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.
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.
