Pine Bark Extract Enzogenol Attenuated Tumor Necrosis Factor-α-Induced Endothelial Cell Adhesion and Monocyte Transmigration

Abstract

The transmigration and extravasation of leukocytes across the endothelium that lines the vessel wall occurs in distinct multisteps first comprising rolling of the leukocytes over the endothelial cells, resulting in a tightly controlled and very complex system of leukocyte trafficking and transmigration. Vascular endothelial cells are an important target of proinflammatory cytokines modulating many genes involved in cell adhesion, thrombosis, and inflammatory responses. This study examined whether enzogenol blunts transendothelial migration of monocytes through tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC). HUVECs were incubated with 10 ng/mL TNF-alpha for 6 h in the absence and presence of 5-50 microg/mL enzogenol. Expression of protein and mRNA of adhesion molecules in HUVEC were measured with Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) assay. Monocytic THP-1 cell adhesion and transmigration were examined by calcein AM-staining and matrix metalloproteinase-9 (MMP-9) activity measured by gelatin zymography. Intracellular localization of nuclear factor-kappa B (NF-kappaB) p65 revealed involvement of NF-kappaB signaling. TNF-alpha markedly induced protein expression of cell adhesion molecule and E-selectin with increasing mRNA levels in HUVEC. Nontoxic enzogenol at 5-25 microg/mL attenuated the expression of all adhesion molecules in a dose-dependent fashion. Consistently, enzogenol suppressed the enhanced THP-1 cell adhesion onto TNF-alpha-activated HUVEC through diminishing integrin beta2 induction. In TNF-alpha-activated HUVEC were observed IkappaB dissociation and NF-kappaB nuclear translocation, which was ameliorated by enzogenol. Furthermore, enzogenol hampered the transendothelial migration of THP-1 cells by increasing MMP-9 secretion and activity. Blunting induction of cell adhesion molecules by enzogenol was mediated by their interference with the NF-kappaB-dependent transcription pathways. Thus, enzogenol may have therapeutic potential targeting inflammatory response-associated atherosclerosis.