Abstract：
OBJECTIVE: Vein grafts are used for many indications, including bypass graft surgery and arterio-venous fistula (AVF) formation. However, patency following vein grafting or AVF formation is suboptimal for various reasons, including thrombosis, neointimal hyperplasia and adverse remodeling. 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.
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, 6 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 to 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 to controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56±1.66 versus 4.26±1.71mm, 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 versus 0.45±0.19mm, p<0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (p<0.05).
CONCLUSIONS: 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.
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, 6 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 to 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 to controls, SMAD3 knockdown was associated with an increase in the minimum diameter of the venous limb of the AVF (1.56±1.66 versus 4.26±1.71mm, 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 versus 0.45±0.19mm, p<0.05). Furthermore, endothelial integrity (the proportion of luminal cells expressing endothelial markers) was improved in the SMAD3 knockdown group (p<0.05).
CONCLUSIONS: 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.
摘要：
目的：静脉移植用于许多适应症，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形成后的通畅性欠佳，包括血栓形成,新生内膜增生和不良重塑。最近,发现内皮-间质转化(EndMT)有助于小鼠静脉移植物中的新生内膜增生。我们旨在评估抑制EndMT的临床潜力，并开发了第一个专门的临床前模型，以研究AVF创建前局部EndMT抑制的功效。
结果：我们首先进行了初步研究，以优化猪股骨AVF的产生，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产生之前在股静脉中留置含有SMAD3shRNA的慢病毒构建体来实现局部体内SMAD3敲低的方法。接下来,在第1阶段中,6只猪被随机分配给SMAD3敲低或对照慢病毒,以在AVF产生8天后评估SMAD3敲低和EndMT抑制的有效性。在第2阶段，将16只猪随机分为SMAD3敲低或对照慢病毒，并进行评估以评估对AVF直径的长期影响。AVF创建后30天的通畅性和相关措施。在第一阶段，与对照相比，SMAD3敲低表达SMAD3的CD31+内皮细胞的比例降低了75%(p<0.001)，以及EndMT程度的显著降低(p<0.05)。在第二阶段，与对照组相比，SMAD3敲低与AVF静脉肢体最小直径的增加有关（1.56±1.66对4.26±1.71mm，p<0.01）和狭窄程度降低（p<0.01）。与此一致，SMAD3敲低组的新内膜厚度减少（0.88±0.51对0.45±0.19mm，p<0.05)。此外，SMAD3敲低组的内皮完整性(表达内皮标志物的腔内细胞比例)得到改善(p<0.05).
结论：在临床前AVF模型中，通过基因疗法局部SMAD3敲低的EndMT抑制导致新生内膜增生减少，内皮化增加和AVF狭窄程度减少。这为采用这种方法作为改善AVF和其他静脉移植物的通畅性的临床策略提供了重要的概念证明。
结果：我们首先进行了初步研究，以优化猪股骨AVF的产生，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产生之前在股静脉中留置含有SMAD3shRNA的慢病毒构建体来实现局部体内SMAD3敲低的方法。接下来,在第1阶段中,6只猪被随机分配给SMAD3敲低或对照慢病毒,以在AVF产生8天后评估SMAD3敲低和EndMT抑制的有效性。在第2阶段，将16只猪随机分为SMAD3敲低或对照慢病毒，并进行评估以评估对AVF直径的长期影响。AVF创建后30天的通畅性和相关措施。在第一阶段，与对照相比，SMAD3敲低表达SMAD3的CD31+内皮细胞的比例降低了75%(p<0.001)，以及EndMT程度的显著降低(p<0.05)。在第二阶段，与对照组相比，SMAD3敲低与AVF静脉肢体最小直径的增加有关（1.56±1.66对4.26±1.71mm，p<0.01）和狭窄程度降低（p<0.01）。与此一致，SMAD3敲低组的新内膜厚度减少（0.88±0.51对0.45±0.19mm，p<0.05)。此外，SMAD3敲低组的内皮完整性(表达内皮标志物的腔内细胞比例)得到改善(p<0.05).
结论：在临床前AVF模型中，通过基因疗法局部SMAD3敲低的EndMT抑制导致新生内膜增生减少，内皮化增加和AVF狭窄程度减少。这为采用这种方法作为改善AVF和其他静脉移植物的通畅性的临床策略提供了重要的概念证明。
