ABSTRACT
Both atherosclerosis and arterial interventions induce oxidative stress mediated in part by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that have a pivotal role in the development of neointimal hyperplasia and restenosis. For small interfering RNA (siRNA) targeting of the NOX2 (Cybb) component of the NADPH oxidase to prevent restenosis, gene transfer with viral vectors is effective, but raises safety issues in humans. We developed a new approach using the amino-acid-based nanoparticle HB-OLD7 for local delivery of siRNA targeting NOX2 to the arterial wall. siRNA-nanoparticle complexes were transferred into the regional carotid artery walls after angioplasty in an atherosclerotic rat model. Compared with angioplasty controls, Cybb gene expression (measured by quantitative reverse transcriptase-PCR) in the experimental arterial wall 2 weeks after siRNA was reduced by >87%. The neointima-to-media-area ratio was decreased by >83%, and the lumen-to-whole-artery area ratio was increased by >89%. Vital organs showed no abnormalities and splenic Cybb gene expression showed no detectable change. Thus, local arterial wall gene transfer with HB-OLD7 nanoparticles provides an effective, nonviral system for efficient and safe local gene transfer in a clinically applicable approach to knock down an NADPH oxidase gene. Local arterial knockdown of the Cybb gene significantly inhibited neointimal hyperplasia and preserved the vessel lumen without systemic toxicity.
Subject(s)
Atherosclerosis/therapy , Membrane Glycoproteins/antagonists & inhibitors , NADPH Oxidases/antagonists & inhibitors , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , Animals , Atherosclerosis/genetics , Atherosclerosis/pathology , Genetic Vectors/administration & dosage , Hyperplasia/metabolism , Hyperplasia/pathology , Male , Membrane Glycoproteins/genetics , Mice , Models, Animal , NADPH Oxidase 2 , NADPH Oxidases/genetics , Neointima/metabolism , Neointima/pathology , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , RecurrenceABSTRACT
A highly purified erythropoietin (ESF) preparation (12,000 units per milligram of protein) was labeled with Na'125I using the Chloramine-T method. Undamaged immunoreative labeled ESF was separated from the damaged, nonimmunologically receiveESF by Sephadex G-150 fractionation. This undamaged immunreactive ESF was usedin radioimmunoassay for human erythropoietin. Separation of bound from free antigen was acheived using the double-antibody technique. Approximately 55 per cent binding wasobserved at an antiserum dilution of 1:1500. This assay appears to be sensitive enough to detect as little as 0.025 milliunits of the International Reference Preparation erythropoietin. The estimated levels of thid hormone in normal and anemic uremic human subjects suggests that immunoreactive serum erythropoietin levels are elevated above normal in anemia of uremia.
