A role for VEGFR2 activation in endothelial responses caused by barrier disruptive OxPAPC concentrations.
BACKGROUND
Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation.


METHODS
EC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC-induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown.


RESULTS
Low OxPAPC concentrations (5-20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50-100 µg/ml) caused a rapid increase in permeability; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y(731). We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction.


CONCLUSIONS
This study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation.
Section of Pulmonary and Critical Medicine, Department of Medicine, Lung Injury Center, University of Chicago, Chicago, Illinois, United States of America.
Anna A
AA
Birukova
Sangderk
S
Lee
Vitaliy
V
Starosta
Tinghuai
T
Wu
Tiffany
T
Ho
Jin
J
Kim
Judith A
JA
Berliner
Konstantin G
KG
Birukov
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001011:Aorta / Q000166:cytology; D015820:Cadherins / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D011651:Pulmonary Artery / Q000166:cytology; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D018994:Myosin Light Chains; D010713:Phosphatidylcholines; D040301:Vascular Endothelial Growth Factor Receptor-2; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D000099152:Cadherin 5; C472349:oxidized-L-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine