Induction of Inflammatory Cytokine Release From Human Umbilical Vein Endothelial Cells by Agonists of Proteinase‐activated Receptor‐2

Abstract

1. Human endothelial cells express proteinase-activated receptor-2 (PAR-2), inflammatory cytokines and trypsin (EC 3.4.21.4). However, little is known about the mechanism through which trypsin induces cytokine release from endothelial cells. 2. In the present study, we investigated the effect of trypsin on cytokine release from primary cultures of human umbilical vein endothelial cells (HUVEC) using an antibody based protein microarray and ELISA. 3. The results showed that 1 microg/mL trypsin induced release of 32 different inflammatory factors, whereas 100 micromol/L Ser-Leu-Ile-Gly-Lys-Val-NH2 (SLIGKV-NH2) only stimulated secretion of 16 inflammatory factors from HUVEC, as assessed by an antibody based protein microarray. Because the release of interleukin (IL)-1a, IL-8, IL-10 and IL-12 was markedly increased following PAR-2 activation, their release was investigated further using ELISA. Increases in release of up to approximately 4.8-, 4.3-, 4.1- and 1.8-fold were observed for IL-1a, IL-10, IL-12 and IL-8, respectively, when HUVEC were challenged with trypsin for 16 h. Agonist peptides of PAR-2, namely SLIGKV-NH2 and trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 (tc-LIGRLO-NH2), also provoked significant release of IL-8. Trypsin-induced cytokine release was inhibited by its inhibitors soybean trypsin inhibitor, alpha1-antitrypsin and the inhibitor peptide of PAR-2 Phe-Ser-Leu-Leu-Arg-Tyr-NH2 (FSLLRY-NH2). 4. These data indicate the action of trypsin on HUVEC is most likely through activation of PAR-2, suggesting that PAR-2-related mechanisms are involved in the inflammatory process in humans.

Expression of Proteinase-Activated Receptor-2 in the Intervertebral Disc

“…Western blot analysis revealed that treatment with 2-furoyl-LIGRLO-NH 2 (0.1 and 1.0 mol/L) significantly increased IL-1␤ release into the medium from rat AF cells. Our results are consistent with previous reports that PAR-2 activation induced the release of pro-inflammatory cytokines by endothelial cells 34 and human peripheral blood monocytes. 35 Our study has also shown that treatment with 2-furoyl-LIGRLO-NH 2 alone induced up-regulation of the ADAMTS-4 mRNA level, but not those of MMP-3 and MMP-13.…”

The Recovery Time Course of the Endothelial Cell Glycocalyx In Vivo and Its Implications In Vitro

“…These enzymes very likely cleave proteins that anchor the glycocalyx to the endothelial-cell surface. It is also known that this process inflames the cells as well, since cultured endothelial cells are subjected to an inflammatory stimulus at passage via their protease activated receptors33. Therefore, when cells are first plated they are likely to be severely inflamed with little or no endothelial glycocalyx on their surface.…”

Protease-Activated Receptor 2 Deficiency Reduces Cardiac Ischemia/Reperfusion Injury

“…One of them is endothelial cells. It has been demonstrated that activation of PAR-2 on endothelial cells induces the expression of IL-1β, IL-6 and IL-8 (human homolog of mouse KC)39, 40. The production of chemokines leads to the recruitment of leukocytes, including neutrophils and monocytes, into the injured myocardium41.…”