Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo
| Autor: | Frank W. LoGerfo, Christiane Ferran, Monica Jain, Atish Chopra, Leena Pradhan, Junaid Y. Malek, Thomas S. Monahan, Nicholas D. Andersen |
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| Rok vydání: | 2010 |
| Předmět: |
Small interfering RNA
Endothelium Genetic enhancement Transfection Article Tissue Culture Techniques 03 medical and health sciences 0302 clinical medicine RNA interference medicine Humans Gene silencing Saphenous Vein Gene Silencing RNA Small Interfering 030304 developmental biology Air Pressure 0303 health sciences Gene knockdown business.industry Anatomy Molecular biology Endothelial stem cell medicine.anatomical_structure 030220 oncology & carcinogenesis RNA Interference Surgery Endothelium Vascular business Cardiology and Cardiovascular Medicine Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) |
| Zdroj: | Journal of Vascular Surgery. 52(6):1608-1615 |
| ISSN: | 0741-5214 |
| DOI: | 10.1016/j.jvs.2010.06.169 |
| Popis: | BackgroundEndothelial 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.MethodsControl 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.ResultsDistending 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.ConclusionEndothelial 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.Clinical RelevanceVein bypass graft failure due to intimal hyperplasia and restenosis continues to be an obstacle to long-term vein graft durability. Currently, there are no agents available that can be applied to vein grafts to reduce the rate of failure. This work demonstrates the feasibility of intraoperative siRNA therapeutics directed at the endothelium. If developed further, siRNA cocktails could be designed that provide a protective effect by silencing endothelial gene expression that leads to intimal hyperplasia. In addition, endothelial gene silencing could be used to induce favorable effects on the vasculature in other realms of vascular surgery. |
| Databáze: | OpenAIRE |
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