Nitric Oxide in Hemodialysis AV Fistula Maturation

描述(申请人提供)：动静脉(AV)内瘘为终末期肾病(ESRD)患者的血液透析提供了最佳的血管通路，但近50%的内瘘在手术构建后不久失败，部分原因是静脉平滑肌细胞(SMC)的内膜增生堵塞了瘘管的管腔。氧化应激和一氧化氮(NO)生物利用度降低是ESRD伴发的加速血管疾病的重要因素，越来越多的数据表明，同样的因素也是导致静脉内膜增生侵袭性发展的原因。然而，尿毒症时，尤其是血液透析动静脉瘘的静脉流出时，潜在的内膜增生过程还没有被描绘出来。该建议的中心假设是潜在可逆的氧化剂诱导的NO对血管SMC迁移和增殖的抑制作用的损害，在瘘管形成时存在，导致病理性新生内膜增生和成熟失败。在R21的应用中，我们提出了概念验证和可行性研究，为更明确地研究这一假说奠定了基础，这应该会导致基于机制的成熟促进干预措施的发展。我们的建议有以下目的：1)评估静脉SMC对NO的反应性与瘘管成熟结局之间的关系。利用静脉SMC的外植体培养，其去分化表型类似于负责新生内膜生长的细胞，我们将评估血清生长因子对增殖和迁移的反应以及NO对它们的抑制，并比较来自静脉的细胞最终成功或不成功成熟的反应。我们将量化NO的释放，测量活性氧物种，确定肌浆/内质网钙ATPase(SERCA)的氧化还原状态，并评估细胞内钙离子。2)评价静脉组织中NO生物活性介质的表达及其与血管平滑肌功能和继发瘘成熟结局的关系。我们将使用实时定量聚合酶链式反应来评估通过非通路调节SMC功能的基因的表达。其中包括eNOS、iNOS、鸟苷酸环化酶、蛋白激酶G、NADPH氧化酶亚型、钙通道和SERCA。基因表达的改变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来证实。我们关注组织和细胞基因的表达，而不是血浆中这些NO介质的浓度，是基于我们的预期，即局部表达与SMC功能和成熟结局更直接相关。3)确定NO反应性受损是否可以通过抗氧化酶的过度表达、氧化剂产生酶的下调或SERCA的过度表达来恢复。我们预计，这些研究的结果将为改善瘘管成熟结局的治疗确定有希望的靶点。公共卫生相关性：动静脉内瘘为终末期肾病患者提供了最佳的血液透析途径，然而，近50%的内瘘在手术构建后不久就失败了，原因是静脉平滑肌增生阻塞了血管管腔。在动静脉瘘手术时，我们将获得静脉碎片，用于血管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的增殖和迁移，可能预测房室瘘的失败，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.