Kristine Jun

Cardiomyopathy of aging in the mammalian heart is characterized by myocardial hypertrophy, fibrosis and a predisposition towards cardiomyocyte apoptosis and autophagy

Aging is associated with an increased incidence of heart failure, but the existence of an age-related cardiomyopathy remains controversial. Differences in strain, age and technique of measuring cardiac function differ between experiments, confounding the interpretation of these studies. Additionally, the structural and genetic profile at the onset of heart failure has not been extensively studied. We therefore performed serial echocardiography, which allows repeated assessment of left ventricular (LV) function, on a cohort of the same mice every 3 months as they aged and demonstrated that LV systolic dysfunction becomes apparent at 18 months of age. These aging animals had left ventricular hypertrophy and fibrosis, but did not have inducible ventricular tachyarrhythmias. Gene expression profiling of left ventricular tissue demonstrated 40 differentially expressed probesets and 36 differentially expressed gene ontology terms, largely related to inflammation and immunity. At this early stage of cardiac dysfunction, we observed increased cardiomyocyte expression of the pro-apoptotic activated caspase-3, but no actual increase in apoptosis. The aging hearts also have higher levels of anti-apoptotic and autophagic factors, which may have rendered protection from apoptosis. In conclusion, we describe the functional, structural and genetic changes in murine hearts as they first develop cardiomyopathy of aging.

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 Alk12f/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.

Novel Brain Arteriovenous Malformation Mouse Models for Type 1 Hereditary Hemorrhagic Telangiectasia

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 crucial for identifying the underlying mechanisms for brain AVM development and for testing new therapies. However, creating HHT1 brain AVM models has been quite challenging because of difficulties related to deleting Eng-floxed sequence in Eng2fl/2fl mice. To create an HHT1 brain AVM mouse model, we used several Cre transgenic mouse lines to delete Eng in different cell-types in Eng2fl/2fl mice: R26CreER (all cell types after tamoxifen treatment), SM22α-Cre (smooth muscle and endothelial cell) and LysM-Cre (lysozyme M-positive macrophage). An adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) was injected into the brain to induce focal angiogenesis. We found that SM22α-Cre-mediated Eng deletion in the embryo caused AVMs in the postnatal brain, spinal cord, and intestines. Induction of Eng deletion in adult mice using R26CreER plus local VEGF stimulation induced the brain AVM phenotype. In both models, Eng-null endothelial cells were detected in the brain AVM lesions, and formed mosaicism with wildtype endothelial cells. However, LysM-Cre-mediated Eng deletion in the embryo did not cause AVM in the postnatal brain even after VEGF stimulation. In this study, we report two novel HHT1 brain AVM models that mimic many phenotypes of human brain AVM and can thus be used for studying brain AVM pathogenesis and testing new therapies. Further, our data indicate that macrophage Eng deletion is insufficient and that endothelial Eng homozygous deletion is required for HHT1 brain AVM development.

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 &mgr;m 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.

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 Alk11f/2f mice (loxP sites flanking exons 4–6) and wild-type mice were injected with 2×107 PFU adenovious-cre recombinase and 2×109 genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 versus 40±13×103; P=0.001), iron deposition (508±506 pixels/mm2 versus 6±49; P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 versus 240±104 Iba1+; P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more &agr;-smooth muscle actin negative vessels (52±9% versus 12±7%, P<0.001), fewer vascular-associated pericytes (503±179/mm2 versus 931±115, P<0.001), and reduced platelet-derived growth factor receptor-&bgr; expression. Conclusion—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.

Bone Fracture Exacerbates Murine Ischemic Cerebral Injury

Background:Bone fracture increases alarmins and proinflammatory cytokines in the blood, and provokes macrophage infiltration and proinflammatory cytokine expression in the hippocampus. We recently reported that stroke is an independent risk factor after bone surgery for adverse outcome; however, the impact of bone fracture on stroke outcome remains unknown. We tested the hypothesis that bone fracture, shortly after ischemic stroke, enhances stroke-related injuries by augmenting the neuroinflammatory response. Methods:Tibia fracture (bone fracture) was induced in mice one day after permanent occlusion of the distal middle cerebral artery (stroke). High-mobility-group box chromosomal protein-1 (HMGB1) was tested to mimic the bone fracture effects. HMGB1 neutralizing antibody and clodrolip (macrophage depletion) were tested to attenuate the bone fracture effects. Neurobehavioral function (n = 10), infarct volume, neuronal death, and macrophages/microglia infiltration (n = 6–7) were analyzed after 3 days. Results:We found that mice with both stroke and bone fracture had larger infarct volumes (mean percentage of ipsilateral hemisphere ± SD: 30±7% vs.12±3%, n = 6, P < 0.001), more severe neurobehavioral dysfunction, and more macrophages/microglia in the periinfarct region than mice with stroke only. Intraperitoneal injection of HMGB1 mimicked, whereas neutralizing HMGB1 attenuated, the bone fracture effects and the macrophage/microglia infiltration. Depleting macrophages with clodrolip also attenuated the aggravating effects of bone fracture on stroke lesion and behavioral dysfunction. Conclusions:These novel findings suggest that bone fracture shortly after stroke enhances stroke injury via augmented inflammation through HMGB1 and macrophage/microglia infiltration. Interventions to modulate early macrophage/microglia activation could be therapeutic goals to limit the adverse consequences of bone fracture after stroke.

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.