Aims Vein grafts are used for many indications, including bypass graft surgery and arteriovenous fistula (AVF) formation. However, patency following vein grafting or AVF formation is suboptimal for various reasons, including thrombosis, neointimal hyperplasia, and adverse remodelling. Recently, endothelial-to-mesenchymal transition (EndMT) was found to contribute to neointimal hyperplasia in mouse vein grafts. We aimed to evaluate the clinical potential of inhibiting EndMT and developed the first dedicated preclinical model to study the efficacy of local EndMT inhibition immediately prior to AVF creation. Methods and results We first undertook pilot studies to optimize the creation of a femoral AVF in pigs and verify that EndMT contributes to neointimal formation. We then developed a method to achieve local in vivo SMAD3 knockdown by dwelling a lentiviral construct containing SMAD3 shRNA in the femoral vein prior to AVF creation. Next, in Phase 1, six pigs were randomized to SMAD3 knockdown or control lentivirus to evaluate the effectiveness of SMAD3 knockdown and EndMT inhibition 8 days after AVF creation. In Phase 2, 16 pigs were randomized to SMAD3 knockdown or control lentivirus and were evaluated to assess longer-term effects on AVF diameter, patency, and related measures at 30 days after AVF creation. In Phase 1, compared with controls, SMAD3 knockdown achieved a 75% reduction in the proportion of CD31+ endothelial cells co-expressing SMAD3 (P < 0.001) and also a significant reduction in the extent of EndMT (P < 0.05). In Phase 2, compared with controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56 ± 1.66 vs. 4.26 ± 1.71 mm, P < 0.01) and a reduced degree of stenosis (P < 0.01). Consistent with this, neointimal thickness was reduced in the SMAD3 knockdown group (0.88 ± 0.51 vs. 0.45 ± 0.19 mm, P < 0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (P < 0.05). Conclusion EndMT inhibition in a preclinical AVF model by local SMAD3 knockdown using gene therapy led to reduced neointimal hyperplasia, increased endothelialization, and a reduction in the degree of AVF stenosis. This provides important proof of concept to pursue this approach as a clinical strategy to improve the patency of AVFs and other vein grafts.

中文翻译：

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抑制大型动物临床前动静脉瘘模型中的内皮-间充质转化可改善重塑和减少狭窄


目的 静脉移植物用于多种适应证，包括旁路移植手术和动静脉瘘 （AVF） 形成。然而，由于各种原因，静脉移植或 AVF 形成后的通畅性并不理想，包括血栓形成、新生内膜增生和不良重塑。最近，发现内皮-间充质转化 （EndMT） 导致小鼠静脉移植物中的新内膜增生。我们旨在评估抑制 EndMT 的临床潜力，并开发了第一个专用的临床前模型来研究在 AVF 产生前即刻局部 EndMT 抑制的疗效。方法和结果我们首先进行了试点研究，以优化猪股骨 AVF 的创建，并验证 EndMT 有助于新内膜形成。然后，我们开发了一种通过在 AVF 产生之前将含有 SMAD3 shRNA 的慢病毒构建体驻留在股静脉中来实现局部体内 SMAD3 敲低的方法。接下来，在第 1 阶段，将 6 头猪随机分配到 SMAD3 敲低组或对照慢病毒组，以评估 AVF 产生后 8 天敲低 SMAD3 和 EndMT 抑制的有效性。在第 2 阶段，16 头猪被随机分配到 SMAD3 敲低或对照慢病毒组，并进行评估以评估 AVF 产生后 30 天对 AVF 直径、通畅性和相关指标的长期影响。在第 1 阶段，与对照组相比，SMAD3 敲低使共表达 SMAD3 的 CD31 + 内皮细胞比例降低 75% （P < 0.001），EndMT 的程度也显著降低 （P < 0.05）。在第 2 阶段，与对照组相比，SMAD3 敲低与 AVF 静脉肢体最小直径的增加 （1.56 ± 1.66 vs. 4.26 ± 1.71 mm，P < 0.01） 和狭窄程度降低 （P < 0.01） 相关。 与此一致，SMAD3 敲低组的新内膜厚度减少 （0.88 ± 0.51 vs. 0.45 ± 0.19 mm，P < 0.05）。此外，SMAD3 敲低组的内皮完整性 （表达内皮标志物的管腔细胞的比例） 得到改善 （P < 0.05）。结论 在临床前 AVF 模型中，使用基因治疗通过局部 SMAD3 敲低抑制 EndMT 导致新内膜增生减少，内皮化增加，AVF 狭窄程度降低。这为将这种方法作为提高 AVF 和其他静脉移植物通畅性的临床策略提供了重要的概念验证。