TLR4 and the microbiome in CCM disease

脑海绵状血管畸形(CCM)是一种累及多种脑血管疾病。 在美国有200,000人，是年轻人中风和癫痫的常见原因 个人。目前还没有治疗CCM病的药物，治疗方法是 仅限于手术切除和抗癫痫药物。我们最近做了 证明了CCM是由于MEKK3信号的获得而产生的，并增加了 KLF2和KLF4转录因子在CCM缺陷脑内皮细胞中的表达 细胞(ECs)。这些研究揭示了影响的下游信号机制。 CCM病，但他们没有确定强有力的治疗靶点或解决这个问题 是什么激活了脑内皮细胞中的MEKK3。我们的初步研究表明，TLR4 是内皮细胞MEKK3-KLF信号的关键上游激活物，它是 CCM的形成，肠道微生物群中的革兰氏阴性菌(GNB)是 小鼠新生CCM模型中这一途径的关键体内配体。遗传分析 我们的合作研究员海伦·金博士独立地 鉴定TLR4及其辅受体CD14为CCM病变数量相关基因， 这表明我们在老鼠身上的发现延伸到了人类CCM疾病。这项提议将 进一步检测血管内皮细胞TLR4信号和肠道微生物群在CCM中的作用 利用小鼠和人类研究疾病的发病机制。目标1将(I)测试 TLR4在成年小鼠CCM形成过程中，(Ii)确定阻断ANT-TLR4和 抗CD14抗体阻止CCM在小鼠模型中的形成(III)使用小鼠 确定TLR4激活MEKK3途径的遗传学方法 CCM的形成，以及(Iv)应用遗传方法完全询问TLR4-MEKK3- 由KRIT1常见突变引起的人类CCM患者的KLF通路。 目的2将通过(I)测试GNB和肠道微生物群在CCM发病机制中的作用 确定CCM损伤是否出现在无菌动物中，(Ii)定义微生物组 使小鼠对CCM疾病具有抵抗力或易感性，以及(Iii)测试 肠道上皮屏障的破坏加速了小鼠CCM的形成。这些 研究将极大地扩展我们对分子和生理的理解 CCM发病机制的基础，并为将这些发现转化为 基于阻断TLR4信号的CCM病的新疗法(FDA为此 批准的制剂已经存在)或改变肠道微生物群。

Cerebral cavernous malformation (CCM) is a cerebrovascular disease that affects over 200,000 individuals in the US and is a common cause of stroke and seizure in young individuals. There are presently no medical therapies for CCM disease, and treatment is limited to surgical resection and anti-seizure medications. We have recently demonstrated that CCMs arise due to a gain of MEKK3 signaling and increased expression of the KLF2 and KLF4 transcription factors in CCM-deficient brain endothelial cells (ECs). These studies reveal the downstream signaling mechanism impacted in CCM disease, but they do not identify strong therapeutic targets or address the question of what activates MEKK3 in brain ECs. Our preliminary studies demonstrate that TLR4 is a critical upstream activator of endothelial MEKK3-KLF signaling that is required for CCM formation, and that gram negative bacteria (GNB) in the gut microbiome are the key in vivo ligand for this pathway in the mouse neonatal CCM model. Genetic analysis of human CCM patients by our co-investigator Dr. Helen Kim has independently identified TLR4 and its co-receptor CD14 as genes associated with CCM lesion number, suggesting that our mouse findings extend to human CCM disease. This proposal will further test the role of endothelial TLR4 signaling and the gut microbiome in CCM disease pathogenesis using mouse and human studies. Aim 1 will (i) test the role of TLR4 during CCM formation in adult mice, (ii) determine whether blocking ant-TLR4 and anti-CD14 antibodies prevent CCM formation in the mouse model, (iii) use mouse genetic approaches to determine the pathway by which TLR4 activates MEKK3 during CCM formation, and (iv) apply genetic approaches to fully interrogate the TLR4-MEKK3- KLF pathway in human patients with CCM disease due to a common mutation in KRIT1. Aim 2 will test the role of GNB and the gut microbiome in CCM pathogenesis by (i) determining whether CCM lesions arise in germ-free animals, (ii) defining microbiomes that confer resistance or susceptibility to CCM disease in mice, and (iii) testing whether breakdown of the gut epithelial barrier accelerates CCM formation in mice. These studies will significantly expand our understanding of the molecular and physiologic basis of CCM pathogenesis, and lay the foundation for translating these discoveries to new therapies for CCM disease based on blocking TLR4 signaling (for which FDA approved agents already exist) or altering the gut microbiome.