Novel Molecular Target to Prevent Maturation Failure of Arteriovenous Fistula

摘要 自体动静脉内瘘（AVF）是血液透析首选的血管通路。然而， 由于流出静脉中的血流不足而导致的成熟失败使得该瘘不能用于血液透析。 新生内膜增生和外向型重构失败是AVF成熟失败的主要原因， 是由于血管平滑肌细胞的炎症、增殖、迁移和表型变化 （VSMCs）和由于增加的基质金属蛋白酶（MMP）引起的细胞外重塑。我们发现 髓样细胞上表达的触发受体-1（TREM-1）、TLR 4和相关蛋白的表达增加 未成熟的动静脉瘘基于我们的新发现，中心假设是血流动力学损伤 在AVF产生过程中诱导炎症以上调TREM-1和TLR 4，从而增强新生内膜 因此，抑制AVF的增殖和血管重塑，并且拮抗TREM-1和TLR 4将增强AVF的成熟。 这一假设将被测试与以下目的：目的1：我们的推论假设预测， 施用TREM-1和TLR 4拮抗剂将防止猪AVF的成熟失败。我们将 检查有效抑制性TREM-1肽在猪AVF模型中的作用。由于TREM-1可以 与TLR 4协同介导AVF成熟失败的病理，有效的TLR 4拮抗剂的作用将 也可用于防止AVF成熟失败。结局参数将包括新生内膜 AVF流入段和流出段增生，AVF血管造影，彩色多普勒超声， 光学相干断层扫描和组织学，免疫染色来分析炎症，各种 介质和巨噬细胞和中性粒细胞浸润，VSMC凋亡和血管重塑。目标二： 我们的推论假设预测TREM-1和TLR 4拮抗作用抑制炎症，从而 通过减少内膜增生和血管生成来防止AVF的成熟失败 重塑主要是由于炎性细胞、组织蛋白酶L、IL-8和MMP-12。这些研究报告将 在来自目的1的猪的股动脉和股静脉的血液和分离的VSMC中进行。 机制研究将检查在IL-8存在下TREM-1和TLR 4抑制对中性粒细胞的作用， 单核细胞分化的巨噬细胞和VSMC，以及组织蛋白酶L介导的弹性蛋白和胶原降解 以及弹性蛋白衍生肽对单核细胞分化为巨噬细胞和VSMC的影响 扩散和迁移。其他机制研究将包括TLR 4和TREM-1之间的联系， 促进基质重塑，从交叉中的中性粒细胞和巨噬细胞释放炎性细胞因子， 诱导VSMCs表型转换和巨噬细胞极化。 这项研究的结果将证实TREM-1是否是治疗干预的新靶点， 开发更好的分子来拮抗TREM-1和设计I期临床试验的知识。

ABSTRACT Autologous arteriovenous fistula (AVF) is the preferred vascular access in hemodialysis. However, high rate of maturation failure due to inadequate blood flow in the outflow vein renders the fistula not useful for hemodialysis. Neointimal hyperplasia and failure of outward remodeling are the major causes of AVF maturation failure which is due to inflammation, proliferation, migration, and phenotypic changes of vascular smooth muscle cells (VSMCs), and extracellular remodeling due to increased matrix metalloproteinases (MMPs). We discovered increased expression of triggering receptor expressed on myeloid cells-1 (TREM-1), TLR4 and related proteins in the immature AV fistula. Based on our novel findings, the central hypothesis is that hemodynamic injury during AVF creation induces inflammation to upregulate TREM-1 and TLR4 to enhance neointimal hyperplasia and vascular remodeling, and antagonizing TREM-1 and TLR4 will enhance AVF maturation. This hypothesis will be tested with the following Aims: Aim 1: Our corollary hypothesis predicts that the administration of TREM-1 and TLR4 antagonists will prevent maturation failure of AVF in swine. We will examine the effect of a potent inhibitory TREM-1 peptide in the AVF model in pigs. Since TREM-1 could synergize with TLR4 to mediate the pathology of AVF maturation failure, effect of a potent TLR4 antagonist will also be examined to prevent maturation failure of AVF. The outcome parameters will include neointimal hyperplasia in the inflow and outflow segments in the AVF, angiography of the AVF, color Doppler ultrasound, optical coherence tomography, and histology, immunostaining to analyze inflammation, expression of various mediators and infiltration of macrophages and neutrophils, VSMC apoptosis, and vascular remodeling. Aim 2: Our corollary hypothesis predicts that the TREM-1 and TLR4 antagonism inhibits inflammation and thus prevents maturation failure of AVF by reducing the development of intimal hyperplasia and vascular remodeling primarily due to inflammatory cells, cathepsin L, IL-8 and MMP-12. These studies will be performed in the blood and isolated VSMCs of femoral artery and femoral vein of the pigs from Aim 1. Mechanistic studies will examine the effect of TREM-1 and TLR4 inhibition in the presence of IL-8 on neutrophils, monocyte-differentiated macrophages and VSMCs, and cathepsin L-mediated elastin and collagen degradation in VSMCs, and the effect of elastin-derived peptides on monocyte differentiation into macrophages and VSMC proliferation and migration. Additional mechanistic studies will include the link between TLR4 and TREM-1 in promoting matrix remodeling, release of inflammatory cytokines from neutrophils and macrophages in the cross- talk inducing phenotype switch in VSMCs and macrophage polarization. The findings from this study will confirm if TREM-1 is a novel target for therapeutic intervention and extend the knowledge to develop better molecules to antagonize TREM-1 and design phase I clinical trials.