Biomedical subjects

Ilia Fishbein

Publications and source records attributed to Ilia Fishbein.

13 recordsLinked to original sources

PubMed2010

Site-specific gene therapy for cardiovascular disease.

Gene therapy holds considerable promise for the treatment of cardiovascular disease and may provide novel therapeutic solutions for both genetic disorders and acquired pathophysiologies such as arteriosclerosis, heart failure and arrhythmias. Recombinant DNA technology and the sequencing of the human genome have made a plethora of candidate therapeutic genes available for cardiovascular diseases. However, progress in the field of gene therapy for cardiovascular disease has been modest; one of the key reasons for this limited progress is the lack of gene delivery systems for localizing gene therapy to specific sites to optimize transgene expression and efficacy. This review summarizes progress made toward the site-specific delivery of cardiovascular gene therapy and highlights selected promising novel approaches.

Animals↗

PubMed2006

Adenoviral gene vector tethering to nanoparticle surfaces results in receptor-independent cell entry and increased transgene expression.

The present studies investigated the hypothesis that affinity immobilization of replication-defective adenoviruses (Ad) on the surfaces of biodegradable nanoparticles (NP) can improve transduction through uncoupling cellular uptake from the coxsackie-adenovirus receptor (CAR). Ad was tethered to the surfaces of polylactide-based NP that were surface-activated using a photoreactive polyallylamine-benzophenone-pyridyldithiocarboxylate polymer, which enabled (via thiol chemistry) the covalent attachment of Ad-binding proteins, either the recombinant D1 domain of CAR or an adenoviral knob-specific monoclonal antibody. Gene transfer by NP-Ad complexes was studied in relation to cellular uptake as a function of cell type and the character of NP-Ad binding. NP-Ad complexes, but not Ad applied with or without control nonimmune IgG-modified NP, significantly increased green fluorescent protein reporter expression in endothelioma and endothelial and arterial smooth muscle cells (SMC) in direct correlation to the extent of NP-Ad internalization. CAR-independent uptake of NP-Ad was confirmed by demonstrating inhibition of free Ad- but not NP-Ad complex-mediated transduction by knob protein. Complexes formulated with an Ad encoding inducible nitric oxide synthase inhibited growth of cultured SMC to a significantly greater extent than those with (GFP)Ad or (NULL)Ad or free vector. It is concluded that Ad-specific affinity tethering to biodegradable NP can significantly increase the level of gene expression via a CAR-independent uptake mechanism.

Adenoviridae↗

PubMed2005

Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents.

The clinical use of metallic expandable intravascular stents has resulted in improved therapeutic outcomes for coronary artery disease. However, arterial reobstruction after stenting, in-stent restenosis, remains an important problem. Gene therapy to treat in-stent restenosis by using gene vector delivery from the metallic stent surfaces has never been demonstrated. The present studies investigated the hypothesis that metal-bisphosphonate binding can enable site-specific gene vector delivery from metal surfaces. Polyallylamine bisphosphonate (PAA-BP) was synthesized by using Michael addition methodology. Exposure to aqueous solutions of PAA-BP resulted in the formation of a monomolecular bisphosphonate layer on metal alloy surfaces (steel, nitinol, and cobalt-chromium), as demonstrated by x-ray photoelectron spectroscopy. Surface-bound PAA-BP enabled adenoviral (Ad) tethering due to covalent thiol-binding of either anti-Ad antibody or a recombinant Ad-receptor protein, D1. In arterial smooth muscle cell cultures, alloy samples configured with surface-tethered Ad were demonstrated to achieve site-specific transduction with a reporter gene, (GFP). Rat carotid stent angioplasties using metal stents exposed to aqueous PAA-BP and derivatized with anti-knob antibody or D1 resulted in extensive localized Ad-GFP expression in the arterial wall. In a separate study with a model therapeutic vector, Ad-inducible nitric oxide synthase (iNOS) attached to the bisphosphonate-treated metal stent surface via D1, significant inhibition of restenosis was demonstrated (neointimal/media ratio 1.68 +/- 0.27 and 3.4 +/- 0.35; Ad-iNOS vs. control, P < 0.01). It is concluded that effective gene vector delivery from metallic stent surfaces can be achieved by using this approach.

