Lipopolysaccharide-mediated oxidative stress and pulmonary vascular injury in Bro

脂多糖介导的氧化应激和肺血管损伤

• 批准号: 8113920
• 负责人: Venkatesh Sampath
• 金额: $ 7.34万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-20 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113920
• 关键词: Angiopoietin-1; Apoptosis; Attenuated; Bacteria; Biology; Blood Vessels; Blood capillaries; Bronchopulmonary Dysplasia; Caring; Cause of Death; Cell Culture Techniques; Cells; Chemicals; Complex; Critical Pathways; Detection; Development; Disease; E-Selectin; Endothelial Cells; Endotoxins; Etiology; Exposure to; Future; Growth; High Pressure Liquid Chromatography; Histologic; Human; Hypoxia; IL8 gene; Immunoprecipitation; In Vitro; Incubated; Inflammation; Inflammatory Response; Injury; Intercellular adhesion molecule 1; Left; Link; Lipopolysaccharides; Lung; Lung diseases; Measures; Mediating; Membrane; Molecular Target; Morbidity - disease rate; NADPH Oxidase; Neonatal; Oxidative Stress; Oxygen measurement, partial pressure, arterial; Pathogenesis; Peptides; Phosphorylation; Premature Infant; Protein Isoforms; Research Project Grants; Role; Signal Transduction; Simulate; Small Interfering RNA; Superoxides; TLR4 gene; Testing; Toxic effect; Vascular Endothelial Growth Factors; Vascular remodeling; angiogenesis; capillary; disability; fetal; human CYBA protein; infancy; matrigel; mortality; novel; prevent; public health relevance; research study

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.

描述（由申请人提供）：尽管新生儿护理取得了进步，支气管肺发育不良（BPD）仍然是婴儿期发病和死亡的主要原因。 BPD 中的血管重塑，其组织学特征是血管生长受阻、树枝化减少和毛细血管变形，可能是由 LPS 诱导的异常人肺微血管内皮 (HPMEC) 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 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，氧张力和脂多糖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有待探索。 本申请将通过以下具体目标研究 LPS 介导的氧化应激和内皮激活对 BPD 血管重塑的作用和机制； 1) 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激活中的作用，以及 ii) 确定胎儿氧张力是否调节 LPS 介导的 HPMEC 中内皮激活、炎症反应和血管生成的毒性。所有实验均使用在胎儿氧张力 (3% O2​​) 或常氧条件下孵育、用 LPS 处理或未处理的培养 HPMEC。通过量化细胞裂解物中 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。将检查基质胶中 HPMEC 的网络形成以及血管生成标记物、VEGF-A、Tie-2、Angiopoietin-1 和 2 的表达，以评估血管生成。通过 HPLC 检测 2-羟基乙锭来量化超氧化物的形成，从而量化氧化应激。将通过免疫荧光评估 P47phox 膜易位，并将进行 TLR4 与 Nox2 或 Nox4 的免疫沉淀，以阐明 Nox 亚型在 LPS 介导的内皮损伤中的作用。将使用化学物质、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 介导的内皮激活和改变的血管生成。将检查 Nox 组装成分的激活、表达或区室化的变化，以阐明胎儿氧张力减轻 HPMEC 中 LPS 诱导的内皮损伤和异常血管生成的机制。 该提案研究了 BPD 发病机制中的一个新范式（LPS - 内皮激活 - 血管生成破坏 - BPD）。这项研究将阐明 BPD 血管重塑的潜在关键途径，将揭示 LPS 与环境氧张力之间相互作用的新机制，可以确定未来药物治疗的分子靶标，并极大地增强我们对 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早产儿出现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。该提案研究了细菌介导的内皮损伤导致未成熟肺部血管异常发育的机制。该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是如何发生的，并可能有助于开发治疗/预防这种疾病的新疗法。