Natriuretic Peptides in Pulmonary Endothelial Cell Barrier Function

描述（由申请人提供）：本提案的目的是确定利钠肽（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传导途径。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至间质和肺泡腔，损害气体交换并引发一系列炎症事件，导致急性呼吸窘迫综合征。 NP（心房、ANP、脑、BNP 和 C 型、CNP）在调节血管内皮细胞通透性方面发挥重要的生理作用。许多研究已经证明它们具有降低近端肺动脉内皮细胞通透性的能力，然而，导致这种作用的受体和信号通路尚未明确定义，并且很少有研究考察纳米颗粒对 PMVEC（负责调节跨肺液体流量的细胞）的影响。申请人实验室的最新研究表明，NP对PMVEC屏障功能有不同的影响，并且鸟苷酸环化酶和非鸟苷酸环化酶连接的受体（分别为NPR-A、NPR-C）具有不同的作用。该提案的总体目标是确定介导 NP 对 PMVEC 屏障功能抑制作用的受体信号传导途径。该提案将测试以下假设：NP 通过 cGMP/PKG 途径减弱凝血酶通过 NPR-A 诱导的 PMVEC 通透性增加，并通过 NPR-C 促进 PMVEC 屏障功能障碍的恢复。此外，该提案假设 NP 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 GTPase RhoA、Rac1 和 cdc42 介导的。该提案旨在通过以下方式测试这些假设：1) 检查 NP、磷酸二酯酶抑制剂和 PKG 抑制剂对 PMVEC 通透性的影响，2) 检查 NPR-A、NPR-C、PKG 和小 GTP 酶表达的改变对 NP 体外防止凝血酶诱导的屏障功能障碍的能力的影响，以及 3) 检查 NP 对有针对性破坏 PMVEC 的小鼠肺水肿形成的影响。 NPR-A 和 NPR-C。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，并确定纳米粒子及其受体是否是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.