Roles of Nogo-B receptor in maintaining the structural integrity of blood vessels

项目摘要 在美国，每200人中就有1人患有脑部海绵状畸形(CCM)，这种疾病会导致 癫痫和脑出血。先前的研究表明，表达减少或功能丧失 CCM家族基因突变(CCM1、CCM2、CCM3)可引起CCM。尽管家族性遗传病的遗传起源 CCMS(20%的病例)具有良好的特征，而散发性CCM(80%的病例)的病因学没有 CCM基因是否存在胚系突变仍有待确定。我们研究的长期目标是 确定Nogo-B受体(NGBR)缺乏是否以及在多大程度上在糖尿病的病因中起作用 零星的CCM。对这一想法的支持来自我们之前由R01资助的研究，在这些研究中，我们不仅阐明了 NGBR与法尼化RAS结合促进血管生成的分子机制 NGBR介导的RAS途径调节转录因子的表达和激活 内皮细胞。有趣的是，这些转录因子在其启动子区域具有结合元件 CCM1和CCM2基因。因此，ngbr缺陷者CCM1和CCM2基因的转录水平降低。 人脑内皮细胞体外培养及内皮细胞特异性基因敲除(ECKO)小鼠卵黄囊内皮细胞 在活体内。对NGBR-CCM联系的额外支持来自组织学研究，表明NGBR 散发性CCM病变内皮细胞免疫染色强度明显降低 组织切片。NGBR ECKO小鼠具有异常的脑血管组装模式，导致 内皮细胞极化有缺陷，血管扩张。这种表型惊人地类似于 CCM病变。此外，NGBR Ecko小鼠血管周围的周细胞层比 产仔对照组小鼠。这意味着内皮NGBR缺乏实际上会损害周细胞募集， 它被认为是维持血管结构完整性所必需的。基于这些发现，我们 假设NGBR-RAS信号通路调节CCM1/2的表达，并扰乱这一过程 信号通路导致脑血管畸形。我们将在三个目标上检验这一假设。目标1： 确定NGBR-CCM1/2轴在调节EC极化和脑血管组装中的作用； 目的2：确定NGBR-CCM1/2轴在维持周细胞募集和结构上的作用 脑血管的完整性；目标3：确定NGBR调节CCM1/2的机制 转录及NGBR-RAS通路缺陷在散发性CCM发病机制中的作用这个 这里提出的研究将在NGBR、CCM1/2和脑血管畸形之间建立明确的联系。 我们的研究结果将为ngbr和ngbr调节因子如何引导CCM1/2提供新的见解。 在脑血管内皮细胞中表达，并维持脑血管结构的完整性。

PROJECT ABSTRACT Cerebral cavernous malformations (CCMs), which can occur in 1 out of every 200 people in the US, cause seizure and cerebral hemorrhage. Previous studies have shown that decreased expression or a loss-of-function mutation of CCM family genes (CCM1, CCM2, CCM3) causes CCMs. Although the genetic origins of familial CCMs (20% of cases) have been well characterized, the etiology of sporadic CCMs (80% of cases) that do not carry a germline mutation in CCM genes remains to be established. The long-term goal of our research is to determine if and the extent to which Nogo-B receptor (NgBR) deficiency plays a causal role in the etiology of sporadic CCMs. Support for this idea comes from our previous R01-funded studies where we not only elucidated the molecular mechanisms by which NgBR binds farnesylated Ras to promote angiogenesis but also identified that the NgBR-mediated Ras pathway regulates the expression and activation of transcription factors in endothelial cells. Interestingly, these transcription factors have their binding elements in the promoter regions of CCM1 and CCM2 genes. Consequently, transcript levels of CCM1 and CCM2 genes decrease in NgBR-deficient human brain endothelial cells in vitro and in the yolk sac of NgBR endothelial cell-specific knockout (ecKO) mice in vivo. Additional support of the NgBR-CCM connection comes from histology studies showing that NgBR immunostaining intensity is significantly decreased in endothelial cells (ECs) of human sporadic CCM lesion tissue sections. NgBR ecKO mice have aberrant patterns of cerebral blood vessel assembly, which results in defective EC polarization and the formation of dilated blood vessels. This phenotype is surprisingly similar to the CCM lesions. In addition, the pericyte layer around blood vessels in NgBR ecKO mice is much thinner than littermate control mice. This means that endothelial NgBR deficiency can actually impair pericyte recruitment, which is known as essential for maintaining the structural integrity of blood vessels. Based on these findings, we hypothesize that the NgBR-Ras signaling pathway regulates CCM1/2 expression, and that disrupting this signaling pathway results in cerebrovascular malformation. We will test this hypothesis in three aims. Aim 1: Determine the role of the NgBR-CCM1/2 axis in regulating EC polarization and cerebral blood vessel assembly; Aim 2: Determine the role of the NgBR-CCM1/2 axis in maintaining pericyte recruitment and the structural integrity of cerebral blood vessels; Aim 3: Determine the mechanisms by which NgBR regulates CCM1/2 transcription and the contributions of NgBR-Ras pathway deficiency to the pathogenesis of sporadic CCM. The studies proposed here will establish clear links between NgBR, CCM1/2, and cerebrovascular malformation. Findings from our studies will provide new insight into how NgBR and NgBR-regulated factors guide CCM1/2 expression in brain ECs, and maintain the structural integrity of cerebral blood vessels.