iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells

•iTRAQ quantitative proteomics identified how thrombin affects protein levels.•MetaCoreTM pathway analysis identified cell adhesion as a dominant phenotype•We showed the feasibility of iTRAQ for evaluating cellular responses to stimulation.

IntroductionThe 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.Materials and methodsHAECs were stimulated with 2 units/mL thrombin for 5 h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCoreTM software.ResultsA total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCoreTM 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.ConclusionsSeveral 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.