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

Cardiac Plasticity of Multipotent Cells Derived from the Human Subcutaneous Adipose Tissue

Einleitung: Ein Myokardinfarkt führt zum Untergang von kontraktilem Gewebe. Auf der Suche nach alternativen Ansätzen für die Behandlung des akuten Myokardinfarkts bietet die Stammzell- und Gentherapie interessante Möglichkeiten. Ziel der vorliegenden Arbeit war es eine kardiomyozytäre Zellfraktion aus dem humanen subkutanen Fettgewebe zu isolieren. Methoden: Unter Verwendung von klonierten lentiviralen Vektoren in denen die kardialen Promotoren pNkx2.5 und pMLC-2v die Expression der Reportergene eGFP und DsRed2 kontrollierten, wurden die humanen ADSCs infiziert. Die infizierten ADSCs wurden durch FACS fraktioniert und unter anderem mittels RT-PCR, Immunzytochemie, Calcium-Imaging, Patch-Clamp und Zellzyklusanalysen charakterisiert. Des Weiteren wurde der Effekt einer Inkubation mit 5-Azacytidine auf die Nkx2.5-gekoppelte eGFP-Expression untersucht. Ergebnisse: Es zeigte sich, dass ein bestimmter Subtyp von eGFPpos/DsRed2pos- Zellen zwischen 0,7-2,5% der ADSCs ausmachte. Diese Zellen zeigten ein von den anderen ADSC-Fraktionen unterschiedliches Genexpressionsprofil. Sie wiesen das höchste Expressionsniveau der kardialen Marker Nkx2.5 und MLC-2v auf. Als einzige Subpopulation exprimierten sie Troponin I. Diese Ergebnisse konnten durch immunzytochemische Analysen bestätigt werden. Die eGFPpos/DsRed2pos- Zellen konnten in vitro expandiert werden, ohne ihre kardiomyozytäre Genexpression einzubüßen. Die molekularen Marker quergestreifter Muskulatur MyoD und Myogenin wurden nicht nachgewiesen. Eine skelettmuskuläre Differenzierung ist somit unwahrscheinlich. Die Präsenz von spannungsabhängigen L-Typ-Calcium- und Kaliumkanälen wurde durch RT-PCR, Immunzytochemie, Calcium-Imaging und Patch Clamp-Analysen bestätigt. Die durchgeführte Zellzyklusanalyse ergab, dass der Großteil der eGFPpos /DsRed2pos-Zellen den Mitosemarker Ki-67 exprimierte. Zwischen 15-25% der eGFPpos/DsRed2pos-Zellen befanden sich in der S-Phase des Zellzyklus. Die Induktionsversuche mit 5-Azacytidine resultierten in einer dosisabhängigen, signifikanten Verringerung der Nkx2.5-gekoppelten eGFP-Exprimierung. Bemerkenswerterweise war die eGFP-Expression ohne 5-Azacytidine-Zusatz im Medium am höchsten ausgeprägt. Diskussion: Die vorgelegten Daten konnten zeigen, dass das humane subkutane Fettgewebe eine Fraktion von spontan differenzierenden kardiomyozytären Zellen enthält, welche mehrere wesentliche Charakteristika von adulten Kardiomyozyten aufwiesen. Unser Ansatz des Gentransfers liefert möglicherweise ein System zur Isolierung von geeigneten humanen adulten kardiomyozytären Zellen für die Therapie des akuten Myokardinfarkts.Introduction: Myocardial infarction (MI) leads to a loss in cardiac tissue. Cardiac repair using cellular therapies is one potential conceivable new therapeutic option. In this study we investigated the potential of human adipose-derived stem cells (ADSCs) to differentiate into cardiomyogenic cells. Methods: For analytical purposes we cloned two HIV-based lentiviral vectors in which Nkx2.5 and MLC-2v promoters controlled the expression of the fluorescent dyes eGFP and DsRed2. ADSCs were infected with both lentiviral vectors simultaneously and 48-72h later analyzed by FACS. EGFPpos/DsRed2pos-cells were investigated using e.g. RT-PCR, immunohistochemistry, Fura-2-calcium imaging, patch clamp and cell cycle analysis. Results: Between 0.7-2.5% among regular ADSCs expressed eGFP and DsRed2 simultaneously. Sorted eGFPpos/DsRed2pos-cells expressed cardiomyocyte-specific mRNAs including: Nkx2.5, MLC-2v, GATA-4, Troponin I and L-type calcium channel alpha-1c subunit (Cav1.2). The skeletal markers MyoD, Myogenin were not detected. 15-20% of eGFPpos/DsRed2pos-cells showed L-type calcium channel specific response and patch clamp analysis revealed cardiac potassium channel presence in some cells. Cell cycle analysis showed that 20% of the cells were in the S-phase and more than 60% expressed mitosis marker Ki-67. Conclusion: Our studies indicate that spontaneously transdifferentiating cardiomyogenic cells can be isolated from ADSCs in vitro using lentiviral fluorescent indicators and FACS. The isolated cells did not resemble a fully differentiated cardiac phenotype yet showed several characteristics of adult cardiomyocytes

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

Instant tough bioadhesive with triggerable benign detachment

© 2020 National Academy of Sciences. All rights reserved. Bioadhesives such as tissue adhesives, hemostatic agents, and tissue sealants have potential advantages over sutures and staples for wound closure, hemostasis, and integration of implantable devices onto wet tissues. However, existing bioadhesives display several limitations including slow adhesion formation, weak bonding, low biocompatibility, poor mechanical match with tissues, and/or lack of triggerable benign detachment. Here, we report a bioadhesive that can form instant tough adhesion on various wet dynamic tissues and can be benignly detached from the adhered tissues on demand with a biocompatible triggering solution. The adhesion of the bioadhesive relies on the removal of interfacial water from the tissue surface, followed by physical and covalent cross-linking with the tissue surface. The triggerable detachment of the bioadhesive results from the cleavage of bioadhesive's crosslinks with the tissue surface by the triggering solution. After it is adhered to wet tissues, the bioadhesive becomes a tough hydrogel with mechanical compliance and stretchability comparable with those of soft tissues. We validate in vivo biocompatibility of the bioadhesive and the triggering solution in a rat model and demonstrate potential applications of the bioadhesive with triggerable benign detachment in ex vivo porcine models

Implications of Mitochondrial Dysfunction for the Anesthetic and Perioperative Management:A Case Report of Spinal Fusion, Genetic Confusion, and a Patient's Perspective

We describe a patient's personal struggle with a symptom complex consisting of profound muscle weakness requiring pyridostigmine, and metabolic abnormalities suggestive of mitochondrial disease. This included a profound sensitivity to opioids, which in the past caused severe respiratory depression during a prior hospital admission. Interestingly, the patient herself is a professor of ethics in genomic sciences, and she and her medical team thus far have not been able to formally diagnose her with mitochondrial disease. The patient now presented for a multilevel lumbar spine fusion and her hospital course and perspective on her medical odyssey are described here

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