Regulatory gene-chemokine networks in the formation of hemodialysis AVF stenosis

项目摘要/摘要： 美国有超过43.5万名患者患有终末期肾病(ESRD)，预计这一人口将翻一番 在接下来的十年。目前这项提案和研究计划的长期目标是改善对 终末期肾病患者，绝大多数采用长期血液透析作为肾脏替代治疗方式 心理治疗。这些患者需要高功能的血管通路才能获得最佳的治疗充分性。 血液透析血管通路失败的常见原因是静脉狭窄继发于新生内膜增生。 (VNH)。 我们的初步数据表明IEX-1/MCP-1通路在VNH中起重要作用。我们证明了：1. 从动静脉瘘患者移除的静脉狭窄中IEX-1染色增加。2.动静脉瘘的静脉狭窄 从IEX-1基因敲除(KO)的慢性肾脏疾病(CKD)小鼠中移除后， 管腔血管面积减少，新生内膜面积减少，细胞凋亡率显著增加，细胞减少 增殖，α-SMA和Ly6C单核细胞染色显著减少，单核细胞趋化蛋白-1显著下降 基质金属蛋白酶-9免疫组化染色。3.单核细胞趋化蛋白-1、成纤维细胞生长因子-1、转化生长因子-β的基因表达均显著降低。 从IEX-1 KO小鼠去除静脉段暗示IEX-1减轻纤维化和炎症 功能。4.聚乳酸-羟基乙酸(PLGA)与骨化三醇组成的纳米粒的外膜给药 在第7天和28天后，IEX-1基因的表达显著降低(P&lt；0.05)。5.基因表达 AvF和CKD WT小鼠去除静脉狭窄后，MCP-1水平显著升高。这些数据 IEX-1在VNH发病机制中对MCP-1表达的调节作用[11，16-22]。6.最后， 在血液透析血管通路放置后，观察到流出静脉的剪应力增加。 [23-26]。总而言之，这些观察结果支持我们的中心假设：动静脉瘘患者的静脉狭窄。 发生的部分原因是内皮细胞上的剪应力增加和机械拉伸 平滑肌细胞导致IEX-1基因表达上调，随后MCP-1表达增加 导致单核/巨噬细胞募集的表达(卡通)。具体目标如下 以下是： 特异性目的1：评价血管内皮细胞(EC)和血管平滑肌(VSMC)IEX-1在体内的作用(S) 慢性肾脏病小鼠动静脉曲张的形成。我们将使用EC和VSMC条件缺失的新型小鼠 IEX-1用于评估单核细胞和巨噬细胞的渗透、增殖、迁移、凋亡、纤维化、管腔 AVF的直径、壁厚及MCP-1、FGF1、TGF-β、MMP9的表达。 假设：IEX-1在ECs和VSMCs中表达降低的小鼠的动静脉曲张将减少 AVF MCP-1表达减少和单核/巨噬细胞减少导致的狭窄形成 渗透。 特异性目的2：评价血管内皮细胞和血管平滑肌细胞对动静脉动静脉瘘单核细胞趋化蛋白-1表达的影响 IEX-1缺失。我们将在EC和VSMC缺失IEX-1的小鼠的AVF中恢复MCP-1的表达 腺病毒技术。我们将评估单核细胞和巨噬细胞的渗透，血管壁细胞的增殖， 迁移、细胞凋亡、纤维化、管腔直径、管壁厚度及MCP-1、FGF-1、TGF-b和 动静脉瘘组织中基质金属蛋白酶-9的表达。 假设：在CKD和EC和VSMCs IEX-1表达降低的小鼠动静脉曲张中，MCP-1的恢复 这种表达将与动静脉动静脉瘘狭窄相关。 特异性目标3：确定骨化三醇(S)在预防和/或减轻慢性萎缩性胃炎猪的VNH形成中的作用 AVFS和CKD。我们将使用患有动静脉瘘和慢性肾脏病的猪来确定骨化三醇对血管外膜的作用。 抑制VNH。 假设：慢性肾脏病猪动静脉动静脉外膜骨化三醇输出量减少 单核/巨噬细胞浸润和随后的VNH，下游IEX-1，MCP-1，FGF-1， 转化生长因子-β、基质金属蛋白酶-9与对照组比较。

PROJECT SUMMARY/ABSTRACT: More than 435,000 patients in the US have end stage renal disease (ESRD), a population expected to double in the next decade. The long-term goal of this current proposal and research program is to improve the care of patients with ESRD, the vast majority of who use long-term hemodialysis as their mode of renal replacement therapy. These patients require highly functioning vascular access for optimal therapeutic adequacy. Hemodialysis vascular access failure is frequently from venous stenosis secondary to neointimal hyperplasia (VNH). Our preliminary data demonstrate an important role for the Iex-1/Mcp-1 pathway in VNH. We show that: 1. IEX-1 staining is increased in venous stenoses removed from patients with AVF. 2. Venous stenoses in AVF removed from a Iex-1 knockout (KO) mice with chronic kidney disease (CKD) have a significant increase in lumen vessel area, decrease in neointima area with a significant increase in apoptosis, decrease in cellular proliferation, significant reduction in α-SMA and Ly6C monocyte staining, and a significant decline in MCP-1 and MMP-9 immunostaining. 