Development and characterisation of a human ex vivo model of aneurysm

背景：世界范围内，主动脉瘤破裂是导致过早死亡的原因之一。为了深入了解动脉瘤的发病机制，已经开发了几种小鼠模型，然而至少15%的动物患有主动脉夹层和猝死。我们的目标是开发和表征一种可重复的离体人动脉瘤模型，与小鼠模型一起使用。方法和结果：从人脐带中分离动脉，置于生物反应器中，暴露于层流（6.5 dynes/cm2） 72小时，含或不含5um血管紧张素- ii (Ang-II)，或含或不含250mm氯化钙（CaCl2）。此外，还研究了金属蛋白酶3组织抑制剂（TIMP-3）、转化生长因子β中和抗体（TGF-B NAb）和MMP-12抑制剂（RXP470）的保护作用。CaCl2预处理降低了脐动脉内侧厚度（44%,p<0.05, n=6）、内侧弹性蛋白含量（33%,p<0.01, n=6）和内侧细胞密度（50%,p<0.05, n=6），但血管面积没有变化。与未治疗的动脉相比，Ang-II刺激增加了总血管面积（90%,p<0.05, n=6）和内侧细胞密度（34%,p<0.05, n=5）。Ang-II降低了内侧厚度（49%,p<0.05, n=6），内侧弹性蛋白含量（61%,p<0.05, n=6），提示动脉瘤形成。与单独使用Ang-II相比，重组TIMP-3、TGF-B NAb或RXP470可显著降低血管扩张（42%、33%、40%,p<0.05, n=5），增加血管内侧厚度（93%、124%、118%,p<0.05, n=5）和弹性蛋白含量（168%、214%、185%,p<0.05, n=5）。结论：我们的新离体模型采用Ang-II输注人脐带动脉，诱导了与动脉瘤形成相关的形态和成分变化，这些变化被外源性TIMP-3、TGF-B Nab或RXP470逆转。总的来说，这些发现支持将该模型用于动脉瘤研究，取代具有伦理挑战性的动物实验。

Background: Aortic aneurysm rupture is a cause of premature mortality worldwide. To gain insight into the pathogenesis of aneurysms, several mouse models have been developed, however at least 15% of animals suffer aortic dissection and sudden death. Our aim is to develop and characterise a reproducible ex-vivo human model of aneurysm, to be used alongside/instead of mouse models.Methods and Results: Arteries were isolated from human umbilical cords, placed within a bioreactor and exposed to laminar flow (6.5 dynes/cm2) for 72 hours, with and without 5 uM angiotensin-II (Ang-II), or with and without 250 mM calcium chloride (CaCl2). Furthermore, the protective effects of tissue inhibitor of metalloproteinases 3 (TIMP-3), transforming growth factor beta neutralising antibody (TGF-B NAb) and an MMP-12 inhibitor (RXP470) were also investigated.CaCl2 pre-treatment of umbilical arteries reduced medial thickness (44%; p<0.05; n=6), medial elastin content (33%; p<0.01; n=6), and medial cell density (50%; p<0.05; n=6), despite no change in vessel area. Ang-II stimulation increased total vessel area (90%; p<0.05; n=6) and medial cell density (34%; p<0.05; n=5), compared to untreated arteries. Moreover Ang-II decreased medial thickness (49%; p<0.05; n=6), and medial elastin content (61%; p<0.05; n=6) suggesting aneurysm formation. Recombinant TIMP-3, TGF-B NAb or RXP470 significantly reduced vessel dilatation (42%, 33%, 40%; p<0.05; n=5), increased medial thickness (93%, 124%, 118%; p<0.05; n=5) and elastin content (168%, 214%, 185%; p<0.05; n=5) compared to Ang-II alone.Conclusions: Our novel ex-vivo model deploying Ang-II infusion of human umbilical cord artery, induced morphological and compositional changes associated with aneurysm formation, which are reversed by exogenous TIMP-3, TGF-B Nab or RXP470. Collectively, these findings support the use of this model for aneurysm studies, supplanting ethically challenging animal experiments.