Inducition Of Inflammatory Cytokine Release From Human Umbilical Vein Endothelial Cells By Agonists Of Proteinase Activated Receptor-2

No longer regarded simply as a barrier in the flow of biological molecules to and from the vessel, vascular endothelial cells between the blood and tissue are now considered active participants in inflammatory responses. During the processes of inflammation, they are able to promote the migration of inflammatory cells such as eosinophils, monocytes, lymphocytes from the blood into the site of inflammation, product inflammatory mediators like IL-1,IL-8,INF-α,MCP-1, regulate the vascular permeability and angiogenesis.Proteinase-activated receptor-2 (PAR-2) belongs to proteinase-activated receptors family. It can control the activities of vascular endothelial cells in inflammatory responses. For example, PAR-2 can interfere with vascular barrier function, increase leukocyte adherence to endothelium, induce retinal neovascularization, and stimulate tissue factor expression. Moreover, tumor necrosis factor-α(TNF-α) and lipopolysaccharide are both able to amplify IL-6 production induced by PAR-2 agonist peptide SLIGKV. Although PAR-2 is involved in many physiological and pathophysiological roles in vascular endothelial cells little is known about the action of PAR-2 mediated by trypsin (EC 3.4.21.4) on the regulation of their inflammatory cytokine release. Vascular endothelial cells express PAR-2 and trypsin, indicating that there is a good chance for trypsin to act on PAR-2. These suggest that trypsin is likely to promote the release of inflammatory cytokines from endothelial cells to regulate their functions.Human umbilical vein endothelial cells (HUVECs) were isolated with collagenase Type I from umbilical veins, cultured in 20% FBS-M199 with endothelial cell growth supplement (12.5μg/mL) and heparin (100μg/mL) and identified using endoglin (CD105),platelet-endothelial cell adhesion molecule-1(PECAM-1, CD31). The expression of PAR-2 protein and mRNA was detected by flow cytometry.Using flow cytometry, the purity of HUVECs was about 95%, as judged by positive stain with anti-CD105 and CD31. The results showed that HUVECs expressed PAR-2 protein and PAR-2 mRNA. Trypsin at 1μg/ml induced release of 32 different inflammatory factors, while Ser-Leu-Ile-Gly-Lys-Val-NH₂ (SLIGKV-NH₂) at 100μM only stimulated secretion of 16 inflammatory factors from HUVECs assessed by an antibody-based protein microarray. Fifteen of 16 inflammatory factor released from HUVECs in response to SLIGKV-NH₂ were also secreted from HUVEC in response to trypsin. Since IL-1α, IL-8, IL-10 and IL-12 appeared dramatically released, they were investigated further with ELISA. The levels of IL-1α,IL-10,IL-12 and IL-8 released from HUVECs were elevated significantly (all P < 0.05), and approximately up to 4.8, 4.3, 4.1 and 1.8-fold increase in release of IL-1α, IL-10, IL-12 and IL-8 was observed, respectively, when HUVECs being challenged with trypsin for 16 h. Agonist peptides of PAR-2 SLIGKV-NH₂ at 10μM and 100μM and trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH₂ (tc-LIGRLO-NH₂) at 10μM also provoked significant release of IL-8 (all P < 0.05).Trypsin-induced cytokine release was inhibited by its inhibitors soybean trypsin inhibitor,α1-antitrypsin, and an inhibitor peptide of Phe-Ser-Leu-Leu-Arg-Tyr-NH₂ (FSLLRY-NH₂). Correlation analysis showed that following the stimulation for 8 and 16 h, the correlation of IL-8 levels between trypsin-induced and SLIGKV-NH₂-induced release was statistically significant (P < 0.05).In conclusion, trypsin are able to induce the release of inflammatory cytokines from HUVECs. The action of trypsin on HUVECs was most likely through activation of PAR-2. Stimulation of cytokine release from HUVECs by trypsin suggests that PAR-2 related mechanisms are involved in the inflammatory process in man.