Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference

Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 07/2012; 425(2):261-5. DOI: 10.1016/j.bbrc.2012.07.078
Source: PubMed


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.
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.
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.
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.

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    • "However, it could be possible that this is a cell-specific effect. We have previously reported differential susceptibility of endothelial and smooth muscle cells to different transfection reagents[23,24]. In the end, the transcriptome of a commonly used control for RNAi experiments is no longer representative of the transcriptome of untreated cells. "
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