Redox Regulation of Vascular cGMP Signaling in Neonatal Lungs

描述（由申请人提供）：支气管肺发育不良（BPD）是早产的常见并发症，影响30%出生体重< 1000 g的婴儿。近年来，肺动脉高压（PH）和右心衰竭被认为是中重度BPD婴儿的并发症。虽然真正的患病率仍然未知，但一个病例系列估计PH发生在高达25%的BPD婴儿中。一旦婴儿出现PH，人们对如何治疗他们知之甚少，发病率和死亡率的风险非常高。BPD治疗的主要支柱之一是氧气（O2），但超生理O2浓度与机械通气相结合会增加活性氧（ROS）的产生，诱导新生儿显著的血管功能障碍。肺血管系统中ROS介导的失调的潜在关键靶点是可溶性鸟苷酸环化酶（sGC）和磷酸二酯酶5（PDE 5）。我们之前已经证明，高氧暴露导致PDE 5表达和活性增加，伴随cGMP降低，我们有初步数据表明，高氧暴露降低sGC表达和活性。因此，如果新生儿早产并暴露于具有超生理氧气的机械通气，则sGC和PDE 5都容易发生调节异常，这可能影响肺血管反应性和血管重塑，导致右心室肥大。我们的研究小组先前已经发表了高氧暴露会增加分离的肺动脉平滑肌细胞（PASMC）中线粒体和细胞质的ROS。线粒体靶向抗氧化剂足以降低PDE 5活性并恢复分离的PASMC中的正常cGMP水平。此外，在未发表的数据中，蛋白激酶G I1（PKGI 1）抑制剂足以阻断ROS介导的PDE 5增加并恢复正常的cGMP水平。我们假设早产与暴露于高氧诱导的线粒体ROS相结合，破坏了肺内关键的sGC-cGMP-PKG-PDE 5信号通路，导致BPD和肺动脉高压婴儿中观察到的异常肺血管生长和RVH。我们将利用已建立的小鼠BPD模型，结合新的技术，包括新生小鼠PASMC，新生儿活肺切片，和比率氧化还原传感器，阐明ROS破坏这一途径的分子机制。此外，我们将利用BPD小鼠模型来测试抗氧化剂或西地那非（一种PDE 5抑制剂）是否足以预防PH（如果与氧气暴露同时给药）或逆转已建立的PH（如果在恢复期给药）。这些研究将为未来的临床前和临床研究提供病理生理学和机制框架，以改善PH的BPD婴儿的预防和药物治疗。PDE 5抑制剂（如西地那非）在临床上可用，足月新生儿的药代动力学数据可用。如果能够证明其使用的合理性，则它们是这些婴儿最直接的治疗选择。 公共卫生相关性：与支气管肺发育不良相关的肺动脉高压是早产的晚期并发症，其导致显著的长期发病率、增加的卫生保健成本和利用，并且在许多情况下，增加死亡率。在本提案中，我们将利用小鼠模型来确定氧暴露如何对这种情况下的可溶性鸟苷酸环化酶-cGMP-磷酸二酯酶-5信号传导产生负面影响，以及抗氧化剂或磷酸二酯酶-5抑制剂西地那非治疗是否可以逆转已建立的疾病。

DESCRIPTION (provided by applicant): Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth affecting 30% of infants with birthweights < 1000 grams. Recently, pulmonary hypertension (PH) and right-sided heart failure have been recognized as complications in infants with moderate or severe BPD. While the true prevalence remains unknown, one case series estimates that PH occurs in up to 25% of BPD infants. Once infants develop PH, little is known about how to treat them, and risk of morbidity and mortality is very high. One of the mainstays of BPD therapy is oxygen (O2), but supraphysiologic O2 concentrations in combination with mechanical ventilation increase reactive oxygen species (ROS) production, inducing significant vascular dysfunction in neonates. Potential key targets for ROS-mediated dysregulation in the pulmonary vasculature are soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We have previously demonstrated that hyperoxia exposure leads to increased PDE5 expression and activity with concomitant decreased cGMP, and we have preliminary data that hyperoxia exposure decreases sGC expression and activity. Thus, if neonates are born prematurely and exposed to mechanical ventilation with supraphysiologic O2, then both sGC and PDE5 are vulnerable to dysregulation that can impact pulmonary vasoreactivity and vascular remodeling, leading to right ventricular hypertrophy over time. Our group has previously published that hyperoxia exposure increases both mitochondrial and cytoplasmic ROS in isolated pulmonary artery smooth muscle cells (PASMC). Mitochondrially-targeted antioxidants are sufficient to decrease PDE5 activity and restore normal cGMP levels in isolated PASMC. Additionally, in unpublished data, protein kinase G I1 (PKGI1) inhibitors are sufficient to block ROS-mediated increases in PDE5 and restore normal cGMP levels. We hypothesize that preterm birth combined with exposure to hyperoxia-induced mitochondrial ROS disrupts the critical sGC-cGMP-PKG-PDE5 signaling pathway within the lung, leading to abnormal pulmonary vascular growth and RVH as seen in infants with BPD and pulmonary hypertension. We will utilize the established mouse model of BPD in combination with novel techniques including neonatal mouse PASMC, neonatal living lung slices, and ratiometric redox sensors, to elucidate the molecular mechanism by which ROS disrupts this pathway. Furthermore, we will utilize the BPD mouse model to test whether antioxidants or sildenafil, a PDE5 inhibitor, are sufficient to either prevent PH if given concurrently with oxygen exposure or to reverse established PH if given during the convalescent phase. These studies will provide the pathophysiologic, mechanistic framework for future pre-clinical and clinical studies to improve prevention and pharmacologic treatment of BPD infants with PH. PDE5 inhibitors, such as sildenafil, are clinically available, and pharmacokinetic data are available for term neonates. They represent the most immediate therapeutic option for these infants if a rationale for their use can be demonstrated. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension associated with bronchopulmonary dysplasia is a late complication of premature birth that results in significant long-term morbidity, increased health care costs and utilization, and in many cases, increased mortality. In this proposal, we will utilize a mouse model to determine how oxygen exposure negatively impacts soluble guanylate cyclase-cGMP-phosphodiesterase-5 signaling in this condition and whether treatment with antioxidants or sildenafil, a phosphodiesterase-5 inhibitor, can reverse established disease.