Control of ECM in AVM formation by hoeobox genes

hoeobox 基因控制 AVM 形成中的 ECM

基本信息

批准号：
8451439
负责人：
Nancy Joan Boudreau
金额：
$ 13.16万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-30 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8451439
关键词：
Address Angiogenic Factor Antibodies Antigens Architecture Behavior Biological Models Blood Vessels Brain Brain Neoplasms Brain Vascular Malformation Cell Line Cells Cellular Morphology Cerebral hemisphere hemorrhage Cerebrum Clinical Research Complex Connective Tissue Consult Cultured Cells Cutaneous Data Dissection Dysplasia Endoglin Endothelial Cells Ephrin-A1 Extracellular Matrix Family Freezing Gene Expression Gene Targeting Genes Genetic Models Growth Hemangioma Hemorrhage Homeobox Homeobox Genes Human Injection of therapeutic agent Integrins Intercellular Junctions Lesion Link Manuscripts Mediating Messenger RNA Modeling Morphology Mus Patients Prevalence Process Proteins Protocols documentation RNA Research Personnel Retrieval Role Sampling Signal Transduction Small Interfering RNA Specimen Staining method Stains Techniques Time Tissue Banking Tissue Banks Tissue Extracts Tissue Sample Tissues Transgenic Mice Vascular Endothelial Growth Factor Receptor-2 Vascular Endothelial Growth Factors Viral Western Blotting Work angiogenesis cell type human disease in vivo mRNA Expression mouse model prevent programs protein expression research study retroviral transduction selective expression thrombospondin 2 transcription factor

项目摘要

in the preliminary data in the last application showing the impact of HoxAS on vascular stability and growth. This work was done using endothelioma cells (bEND and EOMA) in cutaneous tissue.2 Further, we have now extended these studies to examine the impact of restoring HoxAS on growth of transformed endothelial cells in the mouse brain. A manuscript has just been submitted and these data are presented in Section 3.1b (Figs. 4- 7). The purpose of the work was to provide proof-of-concept that proliferative vascular tissue in the brain could be controlled by manipulation of HoxAS. Progress was hampered in part because our first choice of endothelioma cell type (bEND) did not reproducibly form lesions in vivo. The murine EOMA endothelioma cell line formed lesions similar to those induced by the bEND cells, but was phenotypically stable. We subsequently showed that restoring HoxAS could also limit growth of the EOMA tumors in the brain, and this was accompanied by a reduction in the HoxAS target Hifla and an increase in the anti-angiogenic TSP-2 gene. In addition we are currently conducting a clinical study to screen a large number of samples from the AVM tissue bank to document the prevalence of HoxAS dysregulation in a wide spectrum of AVM cases; this is part of Aim1. We previously showed preliminary ISH data (Fig.1) and have now performed this analysis on 7 AVM patient samples. A challenge which has slowed these studies is that our OCT frozen tissue sections are difficult to section due to the complex angio-architecture, thus making quantitative or semi-quantitative analysis by ISH difficult. Moreover, there is currently only one commercially available antibody against HoxAS, and while we have attempted to use this for staining cultured cells and also attempted different antigen retrieval and processing protocols for tissue samples, we have not been successful. I have also consulted with other investigators studying HoxAS who have not been able to apply this antibody for staining. We have now successfully used this for Western blot of tissue lysates (see Fig.8) and can apply this to human BAVM tissue specimens to confirm that HoxAS protein as well as mRNA is reduced. Nonetheless, we have now included new data to show that we can detect changes in HoxAS protein by performing Western blot on tissue lysates (Sec. 3.1, Fig.8) to confirm that changes in HoxAS mRNA are reflected at the level of protein. Importantly, to more accurately quantitate differences in HoxAS expression, we have now adapted a tissue micro-dissection technique that we developed for cutaneous hemangioma tissue to perform real time PCR analysis. We microscopically removed as much of the adjacent connective tissue and extract RNA from the remaining tissue, which is enriched in aberrant vessels. Real time PCR on this tissue has confirmed our ISH results: HoxAS expression is lacking in the vascular nidus of the AVM (Fig.1). We anticipate that we can now complete these experiments within the next several months as additional samples become available from Core C. All reviewers raised concerns regarding the number of different models proposed, the lack of justification for these models, and the paucity of preliminary data to support their use or their relevance to the human disease. Dr. Young has addressed the use of our model systems in the General Introduction. Moreover, the aims in Project 3 have now been revised and simplified to use only one general modelmurine angiogenesis and vascular dysplasiawhich has been well characterized by our group. 1. To first investigate whether HoxAS can impact cerebral angiogenesis, we will use focal VEGF stimulation using AAV-VEGF direct injection into the brain of transgenic mice which selectively expresses HoxAS in PHS 398/2590 (Rev.11/07) Page 266 Continuation Format Page

