VEGF Induces More Severe Cerebrovascular Dysplasia

Authors: Qi Hao Yiqian Zhu Hua Su Fanxia Shen GuoYuan Yang Helen Kim William L Young

Publish Date: 2010/04/13

Volume: 1, Issue: 3, Pages: 197-201

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Abstract

Brain arteriovenous malformations BAVMs are an important cause of intracranial hemorrhage ICH in young adults A small percent of BAVMs is due to hereditary hemorrhagic telangiectasia 1 and 2 HHT1 and 2 which are caused by mutations in two genes involved in transforming growth factorβ signaling endoglin Eng and activinlike kinase 1 Alk1 The BAVM phenotype has incomplete penetrance in HHT patients and the mechanism is unknown We tested the hypothesis that a “responsetoinjury” triggers abnormal vascular dysplasia development using Eng and Alk1 haploinsufficient mice Adenoassociated virus AAV expressing vascular endothelial growth factor VEGF was used to mimic the injury conditions VEGF overexpression caused a similar degree of angiogenesis in the brain of all groups except that the cortex of Alk1+/− mice had a 33 higher capillary density than other groups There were different levels of cerebrovascular dysplasia observed in haploinsufficient mice Eng+/−  Alk1+/− which simulates the relative penetrance of BAVM in HHT patients HHT1  HHT2 Few dysplastic capillaries were observed in AAVLacZinjected mice Our data indicate that both angiogenic stimulation and genetic alteration are necessary for the development of vascular dysplasia suggesting that antiangiogenic therapies might be adapted to slow the progression of the disease and decrease the risk of spontaneous ICHBrain arteriovenous malformations BAVM are an important cause of intracranial hemorrhage ICH in young adults Roughly 2–5 of all brain AVMs is due to hereditary hemorrhagic telangiectasia HHT in which the main subtypes HHT1 and 2 are caused by lossoffunction mutations in two genes involved in transforming growth factorβ signaling endoglin Eng and activinlike kinase 1 Alk1 HHT is an autosomal dominant disorder but the BAVM lesion has incomplete penetrance and the mechanism is unknown 1 The BAVM lesional phenotype includes an active angiogenic and inflammatory component that is inconsistent with a static congenital anomaly 2 suggesting that a “responsetoinjury” may explain incomplete penetrance of BAVM in HHT patients There is important new evidence from extracranial tissues that such responsetoinjury takes place in AVM development 3To explore this general hypothesis we overexpressed vascular endothelial growth factor VEGF an angiogenic factor upregulated in injured tissue and BAVM lesional tissue in the brain of Eng+/− and Alk1+/− mice We showed that vascular dysplasia occurs only after VEGF stimulation and is more pronounced in Eng+/− than in Alk1+/− miceExperimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee of the University of California San Francisco UCSF Eng+/− and Alk1+/− mice were obtained from Dr Douglas Marchuks laboratory at Duke University and bred at UCSFs animal facility These mice have been backcrossed with C57BL mice for more than ten generations in our laboratory Thus they are in C57BL/6 background C57BL wild type WT mice were used as control Adult male Eng+/− Alk1+/− Eng/Alk1+/− Eng and Alk1 double heterozygous knockout and WT mice weighing 25–30 g 8 to 10 weeks old were used for the experimentsMice were anesthetized using ketamine/xylazine 100/10 mg/kg body weight intraperitoneally and placed in a stereotactic frame with a mouth holder David Kopf Instruments Tujunga CA USA A burr hole was drilled in the pericranium 2 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture A 10 µl Hamilton syringe was inserted into the right basal ganglia For cortical injection the needle was inserted 1 mm into the cortex and advanced 2 mm parallel to the surface Two microliters of viral suspension containing 2 × 109 genome copies of adenoassociated virus AAVVEGF or AAVLacZ AAV vectors with cytomegalovirus CMV promoter driving VEGF or LacZ gene expression was slowly injected at a rate of 02 µl per minuteBrain specimens were collected 6 weeks after the vector injection The brain was removed snapfrozen with dry ice and stored at −80°C A series of 20 µm cryostat coronal sections were collected Sections were fixed with 4 paraformaldehyde for 30 min and incubated overnight with fluoresceinlycopersicin esculentum lectin Vector Laboratories Burlingame CA USA 2 µg/ml at 4°CTwo coronal sections located 05 mm rostral to and 05 mm caudal to the injection site were chosen for vessel quantification Pictures were taken from three areas left right and bottom of the injection site of each section using ×10 microscope objective lens Capillary density CD the number of capillaries per ×10 objective field and the dysplasia index the number of capillaries with lumen 15 µm per 200 capillaries were quantified using Image J 4 Two investigators without knowledge of treatment conditions performed the quantification separately

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