iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.
BACKGROUND
The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated.


METHODS
HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software.


RESULTS
A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs.


CONCLUSIONS
Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation.
School of Medicine, China Medical University, Taichung, Taiwan; Division of Cardiology, Department of Medicine, China Medical University Hospital, Taichung, Taiwan.
Huang-Joe
HJ
Wang
Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan.
Sung-Fang
SF
Chen
Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan. Electronic address: drwanyu@gmail.com.
Wan-Yu
WY
Lo
D001011:Aorta / Q000166:cytology* / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D018925:Chemokines / Q000032:analysis / Q000378:metabolism; D019040:E-Selectin / Q000032:analysis / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000032:analysis / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000378:metabolism
D018925:Chemokines; D019040:E-Selectin; D016023:Integrins; D020543:Proteome; C546936:SELE protein, human; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D013917:Thrombin
Isobaric tags for relative and absolute quantitation (iTRAQ); Thrombin; human aortic endothelial cells; quantitative proteomics