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.
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.
|Menden, Heather; Welak, Scott; Cossette, Stephanie et al. (2015) Lipopolysaccharide (LPS)-mediated angiopoietin-2-dependent autocrine angiogenesis is regulated by NADPH oxidase 2 (Nox2) in human pulmonary microvascular endothelial cells. J Biol Chem 290:5449-61|
|Menden, Heather; Tate, Everett; Hogg, Neil et al. (2013) LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-? phosphorylation. Am J Physiol Lung Cell Mol Physiol 304:L445-55|