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.

Public Health Relevance

Endothelial dysfunction results in disruption of cell-cell adhesions leading to vascular damage and edema formation. Our research indicates that AMPK?1 functioning in tandem with N-cadherin forms a rapid response system necessary for endothelial barrier repair. Insight gained from this research can be directly applied to development of clinically applicable vascular therapies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL102296-05
Application #
8448139
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
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$276,147
Indirect Cost
$87,651
Name
University of Alabama Birmingham
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Jian, Ming-Yuan; Liu, Yanping; Li, Qian et al. (2016) N-cadherin coordinates AMP kinase-mediated lung vascular repair. Am J Physiol Lung Cell Mol Physiol 310:L71-85
Zhao, Xiangmin; Zhang, Wei; Xing, Dongqi et al. (2013) Endothelial cells overexpressing IL-8 receptor reduce cardiac remodeling and dysfunction following myocardial infarction. Am J Physiol Heart Circ Physiol 305:H590-8
Jiang, Shaoning; Park, Dae Won; Stigler, William S et al. (2013) Mitochondria and AMP-activated protein kinase-dependent mechanism of efferocytosis. J Biol Chem 288:26013-26
Creighton, Judy (2013) Factors controlling vascular permeability: transmitting mechanical signals. Focus on ""Mechanical induction of group V phospholipase A? causes lung inflammation and acute lung injury"". Am J Physiol Lung Cell Mol Physiol 305:L279-81
Jian, Ming-Yuan; Alexeyev, Mikhail F; Wolkowicz, Paul E et al. (2013) Metformin-stimulated AMPK-?1 promotes microvascular repair in acute lung injury. Am J Physiol Lung Cell Mol Physiol 305:L844-55
Creighton, Judy (2011) Targeting therapeutic effects: subcellular location matters. Focus on ""Pharmacological AMP-kinase activators have compartment-specific effects on cell physiology"". Am J Physiol Cell Physiol 301:C1293-5
Creighton, Judy; Jian, MingYuan; Sayner, Sarah et al. (2011) Adenosine monophosphate-activated kinase alpha1 promotes endothelial barrier repair. FASEB J 25:3356-65