cAMP Phosphodiesterase and Lung Endothelial Cell Permeability

描述(申请人提供)：在肺中，内皮屏障在维持气体交换以响应生物体的需求方面起着关键作用。肺内皮细胞屏障的破坏会导致肺水肿，因为液体会在间质中积聚，最终聚集在远端的空气中。这会导致气体交换严重受损，最终导致死亡。在培养的肺微血管内皮细胞(PMVECs)和活体动物模型中，由质膜定位的腺苷环化酶产生的cAMP(CAMP)具有屏障保护作用。相比之下，细菌毒素ExoY或可溶性腺苷环化酶在细胞质中产生的cAMP是屏障破坏的。肺动脉内皮细胞(PAECs)形成的屏障比PMVECs具有更高的构成通透性。这种泄漏背后的机制尚未确定。已知，近膜cAMP水平的增加可以防止凝血酶和thapsigargin诱导的肺动脉内皮屏障的破坏。因此，膜附近cAMP水平的增加被认为是屏障保护。PAEC同时表达高亲和力PDE7(Km0.1M)和低亲和力PDE4(Km3M)，具有相似的水解率。我们已经证明，PDE7A在PAECs中的稳定敲除具有屏障保护作用。这些和本文中描述的其他观察结果使我们得出了以下工作假设：高亲和力PDE7的活性降低了cAMP水平，并增加了肺动脉内皮细胞的屏障通透性。为了验证这一假说，我们将使用各种生化和细胞成像技术来分析PDE7活性在体外培养的PAEC中调节cAMP信号的作用。然后，我们将使用药理学和遗传学方法，在体外、体外(在离体肺)和体内检测PDE7活性改变对内皮屏障功能的影响。以下具体目标概述了一项整合细胞信号和系统生理途径的计划，以确定PDE7在调节内皮屏障功能中的生理作用。特异性目的1.测定PDE7活性是否降低培养的大鼠肺动脉内皮细胞膜近膜cAMP水平。特异性目的2.确定抑制PDE7活性在体外、体外和体内的肺动脉内皮细胞中是否具有屏障保护作用。 公共卫生相关性：肺血管内皮细胞通透性增加导致呼吸道顺应性降低，呼吸功增加，呼吸窘迫患者的常见并发症。目前还没有针对防止肺动脉内皮细胞通透性增加的策略。在这里，我们建议确定一种特定蛋白质--磷酸二酯酶7型的药物和分子抑制剂是否可以防止这些动脉内皮细胞通透性的增加，从而使呼吸更容易。

DESCRIPTION (provided by applicant): In the lung, the endothelial barrier plays a critical role in maintaining gas exchange in response to the organism's demands. Disruption of the pulmonary endothelial barrier results in pulmonary edema as fluid accumulates in the interstitium and ultimately in the distal airspaces. This leads to severe impairment in gas exchange and eventually, death. In both cultured pulmonary microvascular endothelial cells (PMVECs) and in vivo animal models, cyclic AMP (cAMP) generated by plasma membrane-localized adenylyl cyclase is barrier protective. In contrast, cAMP generated in the cytosol by a bacterial toxin, ExoY, or a soluble adenylyl cyclase is barrier disruptive. Pulmonary arterial endothelial cells (PAECs) form barriers with higher constitutive permeability than PMVECs. The mechanisms underlying this leakiness have not been identified. It is known that increases in the near- membrane cAMP levels prevent thrombin- and thapsigargin-induced endothelial barrier disruption in pulmonary arteries. As such, increases in near-membrane cAMP levels are considered barrier protective. PAECs express both the high affinity PDE7 (Km ~0.1 ¿M) and low affinity PDE4 (Km ~3 ¿M), with similar hydrolysis rates. We have demonstrated that stable knockdown of PDE7A in PAECs is barrier protective. These and other observations described herein led us to the following working hypothesis: The activity of the high affinity PDE7 lowers cAMP levels and increases barrier permeability of pulmonary arterial endothelial cells. To test this hypothesis we will use a variety of biochemical and cellular imaging techniques to dissect the roles of PDE7 activity in regulating cAMP signals in cultured PAECs in vitro. We will then use both pharmacological and genetic approaches to examine the effects of altering PDE7 activity on endothelial barrier function in vitro, ex vivo (in the isolated lung), and in vivo. The following SPECIFIC AIMS outline a plan to integrate cellular signaling and systems physiological approaches in order to establish the physiological roles of PDE7 in regulating endothelial barrier function. SPECIFIC AIM 1. Determine whether PDE7 activity lowers near-membrane cAMP levels in cultured rat pulmonary arterial endothelial cells. SPECIFIC AIM 2. Determine whether inhibition of PDE7 activity is barrier protective in the pulmonary arterial endothelium in vitro, ex vivo, and in vivo. PUBLIC HEALTH RELEVANCE: Increased endothelial permeability in pulmonary arteries results in decreased airway compliance, increased work of breathing, common complications of patients with respiratory distress. There are currently no strategies directed toward preventing increases in endothelial permeability in pulmonary arteries. Here we propose to determine whether pharmacological and molecular inhibitors of a specific protein, phosphodiesterase type 7, prevent increases in endothelial permeability in these arteries, and hence, make breathing easier.