TRANSFORMING GROWTH FACTOR - BETA MEDIATES EFFECTS OF HYPOXIA IN NEWBORN LUNG

描述（由申请人提供）：慢性缺氧导致发育中的肺异常肺动脉重塑（PAR）和肺泡发育（IAD）的抑制。转化生长因子- β (TGF-)是一种肽生长因子，是肺发育和血管重构的关键调节因子，可能是PAR和IAD发病机制的重要调节因子。TGF-(是作为无活性前体合成的，激活TGF-(是引发生物效应的关键步骤。肺内表达细胞表面糖蛋白Thy-1的成纤维细胞可抑制TGF-（）的激活。我们最近发现，由于诱导显性负TGF- II型受体（DN2RII）而导致TGF-信号中断的新生小鼠的IAD和PAR减弱，这表明TGF-信号在IAD和PAR中很重要。我们发现，在新生小鼠中，缺氧减少了Thy-1并增加了活性TGF-，这表明缺氧诱导的Thy-1的减少可能允许TGF-激活的增加。我们的实验室进行了新的观察，Thy-1缺失的小鼠有IAD，磷酸化- smad2， -平滑肌肌动蛋白，间质胶原，组织肺阻力（但正常气道阻力）增加，肺顺应性降低，表明发育中的肺中缺乏Thy-1导致肺表型类似于支气管肺发育不良（BPD）。我们的初步数据表明，TGF-(的合成可能受到内皮素-1 （ET-1）的调节，内皮素-1是PAR的一种已知介质。本项目的目的是确定肺发育期间缺氧暴露导致TGF-(激活增加以及随后的PAR和IAD的机制。特异性目的1将验证TGF-(信号转导是缺氧诱导PAR和IAD的必要条件。特异性目的2将评估Thy-1在缺氧或缺氧情况下TGF-(激活中的机制作用和IAD的发病机制。特异性Aim 3将验证低氧诱导的ET-1升高刺激TGF-（合成和激活）的特异性假设。采用垂直整合的方法，使用体内模型（新生小鼠从出生起暴露于缺氧或空气中2周）和体外模型（新生小鼠肺动脉平滑肌细胞和成纤维细胞）。除了野生型小鼠外，目前可用的转基因小鼠（Thy-1 null, DN2RII, (6 integrin null)）将用于表征关键信号通路。PAR可导致新生儿持续性肺动脉高压（PPHN），在BPD中均可见到IAD和PAR。尽管在新生儿护理方面取得了进步，但PPHN和BPD的发病率仍然很高，新的范例对于开发新的治疗策略至关重要。在本建议中概述的实验将增加我们对围产期正常和异常肺动脉重塑和肺泡发育的机制的认识。至少，这些研究将确定TGF-作为缺氧诱导的IAD和PAR的关键调节因子的作用。这些实验还将确定缺氧调节TGF-信号通路的成分，以及缺氧条件下TGF-激活因子。项目描述：新生儿死亡和发病的两个主要原因是新生儿持续性肺动脉高压（PPHN）和支气管肺发育不良（BPD），其特征是肺动脉异常增厚、肺动脉高压和肺泡发育抑制。转化生长因子- β（一种在正常肺发育中很重要的蛋白质）的过度激活可能导致肺发育异常，见于PPHN和BPD。该提案将确定转化生长因子- β在肺异常发育中的作用以及转化生长因子- β被激活的机制，并可能导致PPHN和BPD的新治疗方案的开发。

DESCRIPTION (provided by applicant): Chronic hypoxia leads to abnormal pulmonary arterial remodeling (PAR) and inhibition of alveolar development (IAD) in the developing lung. Transforming growth factor-beta (TGF-(), a peptide growth factor that is a key regulator of lung development and vascular remodeling, may be a crucial mediator in the pathogenesis of PAR and IAD. TGF-( is synthesized as an inactive precursor, and activation of TGF-( is the critical step necessary to elicit biological effects. TGF-( activation is inhibited by fibroblasts in the lung which express the cell surface glycoprotein Thy-1. We have recently shown that newborn mice with disruption of TGF-( signaling due to inducible dominant negative TGF-( type II receptors (DN2RII) have attenuated IAD and PAR, indicating that TGF-( signaling is important in IAD and PAR. We have shown that hypoxia reduces Thy-1 and increases active TGF-( in newborn mice, suggesting that hypoxia-induced decreases in Thy-1 may permit increased TGF-( activation. Our laboratory has made the novel observations that Thy-1 null mice have IAD and increased phospho-Smad2, (-smooth muscle actin, interstitial collagen, tissue lung resistance (but normal airway resistance), and decreased lung compliance, demonstrating that absence of Thy-1 in the developing lung leads to a lung phenotype similar to bronchopulmonary dysplasia (BPD). Our preliminary data indicate that TGF-( synthesis may be regulated by endothelin-1 (ET-1), a known mediator of PAR. The objective of this project is to determine the mechanisms by which hypoxic exposure during lung development leads to increased TGF-( activation and subsequent PAR and IAD. Specific Aim 1 will test the hypothesis that TGF-( signaling is necessary for hypoxia-induced PAR and IAD. Specific Aim 2 will assess the mechanistic role of Thy-1 in TGF-( activation and the pathogenesis of IAD in the presence or absence of hypoxia. Specific Aim 3 will test the specific hypothesis that hypoxia-induced increases in ET-1 stimulate TGF-( synthesis and activation. A vertically-integrated approach, with in vivo models (newborn mice exposed to hypoxia or air for 2 weeks from birth) and in vitro models (neonatal murine pulmonary arterial smooth muscle cells and fibroblasts) will be used. Currently available transgenic mice (Thy-1 null, DN2RII, (6 integrin null), in addition to wild-type mice will be used to characterize the critical signaling pathways. PAR contributes to persistent pulmonary hypertension of the newborn (PPHN), and both IAD and PAR are seen in BPD. The incidence of PPHN and BPD remains high despite advances in neonatal care, and new paradigms are essential for the development of novel therapeutic strategies. The experiments outlined in this proposal will increase our knowledge of the mechanisms responsible for normal and abnormal pulmonary arterial remodeling and alveolar development in the perinatal period. At a minimum, these studies will determine the role of TGF-( as a key regulator of hypoxia-induced IAD and PAR. These experiments will also identify the components of TGF-( signaling pathway regulated by hypoxia, and TGF-( activators under hypoxic conditions. PROJECT NARRATIVE: Two major causes of death and morbidity in newborn infants are persistent pulmonary hypertension of the newborn (PPHN) and bronchopulmonary dysplasia (BPD), which are characterized by abnormal pulmonary arterial thickening, pulmonary hypertension, and inhibition of alveolar development. Excessive activation of transforming growth factor-beta, a protein that is important in normal lung development, may lead to the abnormal lung development that is seen in PPHN and BPD. This proposal will determine the role of transforming growth factor-beta in abnormal lung development and the mechanisms by which transforming growth factor-beta becomes activated, and may lead to the development of new treatment options for PPHN and BPD.