The Role of Hypoxia In Venous Neointimal Hyperplasia In Hemodialysis Grafts

缺氧在血液透析移植物静脉新生内膜增生中的作用

• 批准号: 9276718
• 负责人: Sanjay Misra
• 金额: $ 57.38万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276718
• 关键词: Acute monocytic leukemia; Adipose tissue; Affect; Angioplasty; Antibodies; Area; Arteriovenous fistula; Blood Vessels; Cartoons; Cell Line; Cell Therapy; Cell Transplants; Cell physiology; Cells; Chronic Kidney Failure; Clinical; Clinical Trials; Coculture Techniques; Data; Data Aggregation; Dialysis Solutions; Dialysis patients; Dialysis procedure; End stage renal failure; Endothelium; Evaluation; Failure; Family suidae; Fistula; Funding; Future; Gene Expression; Goals; Grant; Hemodialysis; Hyperplasia; Hypoxia; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Intervention; Knock-out; Lead; Literature; Mesenchymal Stem Cells; Modeling; Monocyte Chemoattractant Protein-1; Mus; Outcome; PPAR gamma; Pathologic; Patient Care; Patients; Population; Publishing; Radiology Specialty; Renal Replacement Therapy; Research; Role; Secondary to; Staining method; Stenosis; Stents; Stress; TNF gene; Testing; Therapeutic; Time; Tissue Sample; Tissue Stains; Transplantation; Tunica Adventitia; United States; Up-Regulation; Vascular Endothelial Growth Factors; Vascular remodeling; Veins; Venous; bevacizumab; cytokine; design; experience; experimental study; genetic approach; improved; in vivo; macrophage; monocyte; mouse model; patient population; pre-clinical; programs; protein expression; small hairpin RNA; vascular smooth muscle cell proliferation

PROJECT SUMMARY/ABSTRACT: Nearly 600,000 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 that outflow veins treated with AMSCs plus bevacuzimab have a significant increase in lumen vessel area and a decrease in the ratio of neointima/media + adventitia compared to controls. This effect is associated with a reduction in PPAR-γ gene expression. Furthermore, we show that bevacuzimab localizes to the endothelium and not the adventitia where AMSCs are transplanted and thus will not disrupt AMSC function. VEGF-A staining of this tissue demonstrates a reduction in sub-endothelial VEGF- A where bevacuzimab is localized. We used THP-1 monocytes (cell line from acute monocytic leukemia patients) to simulate the in vivo findings of monocyte to macrophage differentiation under hypoxia, which shows that THP-1 monocytes differentiate to macrophages via an increase in PPAR-γ. When monocytes are co-cultured with AMSCs plus bevacuzimab and subjected to hypoxic stress, there is a decrease in differentiation to macrophages with a reduction in PPAR-γ gene expression compared to controls. In aggregate, these data support the following CENTRAL Hypothesis, which will be tested in the present revised competitive renewal: Adventitial transplantation of AMSCs to the outflow vein of AVF combined with bevacuzimab therapy reduces PPAR-γ gene expression in monocytes leading to decreased macrophage differentiation and subsequent VNH formation. To test our hypothesis, we have developed three specific aims: SPECIFIC AIM 1: Evaluate the efficacy of AMSCs combined with bevacizumab on reducing VNH using murine and porcine models. Optimal bevacizumab combination with AMSCs will be determined in a murine model and next validated in a preclinical porcine model of AVF with CKD. The pathological and radiological evaluation will be carried out in the tissue samples to assess long-term patency and vascular remodeling. SPECIFIC AIM 2: Determine in vitro role(s) of AMSCs combined with bevacuzimab on reducing hypoxia-induced up-regulation of PPAR-γ in monocytes leading to a reduction in macrophage differentiation. Initially, the effect of hypoxia on expression of PPAR-γ in monocytes will be examined. Next, experiments will be conducted using co-culture with AMSCs to override the negative effect of PPAR-γ caused by hypoxia followed by administering bevacuzimab. The outcomes will include the expression of genes responsible for inflammation and vascular remodeling as well as cellular function. These results will be validated in tissue samples from Aim 1. SPECIFIC AIM 3: Assess in vivo role(s) of PPAR-γ using genetic approaches on VNH formation. We will use monocyte specific conditional knockout of PPAR-γ in mice with AVF and CKD and examine the effect on venous stenosis formation with gene and protein expression of pro-inflammatory cytokines. Next, we will determine the role(s) of bevacuzimab plus monocyte specific conditional knockout of PPAR-γ mice on VNH.

项目总结/摘要： 在美国，近60万患者患有终末期肾病（ESRD），预计这一人群将 在未来十年翻一番。目前这项提案和研究计划的长期目标是改善 ESRD患者的护理，绝大多数使用长期血液透析作为其肾透析模式的患者， 替代疗法这些患者需要高功能的血管通路，以获得最佳的治疗效果。 适足性血液透析血管通路失败通常是由于继发于新生内膜的静脉狭窄 增生（VNH）。 我们的初步数据表明，用AMSC加贝伐单抗治疗的流出静脉具有良好的抗肿瘤作用。 管腔血管面积显著增加，新生内膜/中膜+外膜比率降低， 控制。这种效应与PPAR-γ基因表达的减少有关。此外，我们表明， 贝伐单抗定位于内皮，而不是AMSC移植的外膜， 不破坏AMSC功能。该组织的VEGF-A染色显示内皮下VEGF-1表达减少。 贝伐单抗的局部化位置。我们使用THP-1单核细胞（来自急性单核细胞白血病的细胞系 患者）以模拟缺氧条件下单核细胞向巨噬细胞分化的体内发现， 显示THP-1单核细胞通过PPAR-γ的增加分化为巨噬细胞。当单核细胞 与AMSC加贝伐单抗共培养并经受缺氧应激， 与对照相比，在向巨噬细胞分化时，PPAR-γ基因表达降低。在 总的来说，这些数据支持以下中心假设，这将在本修订版中进行检验 竞争性更新：将AMSC外膜移植到AVF流出静脉， 贝伐单抗治疗降低单核细胞中的PPAR-γ基因表达，导致巨噬细胞减少 分化和随后的VNH形成。为了验证我们的假设，我们制定了三个具体目标： 具体目的1：使用鼠模型评估AMSC与贝伐单抗组合在降低VNH方面的功效。 和猪模型。将在鼠模型中确定贝伐珠单抗与AMSC的最佳组合， 接下来在患有CKD的AVF的临床前猪模型中进行验证。病理学和放射学评估将 在组织样本中进行，以评估长期通畅性和血管重塑。 具体目的2：确定AMSC与贝伐单抗组合在减少缺氧诱导的细胞凋亡中的体外作用。 单核细胞中PPAR-γ的上调导致巨噬细胞分化减少。最初，效果 将检查缺氧对单核细胞中的PPAR-γ表达的影响。接下来，实验将使用 与AMSC共培养以克服由缺氧引起的PPAR-γ的负面作用， 贝伐单抗。结果将包括负责炎症和血管的基因的表达 重塑以及细胞功能。这些结果将在目标1的组织样本中得到确认。 具体目的3：使用遗传方法评估PPAR-γ对VNH形成的体内作用。我们将使用 单核细胞特异性条件性敲除PPAR-γ在AVF和CKD小鼠中的作用， 静脉狭窄形成与促炎细胞因子的基因和蛋白表达。接下来我们就 确定贝伐单抗加PPAR-γ小鼠的单核细胞特异性条件性敲除对VNH的作用。