3. Gene expression of Mcp-1, Fgf-1, and Tgf-β are all significantly decreased in venous segments removed from Iex-1 KO mice implying that Iex-1 decreases fibrotic and inflammatory function. 4. Adventitial delivery of nanoparticles composed of polylactic-co-glycolic acid (PLGA) with calcitriol significantly reduces Iex-1 gene expression at day 7 and VNH 28 days later (P<0.05). 5. Gene expression of Mcp-1 is significantly increased in venous stenosis removed from WT mice with AVF and CKD. These data implicate Iex-1 in the regulation of Mcp-1 expression in the pathogenesis of VNH [11, 16-22]. 6. Finally, increased shear stress at the outflow vein has been observed after hemodialysis vascular access placement [23-26]. Taken collectively, these observations support our central hypothesis: Venous stenosis in AVFs occurs in part due to an increase in shear stress upon endothelial cells and mechanical stretch upon smooth muscle cells causing up-regulation of the Iex-1 gene with subsequent increases in Mcp-1 expression which results in recruitment of monocytes/macrophages (cartoon). The specific aims are as follows: SPECIFIC AIM 1: Assess the in vivo role(s) of endothelial (EC) and vascular smooth muscle (VSMC) Iex-1 on VNH formation in AVFs of mice with CKD. We will use novel mice with EC and VSMC conditional deletion of Iex-1 to assess infiltration of monocytes and macrophages, proliferation, migration, apoptosis, fibrosis, lumen diameter, wall thickness and expression of Mcp-1, Fgf-1, Tgf-b, and Mmp-9 in the AVF. Hypothesis: AVFs of mice with decreased Iex-1 expression in ECs and VSMCs will have reduced venous stenosis formation as a result of reduced AVF Mcp-1 expression and reduced monocyte/macrophage infiltration. SPECIFIC AIM 2: Assess the effect of restoration of the Mcp-1 expression in AVFs of mice with EC and VSMC Iex-1 deletion. We will restore Mcp-1 expression in AVFs of mice with EC and VSMC deletion of Iex-1 with adenoviral technique. We will assess monocyte and macrophage infiltration, vessel wall cell proliferation, migration, apoptosis, fibrosis, lumen diameter, wall thickness and the expression of Mcp-1, Fgf-1, Tgf-b, and Mmp-9 in the AVF. Hypothesis: In AVFs of mice with CKD and reduced Iex-1 expression in EC and VSMCs, restoration of Mcp-1 expression will be associated with AVF stenosis. SPECIFIC AIM 3: Determine the role(s) of calcitriol in preventing and/or attenuating VNH formation in pigs with AVFs and CKD. We will use pigs with AVF and CKD to determine the role of adventitial delivery of calcitriol on inhibiting VNH. Hypothesis: Adventitial delivery of calcitriol to the outflow vein of AVF in pigs with CKD will have reduced monocyte/macrophage infiltration and subsequent VNH with downstream reduction in IEX-1, MCP-1, FGF-1, TGF-β, and MMP-9 compared with controls.