Adenoviridae↗

PubMed2005

Site specific gene delivery in the cardiovascular system.

Gene therapy holds great promise for treating both genetic and acquired disorders. However, progress toward effective human gene therapy has been thwarted by a number of problems including vector toxicity, poor targeting of diseased tissues, and host immune and inflammatory activity to name but a few of the challenges. Gene therapy for cardiovascular disease has been the subject of many fewer clinical trials than other disorders such as cancer or cystic fibrosis. Nevertheless, the challenges are comparable. The present paper reports a review of investigations related to our hypothesis that site specific cardiovascular gene therapy represents an approach that can lead to both optimizing efficacy and reducing the impact of gene vector-related systemic adverse effects. We report experimental studies demonstrating proof of principle in three areas: gene therapy for heart valve disease, gene delivery stents, and gene therapy to treat cardiac arrhythmias. Heart valve disease is the second most common indication for open heart surgery and is now only treatable by surgical removal or repair of the diseased heart valve. Our investigations demonstrate that gene vectors can be immobilized on the surface of prosthetic heart valve leaflets thereby enabling a therapeutic genetic modification of host cells around the valve annulus and on the leaflet. Other animal studies have shown that vascular stents used to relieve arterial obstruction can also be used as gene delivery systems to provide therapeutic vector constructs that can both locally prevent post stenting reobstruction, known as in-stent restenosis, and treat the underlying vascular disease. Cardiac arrhythmias are the cause of sudden death due to heart disease and affect millions of others on a chronic basis. Our group has successfully investigated in animal studies localized gene therapy using an ion channel mutation to treat atrial arrhythmias.

Animals↗

PubMed2005

Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries.

Local delivery of antiproliferative drugs encapsulated in biodegradable nanoparticles (NP) has shown promise as an experimental strategy for preventing restenosis development. A novel PDGFRbeta-specific tyrphostin, AGL-2043, was formulated in polylactide-based nanoparticles and was administered intraluminally to the wall of balloon-injured rat carotid and stented pig coronary arteries. The disposition and elimination kinetics within the vessel wall, as well as the antirestenotic potential of the novel drug and delivery system, were evaluated. The efficacy and the local drug elimination kinetics were affected by the size of the NP and the drug-carrier binding mode. Despite similar arterial drug levels 90 min after delivery in rats, small NP were more efficacious in comparison to large NP (90 and 160 nm, respectively). AGL-2043 selectively inhibited vascular SMC in a dose-dependent manner. The antiproliferative effect of nanoencapsulated tyrphostin was considerably higher than that of surface-adsorbed drug. In the pig model, intramural delivery of AGL-2043 resulted in reduced in-stent neointima formation in the coronary arteries over control despite similar degrees of wall injury. The results of this study suggest that locally delivered tyrphostin AGL-2043 formulated in biodegradable NP may be applicable for antirestenotic therapy independent of stent design or type of injury.

Angioplasty, Balloon, Coronary↗

PubMed2003

The incorporation of an ion channel gene mutation associated with the long QT syndrome (Q9E-hMiRP1) in a plasmid vector for site-specific arrhythmia gene therapy: in vitro and in vivo feasibility studies.

