Highly effective non-viral gene transfer into vascular smooth muscle cells of cultured resistance arteries demonstrated by genetic inhibition of sphingosine-1-phosphate-induced vasoconstriction.
The linkage of vascular genes to specific functions will lead to a better understanding of cardiovascular pathophysiology. We developed an experimental model that enables the introduction of one or multiple gene(s) into vascular smooth muscle cells (VSMCs) of isolated resistance arteries. Exposure of the arteries to a green fluorescent protein (GFP)-encoding plasmid in combination with the transfectant Effectene((R)) for 20 h resulted in the expression of GFP in virtually all VSMCs in the arterial wall at fully preserved vascular function. For functional validation of the model, plasmids encoding the specific RhoA inhibitors C3 transferase or N19RhoA were transfected. In subsequent functional tests, inhibition of RhoA-dependent constriction induced by sphingosine-1-phosphate was similar to that in arteries treated with exogenous C3 transferase protein or the Rho kinase inhibitor Y27632. Responses to norepinephrine remained unaffected. This novel transfection technique enables gene function to be assessed in direct conjunction with signalling pathways in vascular tissue and provides, therefore, a new tool for microvascular proteomics.
Bolz
Steffen-Sebastian
SS
Institute of Physiology, Ludwig Maximilians University, Munich, Germany. bolz@lrz.uni-muenchen.de
Pohl
Ulrich
U
D000818:Animals; D001158:Arteries / Q000166:cytology; D002478:Cells, Cultured; D006224:Cricetinae; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006973:Hypertension / Q000503:physiopathology; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008246:Lysophospholipids; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014162:Transfection; D014655:Vascular Resistance / Q000502:physiology*; D014661:Vasoconstriction / Q000187:drug effects / Q000235:genetics*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008246:Lysophospholipids; D049452:Green Fluorescent Proteins; C060506:sphingosine 1-phosphate; D013110:Sphingosine