9/6/2014 Brain AVM Models and Novel Therapeutic Targets I have nothing to disclose. 2 nd UCSF Stroke and Aneurysm Update CME Saturday September 6, 2014 Hua Su, MD. Professor Center for Cerebrovascular Research Department of Anesthesia and Perioperative Care University of California, San Francisco Hua.su@ucsf.edu CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Brain Arteriovenous Malformations (AVMs) Current Treatments Surgery, embolization and radiosurgery • Tangle of abnormal blood vessels (nidus) No specific medical therapy for brain AVM – Arteriovenous shunting The goals of specific medical treatments are: – No intranidal capillary bed – Range of vessel types 1. Stabilize vessel wall • Located randomly throughout brain -reduce spontaneous intracranial hemorrhage and hemorrhagic • Cause of hemorrhagic stroke stroke 2. Reduce brain AVM grow or regrow after invasive treatment Invasive therapy (n=114) 3. Reduce AVM volume Death or Stoke (%) -surgical resection easier -reduce risk of invasive procedures HR=0.27 (95% CI: 0.14-0.54) Medical management (n=109) Months CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 1
9/6/2014 Tissue assays of surgical specimens: “ angiogenesis run amok” “a healing wound” endothelium Identify Specific Targets Notch Ki-67 aVB3 -Analyzing surgical specimens Macrophage VEGF-R & Leukocytes Hashimoto, Neurosurgery 54: 410, 2004 Tie-2 -Modeling brain AVM in animals Shenkar, Neurosurgery 52: 465, 2003 MMP-9 Kilic, Neurosurgery 57: 997, 2005 VEGF Imbalance in Sure, Neurosurgery 55: 663, 2004 HIF-1 α Angiopoietin 1 & 2 Sonstein; J Neurosurg 85:838, 1996 ZhuGe, Q. et al. Brain 2009 astrocyte Notch Murphy, PA. Laboratory Investigation 2009 smooth muscle CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Are brain AVMs heritable? Hereditary Hemorrhagic Telangiectasia (HHT) Rendu-Osler-Weber Syndrome • Sporadic 95-98% no family history • Autosomal dominant disorder • Mucocutaneous telangiectasia • Familial • AVMs in Liver, Lung and Brain – Hereditary Hemorrhagic Telangiectasias (HHT) • 80% of cases have functional heploinsufficiency of – RASA1 (p120 RasGAP, is a Ras GTPase–activating Endoglin (HHT1) or ALK1 (HHT2) protein) capillary malformation-AVM Brain AVMs Liver AVM • Eerola, Am J Hum Genet 73: 1240, 2003 Lung AVM – Non-HHT • 53 patients in 25 families – van Beijnum, et al, JNNP 78: 1213, 2007 – Inoue, et al, Stroke 38: 1368, 2007 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 2
9/6/2014 Eng +/- and Alk1 +/- mice –minimal brain phenotype AdCre – Regional Conditional Deletion of Alk1 Exon 3 Exon 7 Satomi, et al, Stroke 2003;34:783 Exons 4, 5, 6 Corrosion casting and SEM Alk 1 gene Promoter revealed AVMs in 3/10 mice loxp loxp AdCre CMV Promoter Cre recombinase Srinivasan, et al, Hum Mol Genet 12: 473, 2003 Exon 7 Exon 3 In >47 mice, one Alk1 +/- with dilated Exons 4,5,6 are cerebellar vessel deleted from Alk1 Promoter genome loxp CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research VEGF Stimulation is Necessary for Brain Alk1 Regional Conditional Deletion Plus VEGF AVM Formation Stimulation Results in Brain AVM Alk1 -/- AdCre + AAV-VEGF Angiogenesis 8 wks Alk1 -/- /VEGF Alk1 -/- only Alk1 +/+ /VEGF Alk1 +/+ /VEGF Walker et al. Ann Neurology, 2011 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Walker et al. Ann Neurology, 2011 3
9/6/2014 Adult onset AVM models Some Models have AVM in Other Organs skin Chen et al. Translational Stroke Research, 2014 Choi et al., PLOS One, 2014 Chen et al, Stroke, 2014 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Macrophage Infiltration AVM vessels have less smooth muscle cell coverage Chen et al. ATVB, 2013 CCR UCSF center for cerebrovascular research Chen et al. ATVB, 2013 CCR UCSF center for cerebrovascular research 4
