Establishment Of Endothelium Oxidative Damage Analysis System And Application In Drug Discovery

Vascular endothelial cells form a single cell layer that lines allblood vessels and participate in cardiovascular function regulation.Endothelial dysfunction is involved in many cardiovascular diseases.Many studies indicated that oxidative stress plays an important role inendothelial dysfunction. In the present study, we employed diversebiomedical techniques such as cell-based impedance sensing,quantitative image processing, flow cytometry, confocal imaging,immuocytochemistry and live cell labeling to establish a quantitativeanalysis system to investigate endothelial dysfunction induced byoxidative damage. Furthermore, this quantitative system was applied toevaluate the effects of Hyperoside. This system would be helpful to drugdiscovery and biomedical mechanisms investigation.We applied a non-invasive biosensor system referred to as real-timecell electronic sensor (RT-CES) system to monitor the changes inendothelial cell-substrate adhesion induced by monocyte adhesion in adynamic and quantitative manner, while the number of adherent U937 cellsto the endothelial cells was verified bya standard assay. Furthermore,decrease in FAK protein level and F-actin rearrangement in endothelialcells were observed after addition of U937 cells. Our results indicatethat the adhesion of U937 cells to LPS-treated endothelial ceils reducesthe cell adhesiveness to the substrate, and such reduction may facilitateinfiltration of leukocytes.Based on the culture of endothelial ceils and cellularmicrofluorescence image processing technique, a new method was developedto study the adhesion between endothelial ceils and leukocytes undershear stress by simulating the blood flow in vitro. Employing inversemicrofluorescence imaging and image processing technique, theinteraction between monocytes and endothelial cells under flow condition, was studied quantificationally and objectively.The effect of hyperoside on cndothelial cell damage was studied onendothelial cell oxidative damage model induced by H₂O₂ of cultured humanumbilical vein endothelial cells (HUVEC). The results showed thathyperoside could reduce apoptosis, necrosis, the loss of mitochondrialmembrane potential and the ROS content of endothelial cells induced byH₂O₂, decrease the expression of Bax and increase the expression of Bcl-2.These results proved that reduction of apoptosis and necrosis may be oneof important mechanisms of protective effects of hyperoside onehdothelial cells, and inhibiton of Bax expression and increasing Bcl-2expression were important mechanisms of anti-apoptosis effect ofhyperoside on endothelial cells.On endothelial cells damage model induced by oxLDL in vitro, the effectof hyperoside on ICAM-1 and E-Selectin expression andmonocytes-endothelial cell adhesion were investigated. The resultsshowed that hyperoside could decrease the number of adherent monocytes,increase the CI value during monocytes-endothelial cell binding, anddecrease the velocity and number of slow roiling monocytes by decreasingthe expression of ICAM-1 and E-Selectin. Therefore hyperoside may reducethe adherence of monocytes on oxLDL treated-HUVECs. It may be anotherimportant mechanism of protective effect of hyperoside on endothelialcells.In conclusion, our study achieved the following novel findings: 1. Withthe application of RT-CES system, study on endothelial cell-substrateadhesiveness during leukocyte binding was carried out in a label-free,dynamic, convenient and quantitative way. 2. With the application of flowchamber system and image processing technique, the monocytes wererecognized and counted, and themonocytes' rollingvelocity was measured.3. Two above mentioned methods and traditional biochemical methods were employed in this study to estabilish a quantitative system to analyzeendothelial dysfunction induced by oxidative damage. Additionally, theprotective effects of hyperoside on endothelial cells were investigated.The results showed that hyperoside protected endothelial cells againstoxidative damage by inhibiting apoptosis, necorsis and mitochondrialdysfunction, reducing ROS content and Bax expression, increasing Bcl-2expression, and reduced the interaction between monocytes andendothelial cells.