Comparison of gene silencing in human vascular cells using small interfering RNAs.
BACKGROUND
Gene silencing achieved through small interfering RNA (siRNA) transfection represents a promising approach to vascular gene therapy. Here we characterize the behavior of RNA interference (RNAi) in vascular biology by comparing the RNAi response to single- and multigene siRNA transfections in vitro in human vascular cells.


METHODS
The strength and specificity of multigene silencing in cultured human coronary artery smooth muscle and human coronary artery endothelial cells (HCASMC/HCAEC) were assessed by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and Western blot after transfection singly or simultaneously with siRNAs targeting glyceraldehyde-3-phosphate dehydrogenase, the myristoylated alanine-rich C kinase substrate, and cadherin 11. RNAi response to low-dose (0.25 to 10 nM) siRNA transfection was characterized between the two cell types by QRT-PCR and fluorescence-activated cell sorter analysis.


RESULTS
Powerful and specific silencing of all targets was observed in both cell types after multigene siRNA transfections, but with a reduction in effect compared with single-gene siRNA transfections. Multigene messenger RNA (mRNA) reductions in HCAECs exceeded those achieved in HCASMCs, and superior mRNA silencing and siRNA delivery were observed in HCAECs after low-dose siRNA transfections.


CONCLUSIONS
Multigene silencing by siRNA stands as a promising nonviral approach for manipulating gene expression in human vascular cells. Under our in vitro conditions, endothelial cells were more susceptible to siRNA transfection and gene silencing than vascular smooth muscle cells. RNAi technology could potentially find use in the development of siRNA cocktails for application to vein bypass grafts or for modulating endothelial cell function in other forms of vascular disease.
Andersen
Nicholas D
ND
Department of Surgery, Division of Vascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
Monahan
Thomas S
TS
Malek
Junaid Y
JY
Jain
Monica
M
Daniel
Soizic
S
Caron
Lena D
LD
Pradhan
Leena
L
Ferran
Christiane
C
Logerfo
Frank W
FW
D015153:Blotting, Western; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology*; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D012333:RNA, Messenger; D034741:RNA, Small Interfering