Lipopolysaccharide-mediated oxidative stress and pulmonary vascular injury in Bro

描述(申请人提供)：尽管在新生儿护理方面取得了进步，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的血管重塑的组织学特征是血管生长受阻，分枝减少，毛细血管畸形，可由内毒素诱导的人肺微血管内皮细胞(HPMEC)异常激活和血管生成改变所致。尽管有强有力的证据表明早产儿BPD与内毒素暴露有关，但内毒素介导的氧化应激和微血管损伤导致BPD血管重构的确切机制仍不清楚。此外，氧张力和内毒素在介导氧化应激和肺微血管损伤中潜在相互作用的机制仍未被探索。本研究将从以下几个方面探讨内毒素介导的氧化应激和内皮细胞激活在BPD血管重塑中的作用及其机制：1)确定NADPH氧化酶(NOX)依赖的机制在介导内毒素诱导的HPMEC氧化应激和内皮激活中的作用；2)确定胎儿氧分压是否调节内毒素介导的HPMEC内皮激活、炎症反应和血管生成的毒性。培养的HPMEC在胎儿氧分压(3%O2)或常氧条件下孵育，用内毒素处理或不处理，用于所有实验。通过定量检测细胞裂解液中ICAM-1和E-选择素的表达，以及细胞培养上清液中IL-8的表达，来评估内皮细胞的激活。HPMEC在基质细胞中的网络形成和血管生成标志物VEGF-A、Tie-2、Angiopoietin-1和Angiopoietin-2的表达将用于评估血管生成。超氧化物的形成，通过高效液相色谱检测2-羟乙基的定量，将被测量以定量氧化压力。P47Phox膜转位将用免疫荧光法进行评估，TLR4与NOX2或NOX4的免疫沉淀将被用来阐明NOx亚型在内毒素介导的内皮损伤中的作用。NADPH-氧化酶的活性将通过使用化学物质、多肽和siRNA来控制，以确定Nox依赖的机制是否参与了内毒素介导的内皮激活和改变的血管生成。我们将研究NOx装配组分的激活、表达或区域化的变化，以阐明胎儿氧分压减轻脂多糖诱导的HPMEC内皮损伤和异常血管生成的机制。这项建议探讨了BPD发病机制中的一种新范式(内毒素-内皮激活-血管生成受阻-BPD)。本研究将阐明BPD血管重构的一个潜在关键途径，揭示内毒素与环境氧分压相互作用的新机制，为未来的药物治疗提供分子靶点，并极大地提高我们对TLR和NOx生物学的理解。 公共卫生相关性：支气管肺发育不良，一种在早产儿中发展起来的衰弱的肺部疾病，仍然是婴儿期死亡和残疾的主要原因。这项建议探讨了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早产儿支气管肺发育不良的发展过程，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.