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）仍然是婴儿发病和死亡的主要原因。BPD的血管重构在组织学上表现为血管生长受阻、树突减少和毛细血管畸形，这可能是由lps诱导的人肺微血管内皮（HPMEC）异常激活和血管生成改变引起的。尽管有强有力的证据表明内毒素暴露与早产儿BPD有关，但脂多糖介导的氧化应激和微血管损伤促进BPD中观察到的血管重构的确切机制尚不清楚。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机制仍未被探索。本应用程序将通过以下具体目的研究lps介导的氧化应激和内皮细胞激活对BPD血管重构的影响和机制：1)确定NADPH氧化酶（Nox）依赖机制在lps诱导的HPMEC氧化应激和内皮活化中的作用；ii)确定胎儿氧张力是否调节lps介导的HPMEC内皮活化、炎症反应和血管生成的毒性。在胎儿氧张力（3% O2）或常氧条件下培养的HPMEC，用LPS处理或不处理将用于所有实验。内皮活化将通过定量细胞裂解物中ICAM-1和e -选择素的表达以及细胞培养上清中IL-8的表达来评估。将检测基质中HPMEC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。超氧化物的形成，通过高效液相色谱检测2-羟乙基，将测量氧化应激定量。P47phox膜易位将通过免疫荧光评估，TLR4与Nox2或Nox4的免疫沉淀将被执行，以阐明Nox同种异构体在LPS介导的内皮损伤中的作用。nadph氧化酶活性将使用化学物质、多肽和siRNA来确定一氧化氮依赖机制是否参与LPS介导的内皮活化和血管生成的改变。我们将研究nox组装组分的激活、表达或区室化的改变，以阐明胎儿氧张力减弱lps诱导的HPMEC内皮损伤和异常血管生成的机制。本研究探讨了脂多糖-内皮活化-血管生成中断- BPD的发病机制。该研究将阐明BPD血管重构的潜在关键途径，揭示LPS与环境氧张力相互作用的新机制，为未来的药物治疗提供分子靶点，并极大地增强我们对TLR和Nox生物学的理解。

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.