在上次申请中的初步数据显示了HoxAS对血管稳定性和生长的影响。这工作是使用皮肤组织中的内皮瘤细胞（bEND 和 EOMA）完成的。2 此外，我们现在有扩展了这些研究，以检查恢复 HoxAS 对转化内皮细胞生长的影响老鼠的大脑。手稿刚刚提交，这些数据显示在第 3.1b 节中（图 4- 7).这项工作的目的是提供概念证明，证明大脑中的增殖性血管组织可以通过HoxAS的操纵来控制。进展受到阻碍的部分原因是我们的第一选择内皮瘤细胞类型（bEND）在体内不能重复地形成病变。小鼠EOMA内皮瘤细胞细胞系形成的损伤与 bEND 细胞诱导的损伤相似，但表型稳定。我们随后表明恢复 HoxAS 也可以限制大脑中 EOMA 肿瘤的生长，这是伴随着 HoxAS 靶标 Hifla 的减少和抗血管生成 TSP-2 基因的增加。此外，我们目前正在进行一项临床研究，以筛选来自 AVM 的大量样本组织库记录各种 AVM 病例中 HoxAS 失调的患病率；这是一部分目标1。我们之前展示了初步 ISH 数据（图 1），现在对 7 个 AVM 进行了分析患者样本。减缓这些研究的一个挑战是我们的 OCT 冷冻组织切片很困难由于血管结构复杂，需要进行切片，从而通过ISH进行定量或半定量分析难的。此外，目前只有一种市售的针对 HoxAS 的抗体，而我们已尝试使用其对培养细胞进行染色，并尝试了不同的抗原修复和对于组织样本的处理方案，我们尚未成功。我也咨询过其他人研究 HoxAS 的研究人员无法应用该抗体进行染色。我们现在有成功地将其用于组织裂解物的蛋白质印迹（见图8），并且可以将其应用于人类 BAVM 组织样本以确认 HoxAS 蛋白以及 mRNA 减少。尽管如此，我们现在纳入了新数据，表明我们可以通过以下方式检测 HoxAS 蛋白的变化：对组织裂解物进行蛋白质印迹（第 3.1 节，图 8）以确认 HoxAS mRNA 的变化体现在蛋白质水平上。重要的是，为了更准确地定量 HoxAS 表达的差异，我们现在已经采用了我们为皮肤血管瘤组织开发的组织显微解剖技术进行实时 PCR 分析。我们在显微镜下切除了尽可能多的邻近结缔组织从剩余组织中提取 RNA，该组织富含异常血管。该组织的实时 PCR 证实了我们的 ISH 结果：AVM 的血管巢中缺乏 HoxAS 表达（图 1）。我们预计随着更多样本的出现，我们现在可以在接下来的几个月内完成这些实验可从 Core C 获得。所有审稿人都对提出的不同模型的数量、缺乏这些模型的合理性，以及缺乏支持其使用或相关性的初步数据对人类疾病。 Young 博士在概述中阐述了我们模型系统的使用。此外，项目 3 中的目标现已修改和简化为仅使用一种通用模型小鼠血管生成和血管发育不良已被我们小组充分表征。 1. 为了首先研究HoxAS是否会影响脑血管生成，我们将使用局部VEGF刺激使用AAV-VEGF直接注射到转基因小鼠的大脑中，该小鼠在体内选择性表达HoxAS PHS 398/2590 (Rev.11/07) 第 266 页继续格式页