Materials and Methods
Cells: Human aortic endothelial cells (HAEC) were isolated and cultured in MCDB-131 medium, as previously described. (Silverman et al., 1996, Kanda et al., 1998). Throughout this study, cells from passages 3 to 8 were used, without any appreciable differences in either basal or TNFa-stimulable VCAM-1 expression.
Flow-System: In the past, we described a unique cell growth/flow chamber (see Figure 1) in which endothelial cells, grown on coverslips, are exposed to distinct flow fields, such as laminar, perturbed, turbulent, and oscillatory/stagnant (recirculating eddies). The flow field in these chambers has been analyzed with high spatial (< 100 &mico;m) and temporal (10 msec) resolution (Lelkes and Samet, 1991; Samet and Lelkes, 1993,1999; Lelkes, 1999).
Prior to the experiments, circular cover slips (11 mm diameter, Thermanox from Fisher), were placed individually into the wells of a 24 well-plate and coated with human fibronectin (10 µg/ml). Approximately 48 hours after reach ing confluence, the cell-lined cover slips were transferred either into another 24-well plate (for static controls), or placed into the grooves of the flow chamber, and glued to the bottom using 100 µl of biological glue (fibrinogen/thrombin mixture) (Nikolaychik et al., 1994). In preliminary studies, both static and dynamic, we established, that the small quantity of thrombin (< 0.1 U/ml) in the glue (a total volume of 4 x 10 µl, confined to the space underneath the cover slips) did not activate endothelial cell VCAM-1 protein expression (Davis and Lelkes, data not shown).
Unless indicated otherwise, all experiments were carried out (for up to 48 hours) at a flow rate of 100 ml/min with a pulse frequency of approx. 1.8 Hz. Depending on the position of the coverslips in the chamber shear stress values ranged between 0.01-0.56 dynes/cm2. Higher shear stress values were obtained by halving the distance between the top and the bottom of the chamber.
Fluorescent Enzyme-Linked Immunosorben t Assay (ELISA): The expression of VCAM-1 in HAEC exposed to pulsatile flow and in static controls was assessed by a fluorescent, whole cell based ELISA assay, essentially as previously described (Kanda et al., 1998). Fluorescence intensities were normalized to the number of cells per cover slip, as determined by a subsequent fluorescent assay quantifying Hoechst 33258 incorporation into cellular DNA (Papadimitriou and Lelkes, 1993).
Assessment of Signal Transduction Pathways by Immunoprecipitation/Western Blotting: To determine signal transduction pathways involved in the PF-activation of VCAM-1, we analyzed the activation/tyrosine phosphorylation of erk-1 (extracellular receptor activated kinase), a member of the MAPK-signal transduction cascade and of FAK (focal adhesion kinase), a part of the integrin signaling pathway, using previously describe techniques for Immunoprecipitation/Western bloating (Rasouly et al., 1996). For these experiments, coverslips lined with confluent mon olayers of HAEC were exposed for various amounts of time (5 -60 min) to pulsatile flow.
Electrophoretic Mobility Shift Assay (EMSA): Nuclear extracts from HAEC maintained either under static conditions or exposed for 30 min to pulsatile flow were prepared according to Andrews and Faller (1991). The EMSA was carried out according to established protocols (Read et al., 1994), using specific oligonucleotide probes (Promega, Madison WI). In brief, 50 ng of the respective oligonucleotides (1.75pmol/µl)was end-labeled with [gamma32P] ATP (3000Ci/mmol at 10 mCi/ml, from NEN-Dupont, Boston, MA) using T4 Polynucleotide Kinase as indicated by the manufacturer (Promega, Madison WI), then purified through a NucTrap® probe purification column (Stratagene, La Jolla, CA). For each assay, 10 µg of nuclear extract was incubated with approximately 1 ng of radiolabeled oligonucleotide (15,000 cpm) for 30 minutes at room temperature in a gel shift binding buffer (Promega, Madison WI) in a f inal total volume of 25 µl. The binding reactions were electrophoresed on a 6% non-denaturing polyacrylamide gel in 0.5 X TBE buffer for 2 hours at 200 volts. The gels were evaluated by autoradiography.
Statistical analysis: Unless mentioned otherwise, results are expressed as mean ± standard error of the mean for at least three experiments, each performed in triplicate. The significance of variability amongst the means of the experimental groups was determined by one-way analysis of variance (ANOVA), using INSTAT® Version 1.12a software, (GraphPAD Software, San Diego, CA). Differences among experimental groups were considered to be statistically significant when p<0.05.
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|Lelkes, P.I.; Kettlun, C.S.; Wigboldus, J.; Waters, C.R.; Sukovich, D.A.; Rubanyi, G.M.; (1998). Signaling Mechanisms Involved in Endothelial Cell Activation by Perturbed Flow. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/|
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