cAMP Phosphodiesterase and Lung Endothelial Cell Permeability

描述（由申请人提供）：在肺中，内皮屏障在维持机体需要的气体交换中起着关键作用。肺内皮屏障的破坏导致肺水肿，因为液体在间质中积聚并最终积聚在远端空气中。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）和体内动物模型中，由质膜定位的腺苷酸环化酶产生的环AMP （cAMP）具有屏障保护作用。相反，胞质中由细菌毒素ExoY或可溶性腺苷酸环化酶产生的cAMP具有屏障破坏作用。肺动脉内皮细胞（PAECs）形成屏障，具有比PMVECs更高的本构通透性。这种泄漏背后的机制尚未被确定。众所周知，近膜cAMP水平的升高可防止凝血酶和抑素诱导的肺动脉内皮屏障破坏。因此，近膜cAMP水平的增加被认为具有屏障保护作用。PAECs同时表达高亲和度PDE7 （Km ~0.1¿M）和低亲和度PDE4 (Km ~3¿M)，且水解速率相似。我们已经证明PDE7A在paec中的稳定敲低具有屏障保护作用。这些和其他在此描述的观察结果使我们得出以下工作假设：高亲和力PDE7的活性降低cAMP水平并增加肺动脉内皮细胞的屏障通透性。为了验证这一假设，我们将使用多种生化和细胞成像技术来剖析PDE7活性在体外培养paec中调节cAMP信号中的作用。然后，我们将使用药理学和遗传学方法在体外、离体（在离体肺中）和体内检测改变PDE7活性对内皮屏障功能的影响。以下具体目标概述了整合细胞信号和系统生理学方法的计划，以确定PDE7在调节内皮屏障功能中的生理作用。具体目标1。测定PDE7活性是否降低培养大鼠肺动脉内皮细胞近膜cAMP水平。具体目标2。确定PDE7活性的抑制是否在体外、离体和体内对肺动脉内皮具有屏障保护作用。

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.