Abstract
Exposure of endothelial cells (EC) to hypoxia has been associated with increased neutrophil (PMN) adherence to EC and changes in EC monolayer permeability. We examined the possibility that these changes would be related to upregulation of specific proteins depending on the duration of hypoxia. EC were incubated in room air and 5% CO2 and grown to confluence in 6 well plates. The EC monolayers were exposed to hypoxia for varying periods of time(0, 12 and 24hrs) in a plexiglass chamber purged continuously with 5% CO2 and 95% N2. Control cells were exposed to normoxia in a similar manner and duration. At the specified time points the cells were solubilized with a Laemmli sample buffer, boiled and loaded onto a gel for electrophoresis. Cell extracts were analyzed by 12% SDS-PAGE. Equal amounts of protein were added per lane. The gels were silver stained for detection of protein bands. Conditioned media from all groups was analyzed in a similar manner. Cell viability during hypoxia was assessed by adherent cell counts and trypan blue exclusion. Hypoxia was associated with the appearance of three (3) new proteins, Mr 56-58kd, 45-46kd and 28-29kd. The upregulation appeared to increase over time with greater intensity at 24 hr than at 12 hrs. In addition, two (2) proteins appeared to be down regulated with increasing lengths of hypoxia-Mr 49-50kd and 30-32kd. The degree of downregulation was somewhat variable in appearance. Conditioned media did not show any new or upregulated proteins over time. Cell viability remained constant at >97% for all groups (including hypoxia). These data suggest that EC exposed to hypoxia upregulate a specific set of proteins that are unrelated to viability. These proteins may play a role in the observed responses of endothelial cells to hypoxia.
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