The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection.

Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery.

Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

Background: Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure.

Perivascular CLICK-gelatin delivery of thrombospondin-2 small interfering RNA decreases development of intimal hyperplasia after arterial injury.

Bypass graft failure occurs in 20%-50% of coronary and lower extremity bypasses within the first-year due to intimal hyperplasia (IH). TSP-2 is a key regulatory protein that has been implicated in the development of IH following vessel injury. In this study, we developed a biodegradable CLICK-chemistry gelatin-based hydrogel to achieve sustained perivascular delivery of TSP-2 siRNA to rat carotid arteries following endothelial denudation injury.

Integrated single-nuclei and spatial transcriptomic analysis reveals propagation of early acute vein harvest and distension injury signaling pathways following arterial implantation.

Background: Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF.

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.

Lifelong limb preservation: A patient-centered description of lower extremity arterial reconstruction outcomes.

Background: Life expectancy is short for patients with critical limb ischemia (CLI), many of whom may fear amputation more than death. In light of the reduced life expectancy of these patients, the traditional 5-year freedom from amputation (FFA) statistic may not accurately address their concern. We developed a more relevant patient-centered calculation of major amputation risk during a patient's remaining lifetime to better answer the question, Will I ever lose my leg?

Methods: We identified all limbs undergoing first-time intervention for CLI in a large institutional database from 2005 to 2013.

Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA.

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".

pH-responsive scaffolds generate a pro-healing response.

A principal challenge in wound healing is a lack of cell recruitment, cell infiltration, and vascularization, which occurs in the absence of temporal and spatial cues. We hypothesized that a scaffold that expands due to local changes in pH may alter oxygen and nutrient transport and the local cell density, leading to enhanced cell deposition and survival. In this study, we present a pH-responsive scaffold that increases oxygen transport, as confirmed by our finite element model analysis, and cell proliferation relative to a non-responsive scaffold.

Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.

Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds.

Current siRNA targets in the prevention and treatment of intimal hyperplasia.

Intimal hyperplasia (IH) is the leading cause of late vein and prosthetic bypass graft failure. Injury at the time of graft implantation leading to the activation of endothelial cells and dedifferentiation of vascular smooth muscle cells to a synthetic phenotype are known causes of IH. Prior attempts to develop therapy to mitigate these cellular changes to prevent IH and graft failure have failed.

Current siRNA targets in atherosclerosis and aortic aneurysm.

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.

Gene silencing in human aortic smooth muscle cells induced by PEI-siRNA complexes released from dip-coated electrospun poly(ethylene terephthalate) grafts.

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 prosthetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation.

Expression of neuropeptides and cytokines in a rabbit model of diabetic neuroischemic wound healing.

Objective: The present study is designed to understand the contribution of peripheral vascular disease and peripheral neuropathy to the wound-healing impairment associated with diabetes. Using a rabbit model of diabetic neuroischemic wound healing, we investigated rate of healing, leukocyte infiltration, and expression of cytokines, interleukin-8 and interleukin-6, and neuropeptides, substance P, and neuropeptide Y.

Methods: Diabetes was induced in New Zealand White rabbits by administering alloxan while control rabbits received saline.

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

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.

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.

Expression of Neuropeptide Y, Substance P, and their receptors in the right atrium of diabetic patients.

Objective: To investigate the expression of neuropeptides and their receptors that play a role in cardiac homeostasis in the right atrium of nondiabetic and diabetic patients undergoing coronary artery bypass graft surgery.

Background: The cardioactive neuropeptides and their receptors investigated in this study were Neuropeptide Y (NPY), and its receptors, NPY Receptor1 (NPY1R), NPY Receptor2 (NPY2R), NPY Receptor5 (NPY5R) and Substance P (SP) and its receptor, Neurokinin1R (NK1R).

Methods: The gene and protein expression of NPY, NPY1R, NPY2R, NPY5R, SP and NK1R from the atrial tissue of 10 nondiabetic and diabetic patients undergoing coronary artery bypass grafting (CABG) was assessed by Q-RTPCR, immunohistochemistry, Western blot, and ELISA.

Thrombospondin-2 gene silencing in human aortic smooth muscle cells improves cell attachment.

Background: Despite decades of research, anastomotic intimal hyperplasia remains a major cause of delayed prosthetic arterial graft failure. Previously, we reported profound upregulation of thrombospondin-2 (TSP-2) mRNA in neointimal smooth muscle cells after prosthetic arterial bypass graft placement. TSP-2 is an antiangiogenic matricellular protein with specific functions yet unknown.

Diabetic neuropathy and heart failure: role of neuropeptides.

Cardiovascular autonomic neuropathy (CAN), in which patients present with damage of autonomic nerve fibres, is one of the most common complications of diabetes. CAN leads to abnormalities in heart rate and vascular dynamics, which are features of diabetic heart failure. Dysregulated neurohormonal activation, an outcome of diabetic neuropathy, has a significant pathophysiological role in diabetes-associated cardiovascular disease.

Effect of hyperglycemia and neuropeptides on interleukin-8 expression and angiogenesis in dermal microvascular endothelial cells.

Background: Impaired wound healing is a major complication associated with diabetes, involving a dysregulation and impairments in the inflammatory and angiogenic phases of wound healing. Here, we examine the effects of the neuropeptides substance P (SP) and neuropeptide Y (NPY) on dermal microvascular endothelial cell (DMVEC) angiogenesis and interleukin-8 (IL-8) expression, a known effector of the neuropeptide pathways in normal and hyperglycemic conditions in vitro.

Methods: DMVECs are treated with one of four glucose concentrations: 1) 5 mM glucose; 2) 10 mM glucose; 3) 30 mM glucose; or 4) 30 mM mannitol and cotreated with 100 nM NPY, 100 nM SP, or 10 ng/mL IL-8.

High throughput RNAi assay optimization using adherent cell cytometry.

Background: 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).

Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo.

Background: Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room.

Methods: Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mm Hg hyperbaric pressure, or (iii) 120 mm Hg luminal distending pressure.

Use of textile dyeing technology to create an infection-resistant functionalized polyester biomaterial.

Infection is a major complication when utilizing implantable devices. The purpose of this study was to create a functionalized polyethylene terephthalate (polyester) biomaterial with sustained antimicrobial properties using textile-dyeing technology. Polyester was hydrolyzed via exposure to sodium hydroxide (NaOH) to provide two functional sites within the polymeric backbone.