The present studies investigated the cardiac potassium channel missense mutation, Q9E-hMiRP1, for potential use as a gene therapy construct for cardiac arrhythmias. This gene abnormality is one of a number of mutations that can cause the long QT syndrome (LQTS), a hereditary arrhythmia disorder that is associated with sudden death. However, individuals who carry the Q9E-hMiRP1 variant are predisposed to developing the LQTS only after clarithromycin administration. Because the electrophysiologic mechanism of action of Q9E-hMiRP1 (i.e., diminished potassium currents resulting in delayed myocardial repolarization) is comparable to that of class III antiarrhythmic agents, we examined Q9E-hMiRP1 as a candidate gene therapy construct for site-specific treatment of reentrant atrial cardiac arrhythmias. Our rationale was also based on the hypothetical safety of the atrial use of Q9E-hMiRP1 because LQTS characteristically causes ventricular but not atrial arrhythmias. Furthermore, the possible use of clarithromycin to control the conduction effects of overexpressed Q9E-hMiRP1 pharmacologically was another attractive feature. In our studies we investigated the use of two bicistronic plasmid DNA gene vectors with either hMiRP1 or Q9E-MiRP1 and green fluorescent protein (GFP), plus a C-terminus of the hMiRP1 or of the Q9E-hMiRP1 coding region for the FLAG (MDYKDDDDK) peptide. We generated two stable cell lines using HEK293 and SH-SY5Y (human cell lines), overexpressing the genes of interest, confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots. The expected plasma membrane localization of each overexpressed transgene was confirmed by immunofluorescent confocal fluorescent microscopy using anti-FLAG antibody. Patchclamp studies demonstrated that cells transfected with Q9E-hMiRP1 plasmid DNA exhibited significantly reduced potassium currents but only with clarithromycin administration. A novel plasmid DNA delivery system was formulated for use in our animal studies of the hMiRP1 vectors, which was composed of DNA-anti-DNA antibody-cationic lipid (DAC) heteroplexes. In vitro and in vivo studies using DAC heteroplexes containing anti-DNA antibodies with nuclear targeting capability demonstrated significantly increased transfection compared to naked DNA, and to DNA-cationic lipid complexes. Pig atrial myocardial injections of DAC heteroplexes demonstrated 16% of regional cardiac myocytes transfected using the Q9E-hMiRP1 plasmid, and 15% of cells with the hMiRP1 vector. It is concluded that the present studies support the view that site-specific gene therapy for atrial arrhythmias is feasible using plasmid vectors for overexpressing ion channel mutations that have electrophysiologic effects comparable to class III antiarrhythmic agents.

Animals↗

PubMed2003

Systemic depletion of macrophages by liposomal bisphosphonates reduces neointimal formation following balloon-injury in the rat carotid artery.

OBJECTIVES: Macrophage depletion by liposomal clodronate inhibits neointimal formation after balloon-injury. The present study examined bisphosphonates (BPs) potency-effect relationship and the role of systemic versus local monocytes in vascular repair. METHODS AND RESULTS: Liposomal preparations of clodronate, pamidronate, alendronate, and ISA-13-1 inhibited RAW-264 macrophages growth in a dose-response manner. Administration to balloon-injured rats suppressed neointimal growth. Neointima to media ratio (N/M) at 14 days was reduced from 1.35 +/- 0.22 (control) to 0.4 +/- 0.1 and 0.9 +/- 0.17 by liposomal alendronate (1.5 mg/kg, i.v.) and liposomal ISA-13-1 (15 mg/kg), respectively (n = 8-10, P < 0.05). Suppression of neointimal formation was preserved at 30 days. Subcutaneous administration of liposomal BP (LBP) was also effective in suppressing neointimal formation, while short local intraluminal application had no effect. Immunostaining for ED-1 and ED-2 revealed no resident macrophages in the arterial wall, and reduced macrophage infiltration in LBP-treated animals. Arterial PDGF-B chain and PDGF-beta receptor activation were reduced in LBP-treated animals and up-regulation of the PDGF receptor was noted. CONCLUSIONS: Systemic transient inactivation of monocytes and macrophages by LBPs reduced macrophage infiltration and neointimal formation in the rat carotid injury model. The findings demonstrate a BP potency-effect relationship, and highlight the role of circulating monocytes in vascular injury and repair.

Angioplasty, Balloon↗

PubMed2002

Inhibition of aortic allograft vasculopathy by local delivery of platelet-derived growth factor receptor tyrosine-kinase blocker AG-1295.

