High-efficiency transient transfection of endothelial cells for functionalanalysis

The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high effici ency. This investigation was undertaken to develop an efficient technique f or the transfection of endothelial cells for functional analyses. Cells cot ransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (F ACS) based on GFP expression. In the sorted cell population, a 2.5-fold enh ancement in the number of cells expressing the gene of interest was observe d, as confirmed by FAGS analysis and Western blotting. Sorted cells retaine d functional properties, as demonstrated by chemotaxis to the agonist sphin gosine l-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmid s encoding GFP and the carboxyl-terminal domain of the beta -adrenergic rec eptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma -dependent pathway. However, no significant inh ibition was observed in cells transfected with beta ARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cell s that allows the elucidation of crucial mechanisms of endothelial cell act ivation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and oth er functions in these dynamic cells.