Article

Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappa B pathways

Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.6). 05/2006; 281(17):11792-804. DOI: 10.1074/jbc.M509292200
Source: PubMed

ABSTRACT The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.

0 Followers
 · 
67 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.
    PLoS ONE 10/2014; 9(10):e109375. DOI:10.1371/journal.pone.0109375 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to evaluate differences in the effects of Ephedra sinica Stapf and Fructus Schisandrae Chinensis on angiogenesis in the treatment of bleomycin-induced rat idiopathic pulmonary fibrosis. The rat models were created using bleomycin. The animals were divided into six groups: model, control, Ephedra alone, Schisandrae alone, combination of Ephedra and Schisandrae, and hydrocortisone alone. The treatments were administered for 28 days. After 7 and 28 days, the rats were sacrificed for pathological morphology examination, microvascular density determination, and angiogenesis-related cytokine examination. The Ephedra and hydrocortisone groups demonstrated significantly reduced alveolitis and pulmonary fibrosis grades compared with the model group (P < 0.05). The number of blood vessels in the Ephedra group was higher than that in the Schisandrae and combination therapy groups. At 7 days, the expression level of endothelin (ET)-1 in the model group was significantly higher than that in the normal group (P < 0.01). The level of 6-keto-prostaglandin F1α (6-keto-PGF1α) in the treatment group increased, and there were significant differences between the Ephedra group and the combination therapy and normal groups (P < 0.05). Ephedra inhibited the increase in the lung coefficient. The combination therapy prevented pulmonary artery injury and angiogenesis of the arteries by reducing the level of ET-1 and promoting the level of 6-keto-PGF1α in the blood. Ephedra and Schisandrae prevented alveolitis and the development of pulmonary fibrosis.
    Genetics and molecular research: GMR 01/2014; 13(2):3761-71. DOI:10.4238/2014.May.13.3 · 0.85 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Different protease-activated receptors (PARs) activated by thrombin have been shown to implicate in various cardiovascular diseases, via up-regulation of inflammatory proteins including cyclooxygeanse-2 (COX-2). However, the mechanisms underlying thrombin-regulated COX-2 expression in human cardiomyocytes remain unclear.
    British Journal of Pharmacology 06/2014; DOI:10.1111/bph.12794 · 4.99 Impact Factor

Full-text

Download
42 Downloads
Available from
May 22, 2014