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%的出生体重和1000克的婴儿。最近，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。虽然真正的患病率尚不清楚，但一系列病例估计，多达25%的BPD婴儿发生了PH。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治疗的主要手段之一是氧气(O2)，但超生理氧气浓度与机械通气相结合会增加活性氧(ROS)的产生，导致新生儿严重的血管功能障碍。ROS介导的肺血管调节失调的潜在关键靶点是可溶性鸟苷环化酶(SGC)和磷酸二酯酶5(PDE5)。我们先前已经证明，高氧暴露导致PDE5表达和活性增加，伴随着cGMP的降低，我们有初步数据表明，高氧暴露降低了sGC的表达和活性。因此，如果新生儿早产并暴露在机械通气和超肺氧合作用下，那么sGC和PDE5都容易受到调节失调的影响，这可能会影响肺血管反应性和血管重塑，随着时间的推移导致右室肥厚。我们的研究小组此前曾发表文章指出，高氧暴露会增加分离的肺动脉平滑肌细胞(PASMC)线粒体和细胞质ROS。线粒体靶向抗氧化剂足以降低PDE5活性并恢复分离的PASMC中cGMP的正常水平。此外，在未发表的数据中，蛋白激酶G1(PKGI1)抑制剂足以阻断ROS介导的PDE5增加并恢复正常的cGMP水平。我们假设早产和暴露于高氧诱导的线粒体ROS扰乱了肺内关键的sGC-cGMP-PKG-PDE5信号通路，导致肺血管异常生长和RVH，就像BPD和肺动脉高压的婴儿一样。我们将利用已建立的BPD小鼠模型，结合新技术，包括新生小鼠PASMC、新生儿活肺切片和比率氧化还原传感器，来阐明ROS扰乱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PDE5抑制剂西地那非是否足以预防PH，如果与氧气暴露同时给予，或者如果在恢复期给予，则足以逆转已建立的PH。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，以改进对患有PH的BPD婴儿的预防和药物治疗。PDE5抑制剂，如西地那非，可在临床上使用，足月新生儿的药代动力学数据也可用。如果可以证明使用它们的理由，它们代表了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一种晚期并发症，会导致显著的长期发病率、增加的医疗保健成本和利用率，在许多情况下，还会增加死亡率。在这个方案中，我们将利用一个小鼠模型来确定在这种情况下，氧气暴露如何对可溶性鸟苷环化酶-cGMP-磷酸二酯酶-5信号产生负面影响，以及抗氧化剂或磷酸二酯酶-5抑制剂西地那非的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.