Novel Molecular Mechanisms Regulating Postnatal Pulmonary Angiogenesis

描述(申请人提供)：通过血管生成的肺毛细血管的生长是肺泡化的关键，而肺血管生成的中断导致了支气管肺发育不良(BPD)的发病，这是早产最常见的并发症。然而，在发育和疾病过程中协调肺血管生成的分子途径还不完全清楚。这种认识上的差距继续扰乱了开发有针对性的疗法来治疗由血管生成受损引起的肺部疾病的努力，包括BPD。核因子κ-B(核因子κ-B)是炎症反应的关键调节因子，越来越多的证据表明，核因子κB在肿瘤创面愈合过程中调节血管生成。相比之下，人们对核因子κB在发育中的肺中的作用知之甚少。我们最近证实了核因子κB在牙槽骨形成过程中是必不可少的。 NFκB在新生小鼠肺组织中具有结构性活性，但在成年小鼠肺中处于静止状态，抑制NFκB可干扰新生小鼠肺血管生成和肺泡化，但对成年小鼠无影响。阻断新生原代肺内皮细胞中的核因子κB会损害其存活、增殖和体外血管生成。肺内皮细胞中核因子κB的活化与发育中肺血管生成的时期相对应，在肺泡化开始时达到高峰，在肺泡化中期下降到低水平。然而，NFκB在肺内皮细胞中被激活的能力并不是细胞成熟所固有的，因为从早期肺泡肺器官培养中获得的条件培养液强烈地激活了NFκB并促进了成年肺内皮细胞的迁移。整体蛋白质组学分析比较早期肺泡和成人肺分泌体，确定只由早期肺泡(EAL)分泌的因子。为了检测中和这些蛋白对EA-LCM促进NFκB激活和迁移能力的影响，我们在早期肺泡中鉴定了两种可能的NFκB激活剂：转化生长因子诱导蛋白(TGFB)和甲胎蛋白(AFP)。这两种因子在活体肺泡灌洗液中都有表达，但在成人肺中的表达很低，而且这两种因子都被破坏肺泡化的损伤所抑制。因此，我们的数据提出了总体工作假说，即EAL独特分泌的因子通过激活肺内皮细胞中的NFκB来促进肺血管生成和肺泡化，从而解决了两个相互关联的特定目标。在目标1中，我们将检测EAL分泌的TGFBI和AFP是否通过NFκB依赖的途径诱导PEC中促血管生成的表型，并探索TGFBI和AFP是否激活共同或互补的基因表达模式。目的2将利用体内功能获得和功能丧失策略来确定在发育和损伤过程中，干扰TGFFI型或甲胎蛋白介导的NFκB的激活是否会损害肺泡的生长。这些研究的成功完成将确立核因子κB在发育中的肺中的新的生理学作用，并确定可直接转化为治疗策略的新的血管生成因子，以促进以肺血管生成受损为标志的疾病的肺生长和再生。

DESCRIPTION (provided by applicant): Growth of the pulmonary capillaries by angiogenesis is essential for alveolarization, and disruption of pulmonary angiogenesis contributes to the pathogenesis of bronchopulmonary dysplasia (BPD), the most common complication of premature birth. However, the molecular pathways that orchestrate pulmonary angiogenesis during development and disease are not fully understood. This gap in knowledge continues to confound efforts to develop targeted therapies to treat lung diseases caused by impaired angiogenesis, including BPD. Nuclear factor κ-B (NFκB) is a key regulator of inflammation, and growing evidence suggests that NFκB regulates angiogenesis in wound healing in cancer. In contrast, little is known about the role of NFκB in the developing lung. We recently demonstrated that NFκB is essential for alveolarization. NFκB is constitutively active in the neonatal, but quiescent in the adult lung, and inhibiting NFκB disrupts pulmonary angiogenesis and alveolarization in neonatal mice, but has no effect in adults. Blocking NFκB in neonatal primary pulmonary endothelial cells (PEC) impairs survival, proliferation, and in vitro angiogenesis. Activation of NFκB in the PEC corresponds to the period of angiogenesis in the developing lung, peaking at the onset of alveolarization, and decreasing to low levels by mid-alveolarization. However, the capacity for NFκB to be activated in the PEC is not intrinsic to the cell's maturation, as conditioned media obtained from early alveolar lung organ culture (EA-LCM), robustly activates NFκB and enhances migration in adult PEC. Global proteomic analysis to compare the early alveolar and adult lung secretomes identified factors secreted only by the early alveolar lung (EAL). Studies to examine the effect of neutralizing each of these proteins on the ability of the EA-LCM to promote NFκB activation and migration, allowed the identification of two putative activators of NFκB in the early alveolar lung: transforming growth factor-ß induced protein (TGFBI) and alpha-fetoprotein (AFP). Both factors are expressed in the EAL in vivo but minimally expressed in the adult lung, and both are suppressed by injuries that disrupt alveolarization. Therefore, our data suggest the overall working hypothesis that factors uniquely secreted by the EAL promote lung angiogenesis and alveolarization by activating NFκB in the pulmonary endothelium by addressing two inter-related specific aims. In Aim 1, we will examine if TGFBI and AFP secretion by the EAL induces a pro-angiogenic phenotype in the PEC via an NFκB-dependent pathway, and explore if TGFBI and AFP activate common or complementary patterns of gene expression. Aim 2 will utilize in vivo gain and loss of function strategies to determine if disrupting TGFBI- or AFP-mediated activation of NFκB impairs alveolar growth during development and injury. The successful completion of these studies will establish a novel, physiologic role for NFκB in the developing lung, and identify new angiogenic factors that can be directly translated into therapeutic strategies to promote lung growth and regeneration in diseases marked by impaired pulmonary angiogenesis.