Circulating cells as tools to study vascular pathobiology of HHT

描述（由申请人提供）：遗传性出血性毛细血管扩张症（HHT）是一种罕见的疾病（发生率约为1/ 5000），主要由编码TGF - MP受体的ENG或ACVRL1（又称ALK1）突变引起。患者出现血管畸形，包括小的粘膜皮肤毛细血管扩张和/或大的多器官内脏动静脉畸形（AVMs）。临床表现的外显率和表达率在患者之间变化很大，甚至在一个家庭内也是如此。该项目的基本原理是研究血液中循环细胞的生物学和分子特性，特别是但不仅限于循环内皮祖细胞（cEPCs），将为HHT病理生物学的基本细胞和分子机制提供重要的新知识，并有助于开发预后HHT标记物。需要验证的主要假设是：a) HHT患者血液中循环骨髓来源细胞群的细胞组成受到干扰；b) HHT中cEPC数量升高，导致并与包括毛细血管扩张和avm在内的疾病病理相关；c) HHT cEPC中的基因表达网络受到干扰，从而改变其生物学功能，d) HHT基因修饰物调节cEPC的数量和生物学。将测试细胞和分子扰动与疾病类型（HHT1 vs HHT2）和疾病严重程度的关联程度，并使用HHT血液中培养的内皮集落形成细胞（ecfc）研究分子机制。目的1)多色FACS分析将用于评估HHT患者和HHT小鼠模型中循环细胞数量的扰动，重点是cEPCs和单核细胞。目的2)从HHT患者和对照组的血液中分离ecfc，用于研究体外血管生成临床相关参数的改变，以及HHT修饰基因siRNA敲低的生物学效应。一个cepc衍生的iPS细胞库将产生，供HHT社区使用。目的3)比较野生型、Eng+/-和Alk1+/-小鼠的血细胞mRNA和miRNA转录组谱。血液基因表达网络将从遗传异质性小鼠（>100）和人类（>100）群体中现有的转录组学数据中生成。最后，将从人类HHT1、HHT2和对照血液中生成新的转录组学数据，以验证HHT cEPCs和/或单核细胞内基因表达网络重新连接以改变细胞参数的假设，这些参数可以在Aim 2中进行分子探测，从而为HHT疾病机制提供机制见解。

DESCRIPTION (provided by applicant): Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease (affecting ~1/5, 000), caused predominantly by mutations in ENG or ACVRL1 aka ALK1 encoding TGF�MP receptors. Patients develop vascular malformations, including small mucocutaneous telangiectases and/or large visceral arteriovenous malformations (AVMs) in multiple organs. The penetrance and expressivity of clinical manifestations is highly variable between patients, even within a family. The rationale for the project is that investigation of the biological and molecular characteristics of cells circulating within blood, especially, but not onl, circulating endothelial progenitor cells (cEPCs), will provide important new knowledge about basic cellular and molecular mechanisms of HHT pathobiology, and contribute to development of prognostic HHT markers. The overarching hypotheses to be tested are: a) the cellular makeup of circulating bone marrow-derived cell populations is perturbed in the blood of HHT patients; b) there is an elevation of cEPC numbers in HHT that contributes to and correlates with disease pathology including telangiectases and AVMs; c) gene expression networks are perturbed in HHT cEPCs to alter their biological functions, and d) HHT genetic modifiers regulate cEPC numbers and biology. The extent to which cellular and molecular perturbations correlate with disease type (HHT1 versus HHT2) and disease severity will be tested, and molecular mechanisms investigated using cultured endothelial colony forming cells (ECFCs) from HHT bloods. Aim 1) multi-color FACS analysis will be utilized to assess perturbation in circulating cell numbers in HHT patients and HHT mouse models, focusing on cEPCs and monocytes. Aim 2) ECFCs will be isolated from blood of HHT patients and controls, and utilized to investigate alterations in clinically-relevant parameters of in vitro angiogenesis, and biologicl effects of siRNA knock down of HHT modifier genes. A bank of cEPC-derived iPS cells will be generated for use by the HHT community. Aim 3) Blood cell mRNA and miRNA transcriptomic profiles will be compared between wild type, Eng+/- and Alk1+/- mice. Blood gene expression networks will be generated from existing transcriptomic data available from genetically heterogeneous mouse (>100) and human (>100) populations. Finally, new transcriptomic data will be generated from human HHT1, HHT2 and control blood, to test the postulate that gene expression networks are rewired within HHT cEPCs and/or monocytes to alter cellular parameters that can be molecularly probed in Aim 2 in order to provide mechanistic insight into HHT disease mechanisms.