Vascular Endothelial Growth Factor Induces Abnormal Microvasculature in the Endoglin Heterozygous Mouse Brain

Abstract

Summary:Hereditary hemorrhagic telangiectasia (HHT), associated with brain arteriovenous malformations, is caused by a loss of function mutation in either the endoglin (HHT1) or activin receptor-like kinase 1 gene (ALK-1, HHT2). Endoglin heterozygous (Eng +/− )mice have been proposed as a disease model. To better understand the role of endoglin in vascular malformation development, we examined the effect of vascular endothelial growth factor (VEGF) hyperstimulation on microvessels in adult endoglin heterozygous (Eng +/− ) mice using an adenoviral vector to deliver recombinant human VEGF 165 cDNA (AdhVEGF) into basal ganglia. VEGF expression was increased in AdhVEGF mice compared with the AdlacZ and saline group (P < 0.05) and localized to multiple cell types (neurons, astrocytes, endothelial cells, and smooth muscle cells) by double-labeled immunostaining. VEGF overexpression increased microvessel count for up to 4 weeks in both the Eng +/+ and Eng +/− groups (Eng +/+ 185 ± 14 vs. Eng +/− 201 ± 10 microvessels/mm 2 ). Confocal microscopic examination revealed grossly abnormal microvessels in eight of nine Eng +/− mouse brains compared with zero of nine in Eng +/+ mice (P < 0.05). Abnormal microvessels featured enlargement, clustering, twist, or spirals. VEGF receptor Flk-1 and TGF-␤ receptor 1 (T␤R1) expression were reduced in the Eng +/− mouse brain compared with control.Excessive VEGF stimulation may play a pivotal role in the initiation and development of brain vessel malformations in states of relative endoglin insufficiency in adulthood. These observations are relevant to our general understanding of the maintenance of vascular integrity.

Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation

“…Fluorescein-lycopersicin esculentum lectin (Vector Laboratories, Burlingame, CA) staining was used to identify capillaries. We chose lectin staining because our previous studies demonstrated that this method is reproducible and reliable (29,39,48,50). Sections were fixed with 4% paraformaldehyde (PFA) for 30 min, blocked with 5% normal blocking serum for another 30 min, and incubated overnight with 2 g/ml lectin at 4°C.…”

Erythropoietin Promotes Neuronal Replacement Through Revascularization and Neurogenesis After Neonatal Hypoxia/Ischemia in Rats

“…Functional vessels were detected by staining for tomato lectin injected intracardially immediately before euthanasia, which allows for identification of vessels receiving active blood flow 18,21 and precludes nonspecific labeling of microglia. In the CTX of control animals, the number of functional vessels continuously rose through postnatal day 21 ( Figure 1C-D), reflecting ongoing neovascular formation during postnatal development.…”

“…Functional vessels were identified by transcardial administration (5 minutes before euthanasia) of biotinylated-lycopersicin esculentum lectin (tomato lectin, 1.25 mg/kg; Vector Laboratories), which labels endothelial cells only in perfused vessels. 18 Lectin and BrdU stainings were visualized with fluorescence-streptavidin (1:2000; Vector Laboratories) and Cy3-conjugated secondary antibody using a Nikon microscope or Zeiss laser-scanning confocal microscope, respectively. Vessels that showed double labels of lectin and BrdU were counted in a square area (460ϫ460 mm) from the perilesion area (including the ischemic boundary), the distant area (Ͼ500 mm apart from the ischemic boundary), and contralateral area.…”

Doxycycline Suppresses Cerebral Matrix Metalloproteinase-9 and Angiogenesis Induced by Focal Hyperstimulation of Vascular Endothelial Growth Factor in a Mouse Model

“…The model consists of focal hyperstimulation by VEGF 165 in the brain using adenovirusmediated DNA transduction, resulting in enhanced formation of cerebral microvessels. 19,20 Our model is not a model of BAVM, but it is characterized by some of the phenotypic features of BAVM lesional tissue. Therefore, use of this model is a first step to mechanistically examine key angiogenic pathways and their response to pharmacological manipulation.…”