Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4A^G12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels only, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

中文翻译：

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人体动静脉畸形 (AVM) 芯片模型的开发和表征

脑动静脉畸形 (AVM) 是一种疾病，其中异常、扩大的血管将动脉直接连接到静脉，而没有介入的毛细血管床。AVM 是儿童和年轻人出血性卒中的主要原因之一。大多数人类散发性脑 AVM 与KRAS基因中的基因激活突变有关。我们的目标是开发一种体外模型，该模型将允许在 AVM 疾病进展期间实时同时捕获形态和功能表型数据。通过生成具有在大多数人类患者中发现的临床相关突变的人类内皮细胞（激活小 GTPase KRAS中的突变）) 并将它们播种在能够形成血管和灌注的动态微流控细胞培养系统中，我们证明由KRAS4A ^G12V突变内皮细胞 (EC) 形成的血管比野生型 EC 形成的血管床更宽、更渗漏，概括了关键人类 AVM 发病机制的结构和功能特征。免疫荧光染色显示仅突变 KRAS 血管中的粘附连接破裂，导致血管通透性增加，这是出血性中风的标志。最后，MEK 激酶活性的药理学阻断，而不是 PI3K 抑制，改善了内皮屏障功能（降低通透性）而不影响血管直径。总的来说，我们的研究描述了人类 KRAS 依赖性 AVM 样血管的产生在适合表型观察和药物递送的自组装微血管平台中进行体外试验。