Chiparray-based identification of gene expression in HUVECs treated with low frequency electric fields

Purpose: After high-voltage electric injury, patients often show progressive tissue necrosis and thrombosis of blood vessels even remote from the entry and exit sites of electrical current. Recently, we were able to demonstrate in vivo and vitro the release of several prothrombotic factors. In this study, we report on in vitro studies performed to characterize gene expression profiles using a DNA-microarray in HUVECs (human umbilical vein endothelial cells) exposed to low frequency electrical current. Methods: HUVECs were plated and grown to confluence in a culture chamber. They were exposed to 25 periods of 50 Hz sinusoidal waves. The periods had field strength of 60 V/cm and duration of 100 ms. Periods were interrupted by 10-second intervals to prevent significant joule heating. Control HUVECs were treated identically except that no electric field was applied. Samples from control and treated cells were taken after six and 24 hours. A PIQOR(TM) Immunology Array (Milteny Biotech) containing 1076 cDNAs was used for gene expression analysis. Hybridization of Cy3- and Cy5-labelled samples, image capture, and signal quantification of hybridized arrays were performed. Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Cy5/Cy3. The ratios were normalized to the median of all ratios and the mean of the ratios of four corresponding spots was computed. More than two-fold increases or decreases of the gene expression were regarded as relevant. Results: A total of 413 genes (1 s + s) respectively 345 genes (2 s + s) could be detected. The results obtained display a distinct expression pattern of up-regulated genes known to be important for hemostasis (e.g. UPA, UPAR, ECE1, PAFAH1B1, PGT, INOS, ENOS, TPA, ICAM1, VCAM1, PAI1, PA12, VWF, PTGDR, F3, THBD), which was most evident after 24 hours. This expression profile might lead to a hypercoagulated state. Furthermore, the expression of genes involved in angiogenesis was reduced whereas the expression of those involved in platelet formation was increased. Conclusion: Our results indicate that low frequency electrical fields induce a distinct signature of differential gene expression in exposed HUVECs. This might explain the clinical observation of thrombosis and progressive tissue necrosis after electrical injury.