Regulation of phosphodiesterases and cAMP signaling during the host-pathogen interaction in the pulmonary endothelium

项目摘要/摘要 急性呼吸窘迫综合征(ARDS)是一种炎症性肺部疾病，与高 发病率/死亡率和治疗选择有限。肺内皮细胞屏障功能崩溃， 导致浮肿和肺功能受损，是ARDS的一个特征。公认的是，刺激 CAMP合成，如β肾上腺素受体激动剂，增强内皮屏障功能并具有保护作用 在无菌肺损伤的临床前模型中，例如在内毒素注射时。然而，临床试验探索 β激动剂在急性呼吸窘迫综合征中的应用已经失败，原因尚不清楚。环核苷酸 磷酸二酯酶(PDE)是一种降解和失活cAMP的酶，在 细胞内cAMP水平的调节，这些信号的亚细胞区隔，从而内皮细胞 细胞功能。我们的初步数据表明，单一的类型4 PDE亚型，PDE4D，贡献了 肺内皮细胞中cAMP水解力的主要部分，并受到严格的 生理条件。铜绿假单胞菌(PA)是医院获得性肺炎的常见原因，可发展为 败血症和ARDS。我们观察到，在宿主-病原菌相互作用过程中，不同的PA毒力因子诱导 PDE4D激活，导致内皮细胞cAMP信号的抑制和失调。具体地说， 细菌外毒素环化酶ExoY促进PKA介导的PDE4D的磷酸化和激活，从而 改变内皮细胞cAMP信号。此外，PA感染可诱导PKA非依赖性，但3型 分泌系统和鞭毛蛋白依赖的PDE4D调节，导致激活和亚细胞 酶的重新定位。有了这个提议，我们将定义PA毒力因子的途径 改变PDE4D功能及其在内皮屏障破坏和肺损伤中的作用。我们将测试这一想法 PA诱导的PDE4D激活与PA相关ARDS患者的健康结局相关，以及 相反，抑制PDE4具有内皮屏障保护作用。我们将测试异常PDE4的想法 激活限制了β激动剂在PA肺部感染和其他原因中的治疗效果 阿兹。PDE4家族由四个基因组成，非选择性的PDE4抑制剂已经确立了治疗作用 在ARDS临床前模型中的作用，但也会引起副作用，如呕吐和恶心，这些副作用限制了他们的 临床应用。鉴于每个PDE4独特且不重叠的生理和病理生理学作用 针对单个PDE4蛋白的异构体，可以从侧面剖析治疗上的好处 迄今为止可用的PAN-PDE4抑制剂的效果。为此，我们将确定选择性消融是否 PDE4D在体外和体内PA肺损伤模型中具有保护作用，为发展 与非PDE4D选择性抑制剂相比，PDE4D选择性抑制剂用于ARDS治疗具有更好的安全性 迄今可用的选择性PDE4抑制剂。

PROJECT SUMMARY/ABSTRACT Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease associated with high morbidity/mortality and limited treatment options. A breakdown of pulmonary endothelial barrier function, leading to edema and impaired lung function, is a hallmark of ARDS. It is well established that stimulation of cAMP synthesis, such as with β-adrenoceptor agonists, enhances endothelial barrier function and is protective in preclinical models of sterile lung injury such as upon LPS administration. However, clinical trials probing the utility of β-agonists in ARDS have failed and the reasons remain unclear. Cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, play a critical role in the regulation of cellular cAMP levels, the subcellular compartmentalization of these signals, and hence endothelial cell functions. Our preliminary data indicate that a single Type-4 PDE isoform, PDE4D, contributes the predominant portion of cAMP-hydrolytic capacity in the pulmonary endothelium and is tightly regulated under physiologic conditions. P.aeruginosa (PA) is a common cause of nosocomial pneumonia that can progress to sepsis and ARDS. We observed that during the host-pathogen interaction, distinct PA virulence factors induce PDE4D activation, resulting in a suppression and dysregulation of endothelial cAMP signals. Specifically, the bacterial exotoxin cyclase ExoY promotes a PKA-mediated phosphorylation and activation of PDE4D that alters endothelial cAMP signaling. In addition, PA infection can induce a PKA-independent, but type-3 secretion system- and flagellin-dependent PDE4D regulation that results in activation and subcellular relocalization of the enzyme. With this proposal, we will define the pathways by which PA virulence factors alter PDE4D functions and its contribution to endothelial barrier disruption and lung injury. We will test the idea that PA-induced PDE4D activation correlates with health outcomes in patients with PA-associated ARDS, and that, conversely, inhibition of PDE4 is endothelial barrier protective. We will test the idea that aberrant PDE4 activation limits the therapeutic efficacy of β-agonists in settings of PA-lung infection as well as other causes of ARDS. The PDE4 family comprises four genes and non-selective PDE4 inhibitors have established therapeutic effects in preclinical models of ARDS, but also induce side effects, such as emesis and nausea, that limit their clinical utility. Given the unique and non-overlapping physiological and pathophysiological roles of each PDE4 isoform, targeting individual PDE4 proteins can serve to dissect the therapeutically beneficial from the side effects of the PAN-PDE4 inhibitors available to date. To this end, we will determine whether selective ablation of PDE4D is protective in ex vivo and in vivo models of PA-lung injury, paving the way for development of PDE4D-selective inhibitors as ARDS therapeutics with an improved safety profile compared to the non- selective PDE4 inhibitors available to date.