Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo.
BACKGROUND
Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room.


METHODS
Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mm Hg hyperbaric pressure, or (iii) 120 mm Hg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry.


RESULTS
Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared with hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods.


CONCLUSIONS
Endothelial gene silencing is possible within the time frame and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease.
Andersen
Nicholas D
ND
Department of Vascular Surgery Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.
Chopra
Atish
A
Monahan
Thomas S
TS
Malek
Junaid Y
JY
Jain
Monica
M
Pradhan
Leena
L
Ferran
Christiane
C
LoGerfo
Frank W
FW
D000399:Air Pressure; D004730:Endothelium, Vascular / Q000166:cytology*; D020868:Gene Silencing; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012501:Saphenous Vein / Q000166:cytology; D046509:Tissue Culture Techniques; D014162:Transfection
D034741:RNA, Small Interfering; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
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