The Effect Of Shear Stress On The Expression Of The Cytokines And Vascular Active Factors In Vascular Endothelial Cells

The objective of our research is to reveal the effects of different shear stress on expression of cytokines and vascular active factors. We chose cultured human umbilical vein endothelial cells (HUVEC) as the object of our study. We established a modified parallel plate flow chamber system to control the strength and duration of shear stress on HUVEC. After pre-treating HUVEC with shear stress for different periods of time, we utilized radiation-immunoassays to assay IL-8 and ET expression, used Fluo-3 AM staining to detect intracellular Calcium (Ca2+) and Nitric acid deoxidize enzyme technique to measure the concentration of the Nitric Oxide(NO). This research provides data to understand the mechanism of the contribution of hemodynamic forces to the function and metabolism of the endothelial cells.The human umbilical venous endothelial cells (HUVEC) were inoculated in culture vessels precoated with 1% gelatin. The cells were incubated in the environment of 37 C in temperature and with 5% CO2. The growth medium was replaced every 2-3 days. The cells could be used to perform the experiment after 3-5 days when the merged mono layer cells expanded to over 2/3 of the surface area of the culture vessel.We developed this parallel plate flow chamber with reference to Stephen's experiment device (Fig. 2). The chamber was composed of two very thin stainless steel sheets and a slide covered with endothelial cells and can be inserted between the steel sheets. The experiment was conducted in an incubator of 37 C in temperature. The perfusate was made up of culture medium. We could regulate the force of shear stress by controlling flux of the circular device that was made up of parallel plate flow chamber, a pump and a collection bottle. We applied 5dynes/cm2, 10 dynes/cm2 and 15 dynes/cm2 in shear stress force respectively in our experiment. We extracted 200ul perfusate in five phases in order to exam the concentration of IL-8, ET-1 and NO. The glass slides were removed after they were exposed to fluid shear stress. Then we analyzed the concentration of Ca2+ in the cell on the slide by staining the cells with Fluo-3 AM.We found that the intensified expression of IL-8 was time-dependence under different force and duration of shear stress. The IL-8 level was higher than that of the two control groups with 5dyne/cm2 and 10dyne/cm2 shear stress applied. However, the IL-8 level had a restrained trend compared with the control group with 15dyne/cm2 shear stress applied. In our experiment, the level of the ET-1 with 10dyne/cm2 shear stress reached the peak at 120min, then it began to decline and became lower than that of the control group upon SOOmin. We also found that the level of the ET-1 with 5dyne/cm2 shear stress was lower than that of 10dyne/cm2 shear stress until 300min. The expression of NO had a similar undulation under 5dyne/cm2 and 10dyne/cm2shear stress. It reached the peak at 120min, then began to decline to basal level(the same as the control group). We found that the concentration of intracellular Ca2+ was higher than that of the control groups under 5 dyne/cm2 and 10dyne/cm2 shear stress.The mechanotransduction of signals in cellular membrane is very complex under the shear stress applied to the endothelial cells. Plenty of genes are involved in the process. In a broad sense, the activation of the secondary messenger such as Calcium via signals in or out of cells would lead to expression of cytokines. Through the above experiments, we can deduce the following conclusions: Shear stress can modulate endothelial cell morphology and function; Shear stress can activate and upmodulate Ca2+ by changing cytoskeleton or activating certain biochemical response, and Ca2+ in turn can modulate gene transcription and expression of the vascular active factors, such as NO and ET-1, by activating protein kinase. All of these are very important to keep integrality of the cell modality and function.