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.
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。