TGFB and Nitric Oxide Signaling in Pediatric Pulmonary Vascular Disease

 描述（由申请人提供）：我们的研究重点是识别和测试破坏血管环鸟苷酸信号传导的新机制，以开发保护受伤新生儿肺的肺部发育的新疗法。小儿肺血管疾病（PVD）可导致新生儿和婴儿严重残疾和死亡。虽然现有的治疗方法可以缓解在患有这种疾病的患者中观察到的肺动脉高压，但没有预防PVD的治疗方法。目前，没有有效的治疗方法直接解决肺动脉平滑肌细胞（PASMC）增生和肺成纤维细胞活化，这是PVD的根本原因，并将其与成人肺动脉高压区分开来。通过刺激可溶性鸟苷酸环化酶（sGC）或通过减少cGMP降解来增加cGMP信号传导的分子抑制培养的PASMC和成纤维细胞中的PVD样变化。然而，它们在预防肺损伤新生儿PVD方面的疗效有限。怀疑在大多数新生儿肺损伤模型中观察到的sGC表达降低极大地限制了这些药物的治疗前景。令人惊讶的是，关于在损伤的新生儿肺中下调sGC表达的机制知之甚少，并且不知道sGC表达的保护是否会增强sGC激动剂的有益活性。使用PVD的小鼠幼鼠模型、活性TGF-β靶向抗体和我们最近开发的分离外周肺小鼠幼鼠血管细胞的方法，我们确定TGF-β下调受损肺和PASMC中sGC的表达，导致PASMC去分化，并减少肺微血管发育和肺泡化。此外，使用sGCa 1敲除小鼠幼崽和我们的新技术来量化外周肺成纤维细胞肌原性激活和肺泡化，我们最近确定sGC活性降低本身会导致PVD，刺激肺肌成纤维细胞转化，并在非常轻微的肺损伤情况下抑制肺泡化。基于这些结果，我们推测TGF-β和sGC信号传导串扰破坏了损伤新生儿肺中的肺发育，因此提出了预防PVD的新治疗靶点。该项目的中心假设是，在损伤的新生儿肺中抑制TGF-β调节的sGC下调将促进cGMP刺激的PASMC和肺成纤维细胞分化，并改善肺发育。在目的1中，我们提出首次鉴定TGF-β下调小鼠幼仔PASMC中sGC表达的细胞内途径，以鉴定新的治疗靶点。在目标2中，我们将测试下调的sGC表达如何控制TGF-β介导的小鼠幼仔PASMC和指示PVD的肺成纤维细胞表型转换。在目的3中，我们将确定如何通过TGF-β抑制拯救sGC表达增强sGC激动剂在预防PVD中的保护活性。该项目的成功完成将确定下调sGC信号传导的新机制，并促进sGC补救疗法的快速发展，改善肺损伤期间的肺成熟并预防PVD。

 DESCRIPTION (provided by applicant): Our research focus is to identify and test novel mechanisms that disrupt vascular cGMP signaling in order to develop new therapies that protect pulmonary development in the injured newborn lung. Pediatric pulmonary vascular disease (PVD) causes important disabilities and death in newborns and infants with lung injury. Although available therapies can relieve the pulmonary hypertension observed in patients that have acquired this disease, there is no treatment that prevents PVD. Currently, no effective therapy directly addresses the pulmonary artery smooth muscle cell (PASMC) hyperplasia and lung fibroblast activation that are the root cause of PVD and distinguish it from adult pulmonary hypertension. Molecules that increase cGMP signaling by stimulating soluble guanylate cyclase (sGC), or by decreasing cGMP degradation, inhibit PVD-like changes in cultured PASMC and fibroblasts. However, they exhibit limited efficacy in preventing PVD in newborns with lung injury. It is suspected that the decreased sGC expression observed in most newborn lung injury models greatly limits the therapeutic promise of these agents. Surprisingly, very little is known about the mechanisms that down-regulate sGC expression in the injured newborn lung and it is unknown whether protection of sGC expression will enhance the salutary activities of sGC agonists. Using a mouse pup model of PVD, active TGF-ß-targeting antibodies, and our recently developed method to isolate peripheral lung mouse pup vascular cells, we determined that TGF-ß down-regulates sGC expression in the injured lung and PASMC, causes PASMC dedifferentiation, and decreases pulmonary microvascular development and alveolarization. Moreover, using sGCa1 knock out mouse pups and our new techniques to quantify peripheral lung fibroblast myogenic activation and alveolarization, we recently determined that decreased sGC activity in itself causes PVD, stimulates lung myofibroblast conversion, and inhibits alveolarization in the setting of very mild lung injury. Based on these results, we speculate that TGF-ß- and sGC-signaling crosstalk disrupts pulmonary development in the injured newborn lung and therefore presents a new therapeutic target to prevent PVD. The central hypothesis of this project is that inhibiting TGF-ß-regulated sGC down-regulation in the injured newborn lung will promote cGMP-stimulated PASMC and lung fibroblast differentiation, and improve pulmonary development. In Aim 1, we propose to identify for the first time the intracellular pathways through which TGF-ß down-regulates sGC expression in mouse pup PASMC to identify new therapeutic targets. In Aim 2, we will test how down-regulated sGC expression controls TGF-ß-mediated mouse pup PASMC and lung fibroblast phenotype switching indicative of PVD. In Aim 3, we will determine how rescuing sGC expression through TGF-ß inhibition potentiates the protective activity of sGC agonists in preventing PVD. Successful completion of this project will identify new mechanisms that down-regulate sGC signaling and promote rapid development of sGC-rescue therapies that improve pulmonary maturation during lung injury and prevent PVD.