as well as to test new therapeutic modalities. Most cases of HHT are caused by mutations in either endoglin (ENG) or activin receptor like kinase 1 (ACVRL1, also known as ALK1). Both genes are associated with TGFβ/BMP signalling, and loss of function mutations in the co-receptor ENG are causal in HHT1, whilst HHT2 is associated with mutations in the signalling receptor ACVRL1. Significant advances in mouse genetics have provided powerful ways to study the function of Eng and Acvrl1 in vivo, and to generate mouse models of HHT disease. Mice that are null for either Acvrl1 or Eng genes show embryonic lethality due to major defects in angiogenesis. However mice that are heterozygous for mutations in either of these genes develop to adulthood with no effect on survival. Although these heterozygous mice exhibit selected vascular phenotypes relevant to the clinical pathology of HHT, the phenotypes are variable and generally quite mild. An alternative approach using conditional knockout mice allows us to study the effects of specific inactivation of either Eng or Acvrl1 at different times in development and in different cell types. These conditional knockout mice provide robust and reproducible models of arteriovenous malformations, and they are currently being used to unravel the causal factors in HHT pathologies. In this review, we will summarize the strengths and limitations of current mouse models of HHT, discuss how knowledge obtained from these studies has already informed clinical care and explore the potential of these models for developing improved treatments for HHT patients in the future.
    Full-text · Article · Feb 2015
    • These data provide evidence that Alk1 modulates angiogenesis at least in part through modulation of VEGF signaling. This is line with a study showing that treatment with bevacizumab, an antibody that binds and neutralises human VEGF, decreases the number of dysplastic vessels in the brain of mice deficient for Alk1 [44]. In addition, we also show that BMP9/Alk1 signaling results in changes in VEGFR1 expression in ECs.
    [Show abstract] [Hide abstract] ABSTRACT: Age-related macular degeneration (AMD) is the leading cause of blindness in aging populations of industrialized countries. The drawbacks of inhibitors of vascular endothelial growth factor (VEGFs) currently used for the treatment of AMD, which include resistance and potential serious side-effects, require the identification of new therapeutic targets to modulate angiogenesis. BMP9 signaling through the endothelial Alk1 serine-threonine kinase receptor modulates the response of endothelial cells to VEGF and promotes vessel quiescence and maturation during development. Here, we show that BMP9/Alk1 signaling inhibits neovessel formation in mouse models of pathological ocular angiogenesis relevant to AMD. Activating Alk1 signaling in laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) inhibited neovascularization and reduced the volume of vascular lesions. Alk1 signaling was also found to interfere with VEGF signaling in endothelial cells whereas BMP9 potentiated the inhibitory effects of VEGFR2 signaling blockade, both in OIR and laser-induced CNV. Together, our data show that targeting BMP9/Alk1 efficiently prevents the growth of neovessels in AMD models and introduce a new approach to improve conventional anti-VEGF therapies.
    Full-text · Article · Nov 2014
    • Bevacizumab (Avastin) normalized cardiac output in HHT patients harboring liver AVMs [128], and was effective in the treatment of severe epistaxis caused by hemorrhage from small mucosal AVMs (telangiectasias)129130131132133134135136. Importantly, we demonstrated that after establishment of the bAVM phenotype in a conditional Alk1 deletion mouse model [55], intra-peritoneal bevacizumab treatment reduced the number of abnormal vessels, suggesting that maintenance of the bAVM phenotype is dependent on tonic VEGF signaling [137]. However, antibody therapy has many drawbacks, including concerns regarding hemorrhage [138] and the need for prolonged periods of intermittent intravenous (i.v.) infusions.
    [Show abstract] [Hide abstract] ABSTRACT: Patients harboring brain arteriovenous malformation (bAVM) are at life-threatening risk of rupture and intracranial hemorrhage (ICH). The pathogenesis of bAVM has not been completely understood. Current treatment options are invasive, and ≈ 20 % of patients are not offered interventional therapy because of excessive treatment risk. There are no specific medical therapies to treat bAVMs. The lack of validated animal models has been an obstacle for testing hypotheses of bAVM pathogenesis and testing new therapies. In this review, we summarize bAVM model development and bAVM pathogenesis and potential therapeutic targets that have been identified during model development.
    Full-text · Article · Apr 2014
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February 2014 · Methods in molecular biology (Clifton, N.J.) · Impact Factor: 1.29
Brain arteriovenous malformations (bAVM) are tangles of abnormal, dilated vessels that directly shunt blood between the arteries and veins. The pathogenesis of bAVM is currently unknown. Patients with hereditary hemorrhagic telangiectasia (HHT) have a higher prevalence of bAVM than the general population. Animal models are important tools for dissecting the disease etiopathogenesis and for... [Show full abstract]
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February 2014 · PLoS ONE · Impact Factor: 3.23
Endoglin (ENG) is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have a higher prevalence of brain arteriovenous malformation (AVM) than the general population and patients with other HHT subtypes. The pathogenesis of brain AVM in HHT1 patients is currently unknown and no specific medical therapy is available to treat patients. Proper animal models are... [Show full abstract]
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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... [Show full abstract]
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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... [Show full abstract]
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