Lipopolysaccharide-mediated oxidative stress and pulmonary vascular injury in Bro

描述（由申请人提供）：尽管新生儿护理取得了进展，但支气管肺发育不良（BPD）仍然是婴儿期发病率和死亡率的主要原因。BPD中的血管重塑在组织学上以血管生长停滞、树枝状化减少和畸形毛细血管为特征，可由LPS诱导的异常人肺微血管内皮细胞（HPMEC）活化和改变的血管生成引起。尽管有强有力的证据将内毒素暴露与早产儿BPD联系起来，但LPS介导的氧化应激和微血管损伤导致BPD中观察到的血管重塑的确切机制仍不清楚。此外，氧张力和LPS在介导氧化应激和肺微血管损伤中的潜在相互作用的机制仍然未被探索。 本申请将通过以下具体目的来研究LPS介导的氧化应激和内皮活化在BPD血管重塑中的作用和机制：1）为了确定NADPH氧化酶（Nox）依赖性机制在介导HPMEC中LPS诱导的氧化应激和内皮活化中的作用，和ii）确定胎儿氧分压是否调节LPS介导的对HPMEC中内皮活化、炎症反应和血管生成的毒性。在胎儿氧分压（3%O2）或常氧条件下孵育、用LPS处理或不处理的培养HPMEC将用于所有实验。通过定量细胞裂解物中ICAM-1和E-选择素以及细胞培养上清液中IL-8的表达来评估内皮活化。将检查HPMEC在基质胶中的网络形成和血管生成标志物VEGF-A、Tie-2、血管生成素-1和2的表达以评估血管生成。将测量超氧化物形成（通过HPLC检测2-羟基噻啶定量），以定量氧化应激。将通过免疫荧光评估P47 phox膜易位，并将进行TLR 4与Nox 2或Nox 4的免疫沉淀以阐明Nox同种型在LPS介导的内皮损伤中的作用。将使用化学品、肽和siRNA操纵NADPH氧化酶活性，以确定Nox依赖性机制是否参与LPS介导的内皮活化和改变的血管生成。将检查Nox组装组件的激活、表达或区室化的改变，以阐明胎儿氧分压减弱HPMEC中LPS诱导的内皮损伤和异常血管生成的机制。 该建议研究了BPD发病机制中的新范式（LPS -内皮活化-破坏的血管生成- BPD）。这项研究将阐明BPD血管重塑的潜在关键途径，将揭示LPS和环境氧张力之间相互作用的新机制，可以为未来的药物治疗确定分子靶点，并大大提高我们对TLR和Nox生物学的理解。 公共卫生相关性：支气管肺发育不良是一种在早产儿中发展的使人衰弱的肺部疾病，仍然是婴儿期死亡和残疾的主要原因。本研究旨在探讨细菌介导的内皮损伤导致未成熟肺血管异常发育的机制。这项研究项目的成功完成将使我们能够更好地了解支气管肺发育不良如何在早产儿中发展，并可能有助于开发治疗/预防这种疾病的新疗法。

DESCRIPTION (provided by applicant): Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a major cause of morbidity and mortality in infancy. Vascular remodeling in BPD, characterized histologically by arrested vascular growth with decreased arborization and dysmorphic capillaries, can result from LPS-induced aberrant human pulmonary microvascular endothelial (HPMEC) activation and altered angiogenesis. Despite strong evidence linking endotoxin exposure to BPD in premature infants, the precise mechanisms by which LPS mediated oxidative stress and microvascular injury contribute to the vascular remodeling observed in BPD remain unknown. Moreover, mechanisms underlying potential interactions between oxygen tension and LPS in mediating oxidative stress and pulmonary microvascular injury remain unexplored. This application will investigate the effect-, and the mechanisms, by which LPS-mediated oxidative stress and endothelial activation contribute to the vascular remodeling in BPD through the following specific aims; 1) To determine the role of NADPH oxidase (Nox)-dependent mechanisms in mediating LPS-induced oxidative stress and endothelial activation in HPMEC, and ii) To determine whether fetal oxygen tension modulates LPS-mediated toxicity on endothelial activation, inflammatory response and angiogenesis in HPMEC. Cultured HPMEC incubated in fetal oxygen tension (3% O2) or normoxia, treated with LPS or left untreated will be used for all experiments. Endothelial activation will be assessed by quantifying expression of ICAM-1 and E-selectin in cell-lysates, and IL-8 in cell culture supernatants. Network formation of HPMEC in matrigel and expression of angiogenic markers, VEGF-A, Tie-2, Angiopoietin-1 and 2 will be examined to assess angiogenesis. Superoxide formation, quantified by HPLC detection of 2-hydroxyethidium, will be measured to quantify oxidative stress. P47phox membrane translocation will be assessed by immunoflourescence, and immunoprecipitation of TLR4 with Nox2 or Nox4 will be performed to clarify the role of Nox isoforms in LPS mediated endothelial injury. NADPH-oxidase activity will be manipulated using chemicals, peptides and siRNA to determine if Nox-dependent mechanisms are involved in LPS mediated endothelial activation and altered angiogenesis. Alterations in the activation, expression or compartmentalization of Nox-assembly components will be examined to elucidate the mechanisms by which fetal oxygen tension attenuates LPS-induced endothelial injury and aberrant angiogenesis in HPMEC. This proposal investigates a novel paradigm (LPS - endothelial activation - disrupted angiogenesis - BPD) in the pathogenesis of BPD. This study will elucidate a potentially critical pathway in the causation of vascular remodeling in BPD, will unveil novel mechanisms of interaction between LPS and environmental oxygen tension, can result in the identification of molecular targets for future pharmacological therapy and greatly enhance our understanding of TLR and Nox biology. PUBLIC HEALTH RELEVANCE: Bronchopulmonary dysplasia, a debilitating lung disease that develops in premature infants, remains a leading cause of death and disability during infancy. This proposal investigates the mechanisms by which bacteria-mediated endothelial injury results in the abnormal development of blood vessels in immature lungs. Successful completion of this research project will enable us to better understand how bronchopulmonary dysplasia develops in premature infants and potentially help in the development of novel therapy to treat/prevent this disease.