Protease activated receptors (PARs) form a distinct group of G-protein coupled receptors with key roles in several pathophysiological processes including inflammation and atherothrombotic disease. This study aimed to investigate the effects of oxidative stress on PAR biology in human endothelial cells. EA.hy926 cells were investigated as an endothelial cell model and subsequently the effect of tert-butylhydroperoxide (tert-BHP) as an oxidative stressor was investigated in terms of PAR expression and activation.Methods
Effects of thrombin, PAR-activating peptide and tert-BHP treatments on EA.hy926 PAR expression were determined by qRT-PCR and activity was assessed by Western blotting for pERK1/2.Results
Both primary HUVECs and EA.hy926 expressed PAR1–3 and thrombin caused a rapid increase in PAR1 and PAR3 gene expression. PAR1 and PAR2 activating peptides caused significant increases in PAR1 and, to a lesser extent, PAR3. Induction of PAR2 via PAR2 activating peptide was not observed.PAR4 activating peptide caused time-dependent increases in PAR1–3. PAR activity increased with PAR activating peptide treatment, however there was no clear relationship between activity and expression of PARs mRNA. Repeated exposure to sub-cytotoxic concentrations of tert-BHP, 1 hr daily for 3 days, caused significant increases in PAR1 and PAR3 expression in EA.hy926 cells.Conclusions
HUVEC PAR expression could be modelled in EA.hy926s. Induction of PAR gene expression in EA.hy926 cells was induced by activating peptides and thrombin. Increased PAR1 expression was often mirrored by increased PAR3 expression suggesting possible crosstalk. EA.hy926 cells treated with tert-BHP under conditions that induce stress-induced premature senescence, displayed significant increases in PAR1 and PAR3 gene expression. Further investigation is required to determine if this increased expression is due to redox changes alone and/or due to induction of a senescence phenotype.