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Polymorphonuclear leukocytes (PMNs) play an important role during inflammation in cardiovascular diseases. Human neutrophil peptides (HNPs) are released from PMN granules upon activation and are conventionally involved in microbial killing. Recent studies suggested that HNPs may be involved in the pathogenesis of vascular abnormality by modulating inflammatory responses and vascular tone. Since HNPs directly interact with endothelium upon release from PMNs in the circulation, we tested the hypothesis that the stimulation with HNPs of endothelial cells modulates the expression of vasoactive by-products through altering cyclooxygenase (COX) activity. When human umbilical vein endothelial cells were stimulated with purified HNPs, we observed a time- and dose-dependent increase in the expression of COX-2, whereas COX-1 levels remained unchanged. Despite an increased expression of COX-2 at the protein level, HNPs did not significantly enhance the COX-2 activity, thus the production of the prostaglandin PGI2. HNPs significantly induced the release of endothelin-1 (ET-1) as well as the formation of nitrotyrosine. The HNP-induced COX-2 and ET-1 production was attenuated by the treatment with the oxygen free radical scavenger N-acetyl-L-cysteine and the inhibitors of p38 MAPK and NF-kappaB, respectively. The angiontensin II pathway did not seem to be involved in the HNP-induced upregulation of COX-2 and ET-1 since the use of the angiotensin-converting enzyme inhibitor enalapril had no effect in this context. In conclusion, HNP may play an important role in the pathogenesis of inflammatory cardiovascular diseases by activating endothelial cells to produce vasoactive by-products as a result of oxidative stress.

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.