Nitric Oxide in Hemodialysis AV Fistula Maturation

描述（由申请人提供）：动静脉（AV）瘘为终末期肾病（ESRD）患者的血液透析提供了最佳血管通路，但近50%的瘘在手术构建后不久就失败了，部分原因是静脉平滑肌细胞（SMC）内膜增生堵塞了瘘腔。氧化应激和一氧化氮（NO）生物利用度降低是伴随ESRD的血管疾病加速的重要因素，积累的数据表明，相同的因素导致静脉内膜增生的侵袭性发展。然而，在尿毒症背景下，特别是在血液透析动静脉瘘的静脉流出中，内膜增生的潜在过程尚未被描述。该建议的中心假设是，潜在的可逆氧化剂诱导的损伤的抑制作用的NO对血管平滑肌细胞迁移和增殖，目前在瘘的创建时间，导致病理性新生内膜增生和成熟失败。在这个R21应用程序中，我们提出了概念验证和可行性研究，为更明确地调查这一假设提供基础，这应该导致基于机制的成熟增强干预措施的发展。我们的建议有以下目的：1）评估静脉SMC对NO的反应性和瘘成熟结果之间的关系。使用静脉SMC的外植体培养物，具有与新创建的瘘管中负责新生内膜生长的细胞类似的去分化表型，我们将评估血清生长因子对增殖和迁移的反应以及NO对其的抑制，并比较最终成熟或未成熟的静脉细胞之间的反应。我们将量化NO释放，测量活性氧，确定肌质/内质网钙ATP酶（SERCA）的氧化还原状态，并评估细胞内Ca 2+。2)评估先前存在的静脉组织中NO生物活性介质的表达及其平滑肌功能和随后的瘘成熟结果之间的关系。我们将使用实时定量PCR来评估通过NO途径调节SMC功能的基因的表达。这些包括eNOS、iNOS、鸟苷酸环化酶、蛋白激酶G、NADPH氧化酶亚型、Ca 2+通道和SERCA。将通过在瘘管形成时固定的额外静脉组织上的免疫组织化学确认基因表达的改变。我们关注组织和细胞基因表达而不是这些NO介质的血浆浓度是基于我们的期望，即局部表达与SMC功能和成熟结果更直接相关。3)确定受损的NO反应性是否可以通过抗氧化酶的过表达、氧化剂产生酶的下调或SERCA的过表达来恢复。我们预计，这些研究的结果将确定有希望的治疗目标，以改善瘘管成熟的结果。公共卫生相关性：动静脉内瘘为终末期肾病患者的血液透析提供了最佳通路，但近50%的内瘘在手术构建后不久就因静脉平滑肌增生阻塞血管腔而失败。在动静脉瘘手术时，我们将获得静脉碎片用于平滑肌细胞培养和免疫组织化学。我们将确定一氧化氮功能、氧化剂产生和细胞内钙控制的异常，这些异常决定了平滑肌细胞增殖和迁移，这些异常可能预测动静脉瘘的失败，从而提供改善预后的治疗靶点。

DESCRIPTION (provided by applicant): Arteriovenous (AV) fistulas provide the optimal vascular access for hemodialysis in patients with end stage renal disease (ESRD), yet nearly 50% of fistulas fail soon after their surgical construction due, in part, to intimal hyperplasia of venous smooth muscle cells (SMC) that occludes the fistula lumen. Oxidative stress and reduced nitric oxide (NO) bioavailability are important contributors to the accelerated vascular disease that accompanies ESRD, and accumulating data suggest the same factors account for aggressive development of venous intimal hyperplasia. However, processes underlying intimal hyperplasia in the setting of uremia, and, specifically, in the venous outflow of hemodialysis AV fistulas, have not been delineated. The central hypothesis of this proposal is that potentially reversible oxidant-induced impairment of the inhibitory action of NO on vascular SMC migration and proliferation, present at the time of fistula creation, leads to pathologic neointimal hyperplasia and maturation failure. In this R21 application we propose proof- of-concept and feasibility studies to provide the groundwork for a more definitive investigation of this hypothesis that should lead to mechanism-based development of maturation-enhancing interventions. Our proposal has the following aims: 1) To evaluate relationships between venous SMC responsiveness to NO and fistula maturation outcomes. Using explant cultures of venous SMC, that have a dedifferentiated phenotype similar to that of cells responsible for neointimal growth in newly created fistulas, we will evaluate proliferation and migration in response to serum growth factors as well as their inhibition by NO, and compare the responses between cells derived from veins that ultimately do or do not mature successfully. We will quantify NO release, measure reactive oxygen species, determine the redox status of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and assess intracellular Ca2+. 2) To evaluate relationships between preexisting venous tissue expression of mediators of NO bioactivity and its smooth muscle functions and subsequent fistula maturation outcomes. We will use real-time quantitative PCR to evaluate expression of genes that regulate SMC function via NO pathways. These include eNOS, iNOS, guanylyl cyclase, protein kinase G, NADPH oxidase isoforms, Ca2+ channels, and SERCA. Alterations in gene expression will be confirmed by immuno-histochemistry on additional vein tissue fixed at the time of fistula creation. Our focus on tissue and cellular gene expression rather than plasma concentrations of these NO mediators is based on our expectation that local expression is more directly relevant to SMC function and maturation outcomes. 3) To determine whether impaired NO responsiveness can be restored by over- expression of antioxidant enzymes, down-regulation of oxidant producing enzymes, or over- expression of SERCA. We anticipate that the findings from these studies will identify promising targets for therapies to improve fistula maturation outcomes. PUBLIC HEALTH RELEVANCE: Arteriovenous fistulas provide the optimal access for hemodialysis in patients with end stage renal disease, yet nearly 50% of fistulas fail soon after their surgical construction due to venous smooth muscle hyperplasia that occludes the vascular lumen. At the time of AV fistula surgery, we will obtain fragments of vein for smooth muscle cell culture and immunohistochemistry. We will identify abnormalities in nitric oxide function, oxidant production, and intracellular calcium control that dictate smooth muscle cell proliferation and migration that may predict failure of the AV fistula, and therefore provide therapeutic targets to improve outcome.