Differential susceptibility of human primary aortic and coronary artery vascular cells to RNA interference
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.
Available from: Leena Pradhan
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ABSTRACT: a b s t r a c t An excessive tissue response to prosthetic arterial graft material leads to intimal hyperplasia (IH), the leading cause of late graft failure. Seroma and abnormal capsule formation may also occur after pros-thetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation. This study evaluates the uptake and release of small interfering RNA (siRNA) from unmodified and surface functionalized electrospun PET graft materials. ePET graft materials were synthesized using electrospinning technology. Subsets of the ePET materials were then chemically modified to create surface functional groups. Unmodified and surface-modified ePET grafts were dip-coated in siRNAs alone or siRNAs complexed with transfection reagents poly-ethyleneimine (PEI) or Lipofectamine RNAiMax. Further, control and TSP-2 siRNAePEI complex treated ePET samples were placed onto a confluent layer of human aortic smooth muscle cells (AoSMCs). Complexation of all siRNAs with PEI led to a significant increase in adsorption to unmodified ePET. TSP-2 siRNA-PEI released from unmodified-ePET silenced TSP-2 in AoSMC. Regardless of the siRNAePEI complex evaluated, AoSMC migrated into the ePET. siRNAePEI complexes delivered to AoSMC from dip-coated ePET can result in gene knockdown. This methodology for siRNA delivery may improve the tissue response to vascular and other prosthetics.
Biomaterials 01/2014; DOI:10.1016/j.biomaterials.2013.12.026 · 8.56 Impact Factor
Available from: Christoph S. Nabzdyk
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ABSTRACT: Atherosclerosis (ATH) and aortic aneurysms (AA) remain challenging chronic diseases that confer high morbidity and mortality despite advances in medical, interventional, and surgical care. RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH and AA. Despite positive results in preclinical and some clinical feasibility studies, challenges such as target/sequence validation, tissue specificity, transfection efficiency, and mitigation of unwanted off-target effects remain to be addressed. In this review the most current targets and some novel approaches in siRNA delivery are being discussed. Due to the plethora of investigated targets, only studies published between 2010 and 2014 were included.
Discovery medicine 05/2014; 17(95):233-46. · 3.63 Impact Factor
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ABSTRACT: Restenosis is pathophysiological process occurring in 10-15% of patients submitted to revascularization procedures of coronary, carotid and peripheral arteries. It can be considered as an excessive healing reaction of the vascular wall submitted to arterial/venous bypass graft interposition, endarterectomy or angioplasty. The advent of bare metal stents, drug-eluting stents and of the more recent drug-eluting balloons, significantly reduced but not eliminated the incidence of restenosis, which remains a clinically relevant problem.
Biomedical research in preclinical animal models of (re)stenosis, despite its limitations, enormously contributed to the identification of processes involved in restenosis progression, going well beyond the initial dogma of a primarily proliferative disease.
Although the main molecular and cellular mechanisms underlying restenosis have been well described, new signalling molecules and cell types controlling the progress of restenosis are continuously discovered. In particular, microRNAs and vascular progenitor cells recently revealed a key role in this pathophysiological process. Also, the advanced, highly-sensitive highthroughput analyses of molecular alterations at transcriptome, proteome and metabolome level occurring in injured vessels in animal models of disease and in human specimens, are serving as a basis to identify novel potential therapeutic targets for restenosis. Molecular analyses are also contributing to the identification of reliable circulating biomarkers predictive of post-interventional restenosis in patients, that could be potentially helpful in the establishment of an early diagnosis and therapy.
This review summarizes the most recent and promising therapeutic strategies identified in experimental models of (re)stenosis and potentially translatable in patients submitted to revascularization procedures.
Clinical science 12/2014; 127(11). DOI:10.1042/CS20140131 · 5.63 Impact Factor
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