BACKGROUND: Signal transduction through the platelet-derived growth factor (PDGF)/PDGF-receptor (PDGFR) system has been linked to vascular smooth muscle cell migration and proliferation leading to allograft vasculopathy. This study describes the effect of the tyrphostin AG-1295, a specific PDGFR tyrosine-kinase inhibitor, on neointimal formation in this disease. METHODS AND RESULTS: Rat aortic allografts transplanted from dark agouti (RT1 ) donors to Wistar-Furth (RT1 ) recipients were assessed in a new treatment model for local drug delivery from polymeric carrier matrices precoated with AG-1295. Matrices were wrapped around the graft immediately after transplantation. The recipients received no background immunosuppression. At day 80 posttransplantation, intimal thickness in AG-1295-treated grafts was reduced when compared to controls (11.8+/-9.1% intimal thickness vs. 23.7+/-6.4% intimal thickness; P=0.042). This finding corresponded to inhibition of intimal PDGFR-beta expression in AG-1295-treated grafts at day 20 posttransplantation (P =0.029 vs. allogeneic controls). CONCLUSIONS: The tyrphostin AG-1295 reduces neointimal formation in aortic allograft vasculopathy by inhibition of PDGFR-beta-triggered tyrosine phosphorylation. Local drug release of specific tyrosine-kinase inhibitors from perivascularly co-implanted polymeric carrier matrices is effective in the prophylaxis of allograft vasculopathy under selected experimental conditions.

Animals↗

PubMed2002

Study of the drug release mechanism from tyrphostin AG-1295-loaded nanospheres by in situ and external sink methods.

The present study focused on in vitro release of polylactide-nanoencapsulated tyrphostin AG-1295, a potential agent for local therapy of restenosis. The drug was formulated in matrix-type nanoparticles, termed nanospheres (NS) using the nanoprecipitation method. AG-1295 is a model for low-molecular weight lipophilic compounds, the release behavior of which cannot be adequately characterized by existing methods. An in vitro release technique suitable for optimizing the nanoparticulate formulation release behavior was developed through a novel external sink method and an in situ release method utilizing the environmental sensitivity of the AG-1295 fluorescence spectrum. Similar tendencies were demonstrated by both methods in drug release studied as a function of selected NS preparation variables. The release properties of the drug fractions varying in their binding mode to the carrier particles were studied by the external sink method. The NS surface-adsorbed drug exhibited a significantly higher release rate compared to the drug entrapped in the polymeric matrix. The in situ release of the encapsulated drug was analyzed using the diffusion models of release from a matrix-type sphere. The release was shown to be a composite process, with a burst phase attributed largely to the rapid dissociation of the surface-bound AG-1295. The diffusion-controlled phase exhibited an alteration in kinetic pattern obviously due to the drug distribution between polymeric matrix compartments differing in their permeability. Drug in vitro release investigation may be effectively used to characterize the drug-carrier interaction and internal carrier structure in nanoparticulate formulations, as well as optimize the release behavior in respect to their therapeutic application.

Chemistry, Pharmaceutical↗

PubMed2002

Lipophilic drug loaded nanospheres prepared by nanoprecipitation: effect of formulation variables on size, drug recovery and release kinetics.

The nanoprecipitation method of nanosphere preparation offers several important advantages, such as readily adjustable and reproducible carrier size in the nanometer range and use of ingredients with low toxic potential, especially important for intravascular delivery. The applicability of the method to encapsulation of strongly lipophilic drugs has not been adequately addressed to date. In this study we applied nanoprecipitation to prepare PLA nanospheres loaded with a lipophilic tyrphostin compound, AG-1295, a potent antirestenotic agent. The effect of several formulation variables on the nanosphere basic properties (carrier size, drug release rate and drug recovery yield) was investigated. The nanosphere size was shown to be readily controlled by modifying the PLA and PLA non-solvent amounts in the organic phase. Carrier size and organic solvents' elimination rate are the main determinants of the drug release rate. The stability and drug recovery yield in the formulation depend on the drug to polymer ratio. Nanoprecipitation protocol modifications were suggested to produce nanospheres combining ultrasmall size (<100 nm) with high drug recovery yield, and to reduce the surfactant amount in the formulation.

Chemical Precipitation↗

PubMed2002

Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits.

