Endothelial repair subsequent to inflammation-induced vascular damage is a poorly understood process. In this application, we propose a novel role for adenosine monophosphate kinase (AMPK) in promoting cadherin adhesion assembly critical for endothelial repair. We hypothesize that AMPK?1 and N-cadherin function in tandem as a rapid response mechanism allowing pulmonary microvascular endothelial cells (PMVECs) to repair barrier disruptions quickly and limit increased capillary permeability. AMPK is most frequently described as a metabolic sensor that maintains ATP levels during periods of metabolic stress. However, recent studies indicate that AMPK also acts as a feedback mechanism that counters the destabilizing effects of elevated [Ca2+]i by promoting protein-protein interactions that strengthen cell-cell junctions. Thus, in this parallel signaling manner, an initially barrier disruptive Ca2+ signal can secondarily activate protective mechanisms. This application expands on this idea of time and location dependent Ca2+ signaling to suggest a novel role for AMPK in Ca2+ dependent cadherin adhesion assembly and barrier repair. Our preliminary results indicate that membrane associated AMPK?1 subsequent to activation by inflammation-induced Ca2+ entry, activates a discrete Ca2+ entry mechanism. This Ca2+ pathway, in contrast to inflammatory Ca2+ signaling, reorganizes the cytoskeleton crucial for N-cadherin adherens'junction assembly and endothelial cell-cell adhesion. Specifically, our preliminary data indicate capillary-derived PMVECs selectively express the AMPK?1 catalytic subunit, and pro-inflammatory challenges increase its expression in the alveolar/capillary segment in-vivo. We utilized shRNA techniques to inhibit AMPK?1 activity and observed an attenuated increase in Ca2+ induced by the inflammatory mimetic thapsigargin suggesting AMPK activated a discrete Ca2+ pathway. Moreover, AMPK inhibition blocked wound resealing in PMVECs. AMPK ?1 co-immunoprecipitates with the adherens junctional protein N-cadherin and co-localizes with N-cadherin to regions of cell-cell contact in PMVECs suggesting AMPK?1 and N-cadherin function in concert to regulate cytoskeletal mechanisms necessary for establishing lung capillary endothelial integrity. This proposal tests the overall HYPOTHESIS that AMPK ?1 forms a functional membrane complex with N-cadherin essential for pulmonary endothelial barrier repair.
Specific Aims test the related hypotheses that: [1] AMPK?1 at the membrane forms a functional complex with N-cadherin necessary for establishing cell-cell adhesion in PMVECs.[2] AMPK?1 activates a discrete Ca2+ entry mechanism that promotes N-cadherin adhesion in PMVECs. [3] Inflammatory stimuli, through Ca2+ signaling, initiate AMPK?1 mediated PMVEC repair.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL102296-06
Application #
8649068
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Colombini-Hatch, Sandra
Project Start
2010-04-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
6
Fiscal Year
2014
Total Cost
$284,269
Indirect Cost
$90,229
Name
University of Alabama Birmingham
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294