EFFICIENT PROTEIN TRANSFECTION OF CULTURED CORONARY VENULAR ENDOTHELIAL-CELLS

Although it is well recognized that microvascular endothelial cells pl ay an important role in the local regulation of tissue perfusion and e xchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of infor mation is partially due to methodological limitations, because pharmac ological approaches that are routinely used in conventional microcircu latory studies produce nonspecific information. The purpose of this st udy was to develop an efficient method of transfecting endothelial cel ls with proteins for functional analysis. TransIT, a polyamine reagent , proved very successful for beta-galactosidase (beta-Gal) protein tra nsfection of bovine coronary venular endothelial cells, because time-c ourse and dose-dependent experiments showed that a transfection effici ency of 88 +/- 7% was possible. In control studies, beta-Gal was detec ted in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furtherm ore, transfection of a cell-impermeable peptide inhibitor of protein k inase C (PKC) resulted in a decrease in PKC activity in comparison wit h control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and consti tutively active or dominant-negative proteins to study the molecular c ontrol of microvascular function.