Modulation of VSMC phenotype through the Insulin Receptor Substrate-1/Kruppel-like factor-4 signal transduction pathway: a Novel Target for AVF Dysfunction

摘要 动静脉瘘(AVF)是永久性透析血管通路的首选方式，因为它具有更好的 与透析移植物和导管相比，可长期存活并降低感染风险。不幸的是，AVF 成熟失败率(定义为透析的直径和血流量不足)在6岁时超过50% 几个月，这导致了多次额外的干预，也延长了隧道透析期 导管依赖及其所有伴随的并发症。因此，AVF的成熟失败会导致非常 严重的发病率、死亡率和经济成本。 我们和其他人以前已经证明，动静脉瘘的成熟失败是由于吻合口周围发生的。 静脉段狭窄的特征是血管平滑肌细胞(VSMC)去分化为 合成表型，继而导致侵袭性静脉新生内膜增生。我们还有 在动静脉瘘成熟生物学(Roy-Chaudhury)和信号方面都有独特的专长 VSMC表型转换(Xi)的信号转导机制。我们现在计划将这一组合应用到 研究导致动静脉瘘成熟失败的信号转导途径的经验和专业知识。 因此，这一提议的首要中心假设是胰岛素的环境调节 受体底物1(IRS-1)信号转导通路在VSMC表型转换中起关键作用 继而导致新生内膜增生和动静脉动静脉瘘成熟失败。我们计划解决这一中心假设 通过三个具体目标。 具体目标1将评估高血糖、尿毒症和遗传疾病的不同组合的影响。 IRS-1和Kruppel样因子4(KLF-4)对信号转导/VSMC表型转换的调控 使用C57BL/6 WT对照小鼠移植的静脉(颈静脉)和动脉(颈)VSMC的通路。特定的 目标2将评估同样的上游操作对信号转导通路的影响，VSMC AVF验证小鼠模型的表型转换与临床、血流动力学和组织学终点 分别于术后第2、7、14天狭窄。最后，《特定目标3》将评估抑制剂Nutlin-3的影响。 MDM2介导的抑制VSMC表型转换的p-53泛素化对体内外的影响 具体目标1和2中分别描述的终点。 如果成功，这种新颖、创新、机制驱动和先发制人的方法将解决 AVF成熟失败可显著降低与此相关的临床发病率和经济成本。 未得到满足的临床需求。

ABSTRACT Arteriovenous fistulae (AVF) are the preferred mode of permanent dialysis vascular access because of better long-term survival and reduced infection risks as compared to dialysis grafts and catheters. Unfortunately, AVFs have a maturation failure rate (defined as inadequate diameter and blood flow for dialysis) of over 50% at 6 months, which results in multiple additional interventions, and also a prolonged period of tunneled dialysis catheter dependency with all of its attendant complications. Thus, AVF maturation failure results in a very significant morbidity, mortality and economic cost. We and others have previously demonstrated that AVF maturation failure occurs due to a peri-anastomotic venous segment stenosis characterized by the de-differentiation of vascular smooth muscle cells (VSMC) into a synthetic phenotype, which then results in an aggressive venous neointimal hyperplasia. We have also developed a unique expertise both in the biology of AVF maturation (Roy-Chaudhury) and in the signal transduction mechanisms involved in VSMC phenotypic switching (Xi). We now plan to apply this combined experience and expertise to study the signal transduction pathways responsible for AVF maturation failure. The overarching central hypothesis of this proposal, therefore, is that environmental modulation of the insulin receptor substrate 1 (IRS-1) signal transduction pathway plays a key role in VSMC phenotype switching which then results in neointimal hyperplasia and AVF maturation failure. We plan to address this central hypothesis through three specific aims. Specific Aim 1 will assess the impact of different combinations of hyperglycemia, uremia and genetic manipulation of IRS-1 and Kruppel like factor 4 (KLF-4) on signal transduction/VSMC phenotypic switch pathways using explanted venous (jugular) and arterial (carotid) VSMC from C57Bl/6 WT control mice. Specific Aim 2 will assess the impact of this same upstream manipulation on signal transduction pathways, VSMC phenotypic switch and clinical, hemodynamic and histological endpoints, in a validated mouse model of AVF stenosis at 2, 7 and 14 days, post-surgery. Finally, Specific Aim 3 will assess the impact of nutlin-3, an inhibitor of MDM2 mediated ubiquitination of p-53 (which inhibits VSMC phenotypic switching) on the in-vitro and in-vivo end points described in Specific Aims 1 and 2 respectively. If successful, this novel, innovative, mechanism driven and pre-emptive approach to the intractable problem of AVF maturation failure, could significantly reduce the clinical morbidity and economic cost associated with this unmet clinical need.