title: Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo.
abstract: 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.
authors.affiliation: Department of Vascular Surgery Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Andersen
authors.forename: Atish
authors.initials: A
authors.lastname: Chopra
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Monahan
authors.forename: Junaid Y
authors.initials: JY
authors.lastname: Malek
authors.forename: Monica
authors.initials: M
authors.lastname: Jain
authors.forename: Leena
authors.initials: L
authors.lastname: Pradhan
authors.forename: Christiane
authors.initials: C
authors.lastname: Ferran
authors.forename: Frank W
authors.initials: FW
authors.lastname: LoGerfo
mesh_terms: 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
chemical_list: D034741:RNA, Small Interfering; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
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