BACKGROUND: Inflammation is critical to vascular repair after mechanical injury. Excessive inflammation enhances neointimal formation and restenosis. We examined whether transient systemic inactivation of macrophages at the time of vascular intervention could attenuate the degree of expected restenosis. METHODS AND RESULTS: Liposomal clodronate (LC) inhibited the growth of cultured macrophages but had no effect on endothelial or smooth muscle cells and suppressed neointimal hyperplasia in hypercholesterolemic rabbits and rats after intravenous administration of LC, with no adverse effects. LC treatment reduced the number of blood monocytes and decreased macrophage infiltration in the injured arteries as well as smooth muscle cell proliferation, interleukin-1beta transcription, and production and matrix metalloproteinase-2 activity. CONCLUSIONS: Macrophages play a pivotal role in vascular repair after mechanical arterial injury. Systemic inactivation and depletion of monocytes and macrophages by LC reduce neointimal hyperplasia and restenosis.

Animals↗

PubMed2002

Metalloproteinase inhibitor attenuates neointima formation and constrictive remodeling after angioplasty in rats: augmentative effect of alpha(v)beta(3) receptor blockade.

Release of matrix metalloproteinases (MMP) from smooth muscle and foam cells following arterial injury facilitates cell migration, neointimal hyperplasia, and vessel wall remodeling. Inhibition of MMP activity using the hydroxamate, zinc-chelating mimicers of collagen, Batimastat and Marimastat, has shown efficacy in reducing constrictive vascular remodeling 6 weeks after experimental angioplasty but not intimal hyperplasia. Vitronectin receptor (alpha(v)beta(3)) blockade interferes with binding of this integrin to MMP-2 and proteolyzed collagen, thereby reducing cell invasion. This study tests the effect of MMP inhibition, with and without vitronectin receptor (alpha(v)beta(3)) blockade, on neointima formation and arterial remodeling in a long-term model (up to 212 months) of balloon injury in vivo. Male Sabra rats were treated with Batimastat (BB-94, British Biotech Pharmaceuticals Ltd., 30 mg/kg, intraperitoneally) and/or the alpha(v)beta(3) receptor inhibiting RGD peptide, G-Pen-GRGDSPCA (GIBCO BRL, 0.1 micromol), administered as a perivascular gel to the common carotid artery after balloon injury. Animals were sacrificed 3, 14, 25, and 75 days (n=21, 23, 22, and 21) after injury. Animals treated with BB-94, peptide, or both had markedly increased absolute luminal area with markedly reduced luminal cross-sectional-area narrowing by neointima and intima-to-media area ratio at all time points except for 3 days after balloon injury versus non-treated, ballooned animals. Combined treatment was significantly more effective than either one alone. Constrictive remodeling, most marked 212 months after balloon injury, was prevented at this time point in all treated animals. The pattern of reduction in luminal narrowing, neointimal formation, and constrictive remodeling across treatment groups correlated very significantly with the reduction in tissue MMP activity as determined by zymography at 3 days. Confirmation of the efficacy of this strategy in larger animals should be the next step toward testing the applicability of this novel approach to the interventional setting.

Angioplasty, Balloon↗

PubMed2002

Activated polyurethane modified with latent thiol groups.

A novel type of modified polyurethane with pendant acetylthio groups (as a latent form of thiol groups) has been proposed for the use in surface modifications with various biomolecules. The polymer was prepared via a modified variant of low-temperature bromoalkylation of urethane hard segments followed by the reaction of pendant bromoalkyl groups with thiolacetic acid in mild conditions. The extent of modification with acetylthio groups can be made as high as 0.45 mmol/g. After deprotection of acetylthio groups and reaction of the resulting thiol groups with an excess of Ellman's reagent, 0.1 nmol/cm(2) of thiol-reactive 3-carboxy-4-nitrophenyldithio groups were detected on the surface of films cast from the modified polymer. A sensitive fluorescent probe--dansyl-L-cysteine was used for the quantification of thiol-reactive groups bound to the surface. The acetylthio-modified polyurethane is sufficiently stable to withstand conditions typical for the high-temperature processing (molding, extrusion) of polyurethanes.

Cysteine↗