A Microphysiological HHT-on-a-Chip Platform Recapitulates Vascular Lesions of Human Patients

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations focally develop in multiple organs. These can be small (telangiectasias) or large (arteriovenous malformations, AVMs) and can compromise tissue perfusion. Critically, they are prone to rupture leading to frequent and uncontrolled bleeding. As a result, HHT patients experience several debilitating clinical manifestations that reduce patient quality-of-life. Most HHT patients are heterozygous for loss-of-function mutations affecting signaling through the Alk1 pathway; yet, the pathogenesis of vascular malformation (VM) in HHT patients remains unclear and there are still few treatment options and no cure. Due to the complex, three-dimensional, and multicellular nature of vascular lesions in HHT, they are traditionally studied in intact genetically-modified mouse and zebrafish models, and studies in these animals have yielded important insights into HHT disease progression. However, intact animal studies can be time-consuming and expensive, and may be difficult to translate to human patients. In this study, we take advantage of our recently developed Vascularized Micro-Organ platform to develop an in vitro fully-humanized HHT-on-a-chip microphysiological model (HHT-VMO) that recapitulates vascular lesions of HHT patients. Using a tunable IPTG-inducible ACVRL1 (Alk1)-knockdown approach, we control the timing and extent of endogenous Alk1 expression in primary human endothelial cells seeded into the HHT-VMO device. We report that Alk1-deficient EC reliably form networks with aberrant morphology that includes lesions reminiscent of both patient telangiectasias and AVMs. HHT-VMO lesions are dependent upon the timing of Alk1 knockdown and their formation is blocked by the Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitor pazopanib. Lastly, we find that AVM-like lesions that form in the HHT-VMO are comprised of mixed Alk1-intact and Alk1-deficient EC suggesting cell non-autonomous effects. Taken together, we report development of a novel HHT-on-a-chip model that appears to faithfully reproduce HHT patient lesions. In the future, we hope to use this model to better understand HHT disease biology and to perform drug screening studies to identify a cure for HHT. Overall design: IPTG inducible shALK1 HUVECs and normal human lung fibroblasts were loaded into the vascularized mirco-organ, a 3D microphysiological system. At Day 0, 3 mm IPTG were added to cell culture medium in the experimental group to knockdown ALK1/ACVRL1 expression. Vessels were allowed to grow until day 10, when devices were harvested and cells sent to sequencing.

遗传性出血性毛细血管扩张症（Hereditary Hemorrhagic Telangiectasia, HHT）是一种罕见的先天性疾病，其特征为多器官局灶性形成脆弱的血管畸形。此类畸形可分为小型（毛细血管扩张症，telangiectasias）与大型（动静脉畸形，arteriovenous malformations, AVMs）两类，可损害组织灌注。尤为关键的是，这类畸形极易破裂，引发频繁且难以控制的出血。因此，HHT患者会出现多种致残性临床表现，严重降低其生活质量。

多数HHT患者携带影响Alk1通路信号转导的功能丧失性杂合突变，但目前HHT患者血管畸形（vascular malformation, VM）的发病机制仍未阐明，且临床治疗手段有限，尚无根治方案。由于HHT患者的血管病变具有复杂的三维结构与多细胞特性，传统研究多借助基因改造的完整小鼠与斑马鱼模型，此类动物研究为阐明HHT的疾病进展提供了重要见解。然而，完整动物模型研究既耗时又昂贵，且难以直接转化应用于人类患者。

本研究依托我们近期开发的血管化微器官平台（Vascularized Micro-Organ platform），构建了一款体外完全人源化的HHT器官芯片微生理模型（HHT-VMO），该模型可重现HHT患者的血管病变特征。我们采用可调控的异丙基-β-D-硫代半乳糖苷（IPTG）诱导型ACVRL1（Alk1）基因敲低策略，对接种于HHT-VMO装置中的原代人内皮细胞的内源性Alk1表达时机与表达水平进行精准调控。研究结果显示，Alk1缺陷型内皮细胞可稳定形成形态异常的血管网络，其病变特征兼具患者毛细血管扩张症与动静脉畸形的典型表现。HHT-VMO模型的病变形成依赖于Alk1敲低的时机，且该病变的形成可被血管内皮生长因子受体（VEGFR）抑制剂帕唑帕尼（pazopanib）阻断。进一步研究发现，HHT-VMO模型中形成的动静脉畸形样病变由混合的Alk1完整型与Alk1缺陷型内皮细胞构成，提示存在细胞非自主效应。

综上，本研究构建了一款新型HHT器官芯片模型，该模型可忠实重现HHT患者的血管病变特征。未来我们拟借助该模型深入解析HHT的疾病生物学机制，并开展药物筛选研究，以期为HHT患者找到根治方法。

【总体实验设计】将IPTG诱导型shALK1人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）与正常人类肺成纤维细胞接种于血管化微器官——一款三维微生理系统中。于第0天向实验组的细胞培养基中添加3 mM IPTG，以敲低ALK1/ACVRL1基因的表达。待血管生长至第10天时，收集装置并对细胞进行测序分析。