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Departments of Surgery and Medical Physiology, Texas A&M University Health Science Center, Temple, Texas 76504
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 
-galactosidase (-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, -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.
COS-7 cells; cell culture; -galactosidase; protein kinase
C
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