Robert S. Meidell

Echocardiographic Destruction of Albumin Microbubbles Directs Gene Delivery to the Myocardium

BACKGROUND The noninvasive, tissue-specific delivery of therapeutic agents to the heart would be a valuable clinical tool. This study addressed the hypothesis that albumin-coated microbubbles could be used to effectively deliver an adenoviral transgene to rat myocardium by ultrasound-mediated microbubble destruction. METHODS AND RESULTS Recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. These bubbles were infused into the jugular vein of rats with or without simultaneous echocardiography. Additional controls included ultrasound of microbubbles that did not contain virus, virus alone, and virus plus ultrasound. One group underwent ultrasound-mediated destruction of microbubbles followed by adenovirus infusion. Rats were killed after 4 days and examined for beta-galactosidase expression. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed nuclear staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside substrate, indicating expression of the transgene. None of the control animals showed myocardial expression of the beta-galactosidase transgene. By quantitative analysis, beta-galactosidase activity was 10-fold higher in the treated group than in controls (P<0.0001). CONCLUSIONS Ultrasound-mediated destruction of albumin-coated microbubbles is a promising method for the delivery of bioactive agents to the heart.

Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene.

BACKGROUND Prostacyclin is an important vasoprotective molecule. It inhibits platelet aggregation, monocyte interaction with endothelium, and smooth muscle cell lipid accumulation. Vascular cyclooxygenase-1 (COX-1) is the rate-limiting step in prostacyclin synthesis. The objective of this study was to determine whether adenovirus-mediated transfer of COX-1 could restore COX-1 activity, augment prostacyclin synthesis, and prevent thrombus formation in a porcine carotid angioplasty model. METHODS AND RESULTS Human COX-1 cDNA driven by a cytomegalovirus promoter was constructed into a replication-defective adenovirus 5 vector by homologous recombination. Recombinant adenovirus without a foreign gene (Ad-RR) and buffer were included as controls. Recombinant Ad-LacZ was used for marking the transfected cells in vivo. In the in vitro experiments, cultured human endothelial cells (ECs) and porcine arterial smooth muscle cells (SMCs) were incubated with Ad-COX-1 for 2 hours and 6-keto-PGF(1 alpha) level and the transgene expression were determined 72 hours after infection. In the in vivo experiments, recombinant adenoviruses were directly instilled into angioplasty-injured porcine carotid arteries for 30 minutes. Cyclic flow changes were monitored for 10 days and thrombus formation was examined histologically thereafter. Transgene expression and prostaglandin I2 (PGI2) synthesis by the injured arteries were determined. Cultured ECs infected with Ad-COX-1 produced a fivefold to eightfold increase in PGI2, and the transgene expression in cultured porcine SMCs was demonstrated by Northern analysis. Direct administration of Ad-COX-1 at a dose of 3 x 10(10) pfu completely inhibited carotid cyclic flow changes and thrombus formation accompanied by a fourfold increase in PGI2 synthesis by the injured arteries 10 days after infection, whereas Ad-COX-1 at a lower dose, 5 x 10(9) pfu, had no antithrombotic effects when compared with Ad-RR vector and buffer controls. CONCLUSIONS Adenovirus-mediated transfer of COX-1 to angioplasty-injured carotid arteries was efficacious in augmenting PGI2 synthesis and was associated with an inhibition of thrombosis when a relatively high titer of adenovirus was instilled.

Genetic modification of the vessel wall. Comparison of surgical and catheter-based techniques for delivery of recombinant adenovirus.

BACKGROUND Gene transfer can potentially alter vessel wall biology and intervene in the pathogenesis of human disease. Although several methods for vector delivery have been described, systematic comparisons of these methods are unavailable. Therefore, this study compared three catheter-based strategies and a surgical technique to assess efficient and selective gene transfer to the vascular wall. METHODS AND RESULTS The common carotid arteries and internal jugular veins of New Zealand White rabbits were infected with recombinant adenovirus encoding either firefly luciferase or a nuclear-localizing variant of beta-galactosidase. Delivery of recombinant virus was achieved by one of four methods: (1) instillation within a surgically isolated vessel segment (dwell), (2) a double-balloon catheter, (3) a perforated balloon catheter (Wolinsky), or (4) an angioplasty balloon catheter coated with a hydrophilic adsorbent polymer (Hydrogel). Vessel segments were analyzed 4 days after infection for luciferase and beta-galactosidase activity and for the extent of injury to the vessel wall. Luciferase activity in vessels infected using the double-balloon method was substantially greater than that achieved by catheter-based methods (P < .05). The dwell and double-balloon methods yielded selective expression in intimal cells, whereas arteries infected using perforated or Hydrogel-coated balloon catheters demonstrated expression primarily in medial cells. Tissue injury was most pronounced with the perforated balloon catheter. CONCLUSIONS Prototype catheters permit relatively efficient direct gene transfer to vascular endothelium; however, delivery methods for targeting the medial cells are inefficient. