Microglia/macrophages as target to prevent intracerebral hemorrhage in KRAS mutation-induced brain arteriovenous malformations

项目概要/摘要 脑动静脉畸形（bAVM）患者发生脑出血（ICH）的风险为50%， 具有严重发病率和死亡率的高风险。虽然手术尝试是一种传统的治疗方法， bAVM，危重bAVM的手术与严重并发症相关，如术中 出血或死亡。我们的长期目标是确定bAVM患者ICH的预防策略。的 导致bAVM破裂的诱发病理生理学尚不清楚。研究表明bAVM的存在 与病灶内和周围脑实质的局部炎症高度相关。 免疫组织化学研究已经确定了密集的脑小胶质细胞和血液单核细胞- 衍生的巨噬细胞（我们称之为巨噬细胞）围绕未破裂的人bAVM。同时，临床 研究发现，62%的散发性bAVM患者血管内皮细胞中存在KRAS突变， （EC）。在这里，我们发现携带KRAS突变的EC（KRAS-EC）诱导炎症反应， 这反过来又破坏了EC连接。这表明M β介导的炎症驱动bAVM 可能导致bAVM破裂/ICH的不稳定。探讨KRAS-EC如何改变M β细胞表型， 为了使bAVM不稳定，我们建立了一种新的bAVM/ICH小鼠模型，该模型使用EC特异性AAV/BR 1， 将突变KRAS（AAV-KRASG 12 V）递送到脑EC（bEC）中。KRASG 12 V/bEC小鼠重现人类 bAVM病理学，包括血管缠结、壁细胞覆盖不完全、自发性ICH和神经系统 缺陷（Park 2021 Ann Neurol）。在KRASG 12 V/bEC小鼠中，我们发现Iba-1+ M受体的数量增加， 未破裂的脑动静脉畸形周围炎性细胞因子、蛋白水解酶的mRNA或蛋白水平， 血管生成介质和粘附分子在KRASG 12 V/bEC小鼠和KRAS-EC本身中增加。 此外，用氯膦酸盐脂质体（以消耗M β）或米诺环素处理的KRASG 12 V/bEC小鼠在早期降低了 微出血我们的初步研究和现有的文献强烈支持这一概念，即激活的M- 介导的炎症驱动bAVM不稳定，可能导致bAVM破裂/ICH。在本提案中，我们将 检验我们假设，KRAS-EC在bAVM区域内募集并激活M β，这些激活的M β 反过来加重（或加重）血管损伤，导致bAVM破裂/ICH，而抑制M β可以避免/延迟 这个过程在目标1中，我们将确定在bAVM中引起M β激活和BBB破坏的机制。在 目的2：探讨脑动静脉畸形（bAVM）破裂和脑出血中M β的临床意义.我们希望成功的 这项临床前研究的完成将揭示M β在bAVM破裂和ICH中的致病作用，并将提供 证据来测试MMP 3调节在预防bAVM相关ICH中的潜力。我们的建议 广泛意义的可能性，因为大多数脑血管疾病通常涉及M β。

Project Summary/Abstract Brain arteriovenous malformation (bAVM) patients have a 50% risk of intracerebral hemorrhage (ICH) which carries a high risk of severe morbidity and mortality. Although a surgical attempt is a conventional treatment for the bAVM, the surgery for critical bAVM is associated with significant complications such as intraoperative hemorrhage or death. Our long-term goal is to identify a preventative strategy for ICH in bAVM patients. The inciting pathophysiology leading to bAVM rupture is unknown. Studies have shown that the presence of a bAVM is highly correlated with local inflammation of intranidal and surrounding brain parenchyma. Immunohistochemistry studies have identified dense concentrations of brain microglia and blood monocyte- derived macrophages (we refer to them as M) surrounding the unruptured human bAVM. Meanwhile, clinical studies found that 62% of human sporadic bAVM patients harbored KRAS mutations in vascular endothelial cells (ECs). Here we found that ECs carrying KRAS mutation (KRAS-EC) induced inflammatory responses toward cultured M that in turn disrupts EC junctions. This suggests that M-mediated inflammation drives bAVM destabilization that may lead to bAVM rupture/ICH. To explore how KRAS-EC alter M phenotype and destabilize bAVM, we established a novel bAVM/ICH mouse model that uses the ECs-specific AAV/BR1 to deliver mutant KRAS (AAV-KRASG12V) into brain ECs (bEC). The KRASG12V/bEC mouse recapitulates human bAVM pathology, including tangled vessels, incomplete mural cell coverage, spontaneous ICH, and neurological deficits (Park 2021 Ann Neurol). In KRASG12V/bEC mouse, we showed that the number of Iba-1+ Mare increased around unruptured bAVM. The mRNA or protein levels for inflammatory cytokines, Proteolytic enzymes, angiogenic mediator, and adhesion molecule are increased in KRASG12V/bEC mice and in KRAS-EC themselves. Furthermore, KRASG12V/bEC mice treated with clodronate liposome (to deplete M) or minocycline reduces early microbleeds. Our preliminary study and existing literature strongly supporting the notion that the activated M- mediated inflammation drives bAVM destabilization that may lead to bAVM rupture/ICH. In this proposal, we will test our hypothesis that KRAS-EC recruit and activate M within bAVM territory and that these activated M in turn exacerbate the (peri)vascular damage causing bAVM rupture/ICH and that inhibition of M can avert/delay this process. In Aim 1, we will determine the mechanism causing Mactivation and BBB disruption in bAVM. In Aim 2, we will evaluate the clinical relevance of M in bAVM rupture and ICH. We expect that the successful completion of this preclinical study will uncover a causative role of Min bAVM rupture and ICH and will provide evidence to test the potential for M modulation in the prevention of bAVM-associated ICH. Our proposal has the potential for broad implications as M are commonly involved in most cerebrovascular diseases.