Targeting the Pial Collateral Circulation for Mitigation of Cerebral Ischemia

描述（由申请人提供）：脑、心脏和下肢的闭塞性血管疾病是美国发病率和死亡率的主要原因。原生（预先存在的）侧支连接相邻的动脉树，并在其中一棵树发生阻塞时作为关键的旁路血管。在过去的25年里，人们一直在关注缺血性疾病中经络外重构的机制。然而，在我们过去三年的工作之前，我们对这些血管的数量和直径（范围）的控制因素一无所知。我们已经证明，由于遗传多态性，小鼠大脑和其他组织中的侧支范围差异很大，导致最近在人类中证实的低侧支范围个体的大脑和其他组织缺血性损伤的风险严重程度大大增加。我们已经发现了主要的遗传位点Candq1，它负责大部分（70%）的这种变异。我们现在试图确定这个位点的多态性基因及其下游调控侧枝形成的效应途径。我们将在初步研究中部分完成的同源菌株集中对Candq1进行精细定位：我们将把C57BL/6“高侧枝”菌株的Candq1位点的信息分区引入到在大脑和其他组织中几乎没有侧枝的BALB/c菌株中。然后对这些同源系在脑和骨骼肌中的侧支程度进行表型分析，然后在缺血性卒中模型中对阳性同源系（+CNG）进行功能分析，该阳性同源系最紧密地分隔了Candq1的致病区域。Aim II-A将通过使用RNA-Seq在Candq1上测定+CNG中的基因表达，以及在整个基因组上鉴定潜在的下游效应物，从而进一步缩小候选基因的范围。Aim II-B将进一步完善候选和靶点，并使用全基因组染色质定位活性DNA调控位点，确定Candq1是否存在顺式/反式调控元件的变异。因此，Aim I和II将确定最优先的候选者，Am III将使用体内基因靶向、细胞分析和功能结果来检测卒中的因果关系。该项目将确定控制本地侧循环形成的主要驱动基因，并对其广泛变化负责。确定Candq1的潜在基因及其下游信号效应物，也将精确定位靶点，以开发治疗方法，旨在诱导很少和/或有急性中风或其他脑缺血状况和疾病风险的成人形成新的侧支。当多态性损害侧枝形成时，确定这一主要等位基因也可以为这些相同的患者群体提供遗传标记。

DESCRIPTION (provided by applicant): Occlusive vascular disease of the brain, heart and lower extremities is the primary cause of morbidity and mortality in the US. Native (pre-existing) collaterals interconnect adjacent arterial trees and function as critical bypass vessels if obstruction occurs in one of the trees. Attention over the past 25 years has been on mechanisms mediating outward remodeling of collaterals in ischemic disease. However, until our work over the last three years, nothing was known about what controls the number and diameter (extent) of these vessels. We have shown that collateral extent in murine brain and other tissues varies widely due to genetic polymorphisms, resulting in a greatly increased risk-severity for ischemic injury of brain and other tissues in individuals having low collateral extenta finding recently confirmed in humans. We have discovered the major genetic locus, Candq1, responsible for most (70%) of this variation. We now seek to identify the polymorphic gene underlying this locus and its downstream effector pathway that regulate collateral formation. Aim I will fine-map Candq1 in a congenic strain-set that we have partly completed generating in preliminary studies: We will introgress informative partitions of the Candq1 locus of the C57BL/6 "high-collateral" strain into the BALB/c strain that has few-to-no collaterals in brain and other tissues. These congenic lines will then be phenotyped for collateral extent in brain and skeletal muscle, followed by functional analysis in a model of ischemic stroke in the positive congenic line(s) (+CNG) that most tightly partitions the causative region of Candq1. Aim II-A will additionally narrow the candidates by determining gene expression in the +CNG using RNA-Seq across Candq1, and also across the whole genome to identify potential downstream effectors. Aim II-B will further refine the candidates and targets, plus determine if a variant in cis/trans regulatory element underlies Candq1, using whole-genome chromatin mapping for active DNA regulatory sites. Aim I and II will thus identify the highest priority candidates that Am III will test for causality using in vivo gene targeting, cellular analyses, and functional outcomein stroke. This project will identify the primary driver gene that controls formation of the native collateral circulation and is responsible for its wide variation. Defining the gene underlying Candq1, and its downstream signaling effectors, will also pinpoint targets to develop as therapies aimed at inducing new collaterals to form in adults with few and/or who are at risk for or have suffered acute stroke or other cerebral ischemic conditions and diseases. Identifying this major allele that when polymorphic impairs collateral formation may also provide a genetic marker for use in these same patient groups.