Endothelial repair subsequent to inflammation-induced vascular damage is a poorly understood process. Inthis application, we propose a novel role for adenosine monophosphate kinase (AMPK) in promoting cadherinadhesion assembly critical for endothelial repair. We hypothesize that AMPK 1 and N-cadherin function intandem as a rapid response mechanism allowing pulmonary microvascular endothelial cells (PMVECs) torepair barrier disruptions quickly and limit increased capillary permeability. AMPK is most frequently describedas a metabolic sensor that maintains ATP levels during periods of metabolic stress. However, recent studiesindicate 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 parallelsignaling 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 forAMPK in Ca2+ dependent cadherin adhesion assembly and barrier repair. Our preliminary results indicate thatmembrane associated AMPK 1 subsequent to activation by inflammation-induced Ca2+ entry, activates adiscrete Ca2+ entry mechanism. This Ca2+ pathway, in contrast to inflammatory Ca2+ signaling, reorganizes thecytoskeleton 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, andpro-inflammatory challenges increase its expression in the alveolar/capillary segment in-vivo. We utilizedshRNA techniques to inhibit AMPK 1 activity and observed an attenuated increase in Ca2+ induced by theinflammatory mimetic thapsigargin suggesting AMPK activated a discrete Ca2+ pathway. Moreover, AMPKinhibition blocked wound resealing in PMVECs. AMPK 1 co-immunoprecipitates with the adherens junctionalprotein N-cadherin and co-localizes with N-cadherin to regions of cell-cell contact in PMVECs suggestingAMPK 1 and N-cadherin function in concert to regulate cytoskeletal mechanisms necessary for establishinglung capillary endothelial integrity. This proposal tests the overall HYPOTHESIS that AMPK 1 forms a functionalmembrane complex with N-cadherin essential for pulmonary endothelial barrier repair.
Specific Aims test therelated hypotheses that:  AMPK 1 at the membrane forms a functional complex with N-cadherin necessaryfor establishing cell-cell adhesion in PMVECs. AMPK 1 activates a discrete Ca2+ entry mechanism thatpromotes N-cadherin adhesion in PMVECs.  Inflammatory stimuli, through Ca2+ signaling, initiate AMPK 1mediated PMVEC repair.
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.
|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|