Development and characterisation of a human ex vivo model of aneurysm

背景：主动脉瘤破裂是世界范围内过早死亡的一个原因。为了深入了解动脉瘤的发病机制，已经建立了几种小鼠模型，然而至少有15%的动物遭受了主动脉夹层和猝死。方法和结果：从人脐带分离动脉，置于生物反应器中，暴露于层流(6.5 dynes/cm2)中72小时，加或不加血管紧张素-II(Ang-II)，或加或不加250 mM氯化钙(CaCl2)。此外，还研究了金属蛋白酶组织抑制因子3(TIMP-3)、转化生长因子-β中和抗体(转化生长因子-βNAB)和基质金属蛋白酶-12抑制剂(RXP470)的保护作用。在血管面积没有变化的情况下，CaCl2预处理使脐动脉中膜厚度(44%；p&lt；0.05；n=6)、中膜弹性蛋白含量(33%；p&lt；0.01；n=6)和中膜细胞密度(50%；p&lt；0.05；n=6)减少。与未经处理的动脉相比，Ang-II刺激增加了血管总面积(90%；p&lt；0.05；n=6)和中层细胞密度(34%；p&lt；0.05；n=5)。此外，Ang-II降低了中膜厚度(49%；p&lt；0.05；n=6)和中膜弹性蛋白含量(61%；p&lt；0.05；n=6)，提示有动脉瘤形成。重组TIMP-3、转化生长因子-βNAB或RXP470显著降低血管扩张(42%、33%、40%；P&lt；0.05；n=5)，增加中膜厚度(93%、124%、118%；P&lt；0.05；n=5)和弹性蛋白含量(168%、214%、185%；P&lt；0.05；P&lt；0.05；结论：人脐动脉注射Ang-II可诱导与动脉瘤形成相关的形态和成分改变，而外源性TIMP-3、转化生长因子-βNAB或RXP470可逆转这种改变。总而言之，这些发现支持将该模型用于动脉瘤研究，取代具有伦理学挑战性的动物实验。

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.