VEGFR2 signalling contributes to increased endothelial susceptibility to TNF-α under chronic non-uniform shear stress.
OBJECTIVE
Vascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli.


METHODS
Endothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence.


RESULTS
siRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective.


CONCLUSIONS
VEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions.
Department of Cardiology and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany.
Katharina
K
Urschel
Christoph D
CD
Garlichs
Werner G
WG
Daniel
Iwona
I
Cicha
D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002448:Cell Adhesion; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D019040:E-Selectin / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D009000:Monocytes / Q000276:immunology; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D034622:RNA Interference; D013314:Stress, Mechanical; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C412373:AG 1879; D002867:Chromones; D019040:E-Selectin; D018836:Inflammation Mediators; D009025:Morpholines; D016328:NF-kappa B; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; C546936:SELE protein, human; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C478783:ZM323881; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D058539:Phosphatidylinositol 3-Kinase; D040301:Vascular Endothelial Growth Factor Receptor-2; D019061:src-Family Kinases