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？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？B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？B在肺发育中的作用知之甚少。我们最近证明了核因子？B在牙槽骨形成过程中是必不可少的。核因子？B在新生小鼠肺组织中呈结构性活性，而在成年小鼠肺中处于静止状态，抑制核因子？B抑制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(PEC)中的核因子？B会损害其存活、增殖和体外血管生成。肺上皮细胞中核因子B的活化与发育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较低水平。然而，肺内皮细胞中激活核因子B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(EA-LCM)获得的条件培养液强烈地激活了核因子B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。为了研究中和这些蛋白对EA-LCM促进核因子B活化和迁移能力的影响，在早期肺泡中鉴定了两种可能的核因子B激活剂：转化生长因子诱导蛋白(TGFBI)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，我们的数据表明，EAL独特分泌的因子通过激活NF？促进肺血管生成和肺泡化。B通过解决两个相互关联的特定目标而在肺内皮细胞中发挥作用。在目标1中，我们将检测EAL分泌的TGFBI和AFP是否通过依赖于NF？B的途径在PEC中诱导促血管生成表型，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干扰TGFBI或AFP介导的核因子？B的激活是否会在发育和损伤过程中损害肺泡的生长。这些研究的成功完成将确立核因子？B在发育中的肺中的新的生理作用，并确定新的血管生成因子，这些因子可以直接转化为治疗策略，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 ?-B (NF?B) is a key regulator of inflammation, and growing evidence suggests that NF?B regulates angiogenesis in wound healing in cancer. In contrast, little is known about the role of NF?B in the developing lung. We recently demonstrated that NF?B is essential for alveolarization. NF?B is constitutively active in the neonatal, but quiescent in the adult lung, and inhibiting NF?B disrupts pulmonary angiogenesis and alveolarization in neonatal mice, but has no effect in adults. Blocking NF?B in neonatal primary pulmonary endothelial cells (PEC) impairs survival, proliferation, and in vitro angiogenesis. Activation of NF?B 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 NF?B 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 NF?B 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 NF?B activation and migration, allowed the identification of two putative activators of NF?B in the early alveolar lung: transforming growth factor-¿ 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 NF? B 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 NF?B-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 NF?B impairs alveolar growth during development and injury. The successful completion of these studies will establish a novel, physiologic role for NF?B 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.