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干扰血管cGMP信号的新机制，以开发保护受损新生儿肺发育的新疗法。儿科肺血管疾病(PVD)是新生儿和婴儿肺损伤后的重要致残和死亡原因。虽然现有的治疗方法可以缓解患上这种疾病的患者所观察到的肺动脉高压，但没有治疗方法可以预防PVD。目前，尚无有效的治疗方法直接针对肺血管平滑肌细胞(PASMC)的增殖和肺成纤维细胞的激活，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环化酶(SGC)或通过减少cGMP降解来增加cGMP信号的分子，抑制了培养的PASMC和成纤维细胞中的PVD样变化。然而，在预防新生儿肺损伤的PVD方面，它们的效果有限。据推测，在大多数新生儿肺损伤模型中观察到的sGC表达下降极大地限制了这些药物的治疗前景。令人惊讶的是，目前对sGC在受损新生肺中表达下调的机制知之甚少，也不清楚保护sGC表达是否会增强sGC激动剂的有益活性。利用PVD小鼠模型、活性的转化生长因子-β靶向抗体，以及我们最近开发的分离外周肺小鼠血管细胞的方法，我们确定转化生长因子-β下调了受损肺和PASMC中sGC的表达，导致PASMC去分化，并减少了肺微血管的发育和肺泡化。此外，利用sGCa1基因敲除小鼠和我们的新技术来量化外周肺成纤维细胞的肌源性激活和肺泡化，我们最近确定，在非常轻微的肺损伤中，sGC活性降低本身就会导致PVD，刺激肺肌成纤维细胞转化，并抑制肺泡化。根据这些结果，我们推测，在受损的新生肺中，转化生长因子和sGC信号串扰干扰了肺的发育，从而为预防PVD提供了一个新的治疗靶点。该项目的中心假设是，抑制新生鼠受损肺组织中由转化生长因子调节的sGC下调将促进cGMP刺激的PASMC和肺成纤维细胞的分化，从而改善肺发育。在目标1中，我们首次确定了细胞内的信号转导途径，即转化生长因子通过下调小鼠PASMC中sGC的表达来确定新的治疗靶点。在目标2中，我们将测试下调的sGC表达如何控制转化生长因子β介导的小鼠PASMC和指示PVD的肺成纤维细胞表型转换。在目标3中，我们将确定如何通过抑制转化生长因子-B来挽救sGC的表达，从而增强sGC激动剂在预防PVD方面的保护活性。该项目的成功完成将确定下调sGC信号转导的新机制，并促进sGC抢救疗法的快速发展，以改善肺损伤时的肺成熟和预防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.