Natriuretic Peptides in Pulmonary Endothelial Cell Barrier Function

描述(申请人提供)：本建议的目的是确定钠尿肽(NP)调节肺微血管内皮细胞(PMVEC)屏障功能和肺水肿形成的受体信号通路。在大多数急性肺损伤病例中，肺水肿是导致肺功能障碍的主要原因。血管液体和蛋白质从肺毛细血管腔渗出到间质和肺泡间隙会损害气体交换，并引发一连串的炎症事件，导致急性呼吸窘迫综合征。NPs(心房、ANP、脑、BNP和C型CNP)在调节血管内皮细胞通透性方面起着重要的生理作用。已有大量研究证明NPs具有降低近端肺动脉内皮细胞通透性的能力，但其受体和信号通路尚未明确，很少有研究研究NPs对PMVECs的影响。PMVECs是一种负责调节跨肺液体流量的细胞。申请人实验室最近的研究表明，NPs对PMVEC屏障功能的影响是不同的，鸟苷酸环化酶连接的受体(分别为NPR-A和NPR-C)具有不同的作用。这项建议的总体目标是确定介导NPs对PMVEC屏障功能抑制作用的受体信号通路。这一提议将检验以下假设：NPs通过cGMP/PKG途径钝化凝血酶通过NPR-A诱导的PMVEC通透性增加，并通过NPR-C促进PMVECs屏障功能障碍的恢复。此外，该方案还假设，NPs对凝血酶诱导的PMVEC通透性增加的抑制作用是通过下游抑制小GTP酶RhoA、rac1和CDc42来实现的。该建议旨在通过1)检测NPs、磷酸二酯酶抑制剂和PKG抑制剂对PMVECs通透性的影响，2)检测NPR-A、NPR-C、PKG和小GTP酶的表达变化对NPs体外保护凝血酶诱导的屏障功能障碍的能力的影响，以及3)通过靶向干扰NPR-A和NPR-C来检测NPs对小鼠肺水肿形成的影响。这些研究的结果将进一步加深我们对调节肺水肿形成的细胞机制的理解，并确定NPs及其受体是否为急性肺损伤的潜在治疗靶点。

DESCRIPTION (provided by applicant): The aim of this proposal is to determine the receptor signaling pathways by which the natriuretic peptides (NP) modulate pulmonary microvascular endothelial cell (PMVEC) barrier function and pulmonary edema formation. Pulmonary edema is the primary cause of lung dysfunction in most cases of acute lung injury. Transudation of vascular fluid and protein from the pulmonary capillary lumen to the interstitial and alveolar space impairs gas exchange and initiates a cascade of inflammatory events that lead to acute respiratory distress syndrome. The NPs (atrial, ANP, brain, BNP, and C-type, CNP) play important physiologic roles in modulating vascular endothelial cell permeability. Numerous studies have documented their ability to reduce permeability of proximal pulmonary artery endothelial cells, however, the receptors and signal pathways responsible for this effect are not well defined and few studies have examined the effect of NPs on PMVECs, the cells that are responsible for regulating trans-pulmonary fluid flux. Recent studies in the applicant's lab have shown that the NPs differentially affect PMVEC barrier function and that the guanylyl cyclase- and non- guanylyl cyclase-linked receptors (NPR-A, NPR-C, respectively) have differing effects. The overall goal of this proposal is to identify the receptor-signaling pathways that mediate the inhibitory effects of the NPs on PMVEC barrier function. This proposal will test the hypotheses that NPs blunt thrombin-induced increases in PMVEC permeability via NPR-A by a cGMP/PKG pathway and facilitate restoration of PMVECs barrier dysfunction via NPR-C. In addition, the proposal hypothesizes that the inhibitory effects of the NPs on thrombin-induced increases in PMVEC permeability are mediated via downstream inhibition of the small GTPases RhoA, Rac1 and cdc42. The proposal aims to test these hypotheses by 1) examining the effect of NPs, phosphodiesterase inhibitors and PKG inhibitors on the permeability of PMVECs, 2) examining the effect of altered expression of NPR-A, NPR-C, PKG and the small GTPases on the ability of NPs to protect against thrombin-induced barrier dysfunction in vitro, and 3) examine the effect of NPs on pulmonary edema formation in mice with targeted disruption of NPR-A and NPR-C. Findings from these studies will further our understanding of cellular mechanism that modulate pulmonary edema formation and determine if the NPs and their receptors are potential therapeutic targets for acute lung injury.