9/6/2014 AVM vessels have less pericyte coverage Microhemorrhage Chen et al. ATVB, 2013 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Chen et al. ATVB, 2013 ALK1 Knockdown Attenuates the Upregulation of PDGFB in ALK1 knockdown in HBMEC impairs the pericyte HBMEC in Response to VEGF Stimulation recruitment B A 3 B A 5 Control Fold Change 2.5 Control Alk1 mRNA Pdgfb mRNA Fold Change shAlk1 4 Control shAlk1 2 3 VEGF 1.5 * 2 1 * shAlk1 * * 0.5 * 1 * VEGF + shAlk1 * * * 0 0 VEGF 0 10 50 100 20 40 60 VEGF 0 10 50 100 (ng /ml) (ng /ml) Average Pericyte Distance µm HBMEC (human brain microvascular endothelial cell) were transfected with control shRNA or shRNA . Cells with >70% reduction of Alk1 gene expression were cultured for 18 h in the presence or absence of VEGF (0, 10, 50, and 100 ng/ml). qRT-PCR was performed for Alk1 (A) and Pdgfb (B) . All data are shown as mean and SD. *p<0.05 vs. control. CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 5
9/6/2014 Reduction of Gene Mutant Endothelial Gene Mutation in Bone Marrow Transmits Cell Reduced GI Hemorrhage and Mortality the Phenotype 50 µm CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Important Factors in AVM Anti-Angiogenesis Therapies Pathogenesis Bevacizumab reverse brain AVM phenotype Anti-angiogenesis Angiogenesis (bevacizumab, sFLT) Improve vascular integrity Impaired mural (Thalidomide,Lenalidomide) cell recruitment BM or monocyte BMDC/Monocyte transfusion Anti-inflammation Inflammation (tetracycline class) Walker et al. Stroke, 2012 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 6
9/6/2014 Developmental onset Anti-Angiogenesis Stereotactic Injection of AAV2-sFLT Inhibited Brain AVM Formation 1. SM22 α -Cre mediated Eng deletion 2. 95% mice have brain AVM at five weeks of age 3. Brain AVM in this model was developed spontaneously without local angiogenic stimulation 4. About 30% mice died between 3 and 6 weeks AAV2-EV 1. Block VEGF that is used for model induction 2. Invasive intra-brain injection AAV2-sFLT02 Choi et al., PLOS One, 2014 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Intravenous Injection of AAV9-sFLT Reversed Brain AVM Phenotype SM22 α Cre; Eng f/ f mice Increase PDGFB 1. 1X10 11 vg AAV9-sFLT 1. AAV-sFLT reverse brain AVM phenotype IV to 5 weeks old mice. 2. Systemicdelivery of AAV-sFLT is feasible 2. Samples were collected 3. AAV-sFLT is effect on spontaneous 4 weeks later developed bran AVM number motality paralyzed final mice number AAV9-GFP 9 3 1 5 Lebrin, et al, Nat Med 16: 420, 2010 AAV9-sFLT 8 2 6 CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 7
9/6/2014 Increase PDGFB Increase PDGFB Thalidomide Treatment Reduced Microhemorrhage Thalidomide Treatment Reduced the Number of Abnormal Vessels CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research Summary Thank You Espen Walker 1. Invasive therapies are associated with considerable risks Wanqiu Chan 2. No specific medical therapy is available Eunjung Choi Fanxia Shen 3. The concept for the treatment of brain AVM is to Yi Guo stabilize vascular tissue and thereby decrease the risk of Lei Mao spontaneous ICH. Marine Camus 4. Novel therapeutic approaches: Mamta Wankhede William L. Young Zhengyi Han A. Anti-inflammation Yue He Mervyn Maze B. Anti-angiogenesis Cameron McDougall Helen Kim C. Improve vascular integrity Funding: Liang Wang Ludmila Pawlikowska D. Correct gene mutation in BM monocyte/progenitors Lei Zhan NIH Michael T. Lawton Shuai Kang AHA Charles E. McCulloch Wan Zhu Michael Ryan Rui Zhang Zodda Foundation Dingquan Zou CCR UCSF center for cerebrovascular research CCR UCSF center for cerebrovascular research 8
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