Document Detail


Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference.
MedLine Citation:
PMID:  22842581     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: RNAi technology is a promising tool for gene therapy of vascular disease. However, the biological heterogeneity between endothelial (EC) and vascular smooth muscle cells (SMC) and within different vascular beds make them differentially susceptible to siRNA. This is further complicated by the task of choosing the right transfection reagent that leads to consistent gene silencing across all cell types with minimal toxicity. The goal of this study was to investigate the intrinsic RNAi susceptibility of primary human aortic and coronary artery endothelial and vascular smooth muscle cells (AoEC, CoEC, AoSMC and CoSMC) using adherent cell cytometry.
METHODS: Cells were seeded at a density of 5000cells/well of a 96well plate. Twenty four hours later cells were transfected with either non-targeting unlabeled control siRNA (50nM), or non-targeting red fluorescence labeled siRNA (siGLO Red, 5 or 50nM) using no transfection reagent, HiPerFect or Lipofectamine RNAiMAX. Hoechst nuclei stain was used to label cells for counting. For data analysis an adherent cell cytometer, Celigo was used.
RESULTS: Red fluorescence counts were normalized to the cell count. EC displayed a higher susceptibility towards siRNA delivery than SMC from the corresponding artery. CoSMC were more susceptible than AoSMC. In all cell types RNAiMAX was more potent compared to HiPerFect or no transfection reagent. However, after 24h, RNAiMAX led to a significant cell loss in both AoEC and CoEC. None of the other transfection conditions led to a significant cell loss.
CONCLUSION: This study confirms our prior observation that EC are more susceptible to siRNA than SMC based on intracellular siRNA delivery. RNAiMax treatment led to significant cell loss in AoEC and CoEC, but not in the SMC populations. Additionally, this study is the first to demonstrate that coronary SMC are more susceptible to siRNA than aortic SMC.
Authors:
Christoph S Nabzdyk; Maggie Chun; Leena Pradhan Nabzdyk; Shun Yoshida; Frank W LoGerfo
Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, N.I.H., Extramural     Date:  2012-07-25
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  425     ISSN:  1090-2104     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-08-27     Completed Date:  2012-12-18     Revised Date:  2013-08-27    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  261-5     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
Affiliation:
Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. cnabzdyk@tuftsmedicalcenter.org
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MeSH Terms
Descriptor/Qualifier:
Aorta / cytology*
Cell Adhesion
Cell Count
Coronary Vessels / cytology*
Endothelium, Vascular / metabolism*
Flow Cytometry
Fluorescence
Humans
Muscle, Smooth, Vascular / cytology,  metabolism*
Myocytes, Smooth Muscle / metabolism*
RNA Interference*
RNA, Small Interfering / genetics,  metabolism*
Transfection*
Grant Support
ID/Acronym/Agency:
5T32 HL007734/HL/NHLBI NIH HHS; R01 HL021796/HL/NHLBI NIH HHS; R01 HL021796-26/HL/NHLBI NIH HHS; R01 HL086741/HL/NHLBI NIH HHS; R01 HL086741-04/HL/NHLBI NIH HHS; T32 HL007734/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/RNA, Small Interfering

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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