RNAi therapy to the wall of arteries and veins: anatomical, physiologic, and pharmacological considerations

Background: Cardiovascular disease remains a major health care challenge. The knowledge about the underlying mechanisms of the respective vascular disease etiologies has greatly expanded over the last decades. This includes the contribution of microRNAs, endogenous non-coding RNA molecules, known to vastly influence gene expression. In addition, short interference RNA has been established as a mechanism to temporarily affect gene expression. This review discusses challenges relating to the design of a RNA interference therapy strategy for the modulation of vascular disease. Despite advances in medical and surgical therapies, atherosclerosis (ATH), aortic aneurysms (AA) are still associated with high morbidity and mortality. In addition, intimal hyperplasia (IH) remains a leading cause of late vein and prosthetic bypass graft failure. Pathomechanisms of all three entities include activation of endothelial cells (EC) and dedifferentiation of vascular smooth muscle cells (VSMC). RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH, AA or IH. Successful RNAi delivery to the vessel wall faces multiple obstacles. These include the challenge of cell specific, targeted delivery of RNAi, anatomical barriers such as basal membrane, elastic laminae in arterial walls, multiple layers of VSMC, as well as adventitial tissues. Another major decision point is the route of delivery and potential methods of transfection. A plethora of transfection reagents and adjuncts have been described with varying efficacies and side effects. Timing and duration of RNAi therapy as well as target gene choice are further relevant aspects that need to be addressed in a temporo-spatial fashion. Conclusions: While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. It might be necessary to achieve simultaneous and/or sequential silencing of multiple, synergistically acting target genes. Some advances in cell specific RNAi delivery have been made, but a reliable vascular cell specific transfection strategy is still missing. Also, off-target effects of RNAi and unwanted effects of transfection agents on gene expression are challenges to be addressed. Close collaborative efforts between clinicians, geneticists, biologists, and chemical and medical engineers will be needed to provide tailored therapeutics for the various types of vascular diseases

The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells

Background: RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a “noncoding RNAi”. However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). Results: Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response. Conclusions: Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and “non coding” siRNA is a hindrance in the development of siRNA based therapeutics. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2267-9) contains supplementary material, which is available to authorized users

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background: To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing. Methods: We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing. Results: All EPC phenotypes except the kinase insert domain receptor (KDR)+CD133+ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34+KDR+ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding. Conclusions: Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34+KDR+ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models

Temporal Network Based Analysis of Cell Specific Vein Graft Transcriptome Defines Key Pathways and Hub Genes in Implantation Injury

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12–24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention

High throughput RNAi assay optimization using adherent cell cytometry

<p>Abstract</p> <p>Background</p> <p>siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC).</p> <p>Methods</p> <p>AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo^®was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell.</p> <p>Results</p> <p>After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs.</p> <p>Conclusion</p> <p>This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate <it>in vitro </it>cell assays and thus save costs.</p

Development of a Composite Electrospun Polyethylene Terephthalate-Polyglycolic Acid Material: Potential Use as a Drug-Eluting Vascular Graft

Intimal hyperplasia (IH), an excessive wound healing response of an injured vessel wall after bypass grafting, typically leads to prosthetic bypass graft failure. In an approach to ameliorate IH, nondegradable poly(ethylene terephthalate) or PET, which has been used in prosthetic vascular grafts for over 60 years, and biodegradable poly(glycolic acid) or PGA were electrospun using different techniques to generate a material that may serve as permanent scaffold and as a drug/biologic delivery device. PET and PGA polymers were electrospun from either a single-blended solution (ePET/ePGA-s) or two separate polymer solutions (ePET/ePGA-d). ePET/ePGA-d material revealed two distinct fibers and was significantly stronger than the single fiber ePET/ePGA-s material. After 21 days of incubation in PBS, ePET-PGA-s showed fiber strand breaks likely due to the degradation of the PGA within the ePET-ePGA-s fiber, while the ePET/ePGA-d material showed intact ePET fibers even after ePGA fiber degradation. The ePET/ePGA- material was able to release red fluorescent dye for at least 14 days. Attachment of human aortic smooth muscle cells (AoSMCs) was similar to both materials. ePET/ePGA-d materials maybe a step towards bypass graft materials that can be custom-designed to promote cellular attachment while serving as a drug delivery platform for IH prevention