Efficient protein transfection of cultured coronary venular endothelial cells.
Although it is well recognized that microvascular endothelial cells play an important role in the local regulation of tissue perfusion and exchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of information is partially due to methodological limitations, because pharmacological approaches that are routinely used in conventional microcirculatory studies produce nonspecific information. The purpose of this study was to develop an efficient method of transfecting endothelial cells with proteins for functional analysis. TransIT, a polyamine reagent, proved very successful for beta-galactosidase (beta-Gal) protein transfection of bovine coronary venular endothelial cells, because time-course and dose-dependent experiments showed that a transfection efficiency of 88 +/- 7% was possible. In control studies, beta-Gal was detected in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furthermore, transfection of a cell-impermeable peptide inhibitor of protein kinase C (PKC) resulted in a decrease in PKC activity in comparison with control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and constitutively active or dominant-negative proteins to study the molecular control of microvascular function.
Tinsley
J H
JH
Departments of Surgery and Medical Physiology, Texas A&M University Health Science Center, Temple, Texas 76504, USA.
Hawker
J
J
Yuan
Y
Y
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology*; D017930:Genes, Reporter; D011506:Proteins / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D011506:Proteins; D001616:beta-Galactosidase