OBJECTIVETo examine interleukin-8 (IL-8) mRNA expression induced by flow shear stress in human umbilical vein endothelial cells (HUVECs) and investigate its transcriptional activation.

METHODSFlow laminar shear stress 4.2 dyne/cm(2) was used for the stimulating experiments. The flow shear stress-induced IL-8 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). pEGFP1 was used to construct IL-8 reporter gene pEGFP1-IL8USCS for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IkappaB phosphorylation and degradation. RT-PCR, Northern blot, immunocytofluorescent staining and laser confocal microscopy were used to determine Toll-like receptor-4 (TLR-4) expression at mRNA and protein levels in the cells.

RESULTSThere was a marked increase in IL-8 mRNA expression in HUVECs after 120 min of exposure to laminar flow shear stress. When exposed to shear stress for 180 min, there was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected endothelial cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that when exposed to the same flow shear stress for 30 or 60 min, the cell nuclei became stained; after 90 or 120 min, the staining became much more pronounced. A significant increase in P-IkappaB in the cell lysates occurred after 10 min of exposure while blot density dramatically dropped after 60 min of exposure. The density of the IkappaB blot dropped with increasing exposure time after 30 min. TLR-4 was present on the surface of HUVECs. HUVECs constitutively expressed TLR-2 and TLR-4 mRNA; when exposed to flow shear stress for 60 min, TLR-4 mRNA expression increased.

CONCLUSIONNF-kappaB activation is involved in flow shear stress-induced IL-8 mRNA expression in human umbilical vein endothelial cells. TLR-4 receptor for innate immunity most likely mediate these events.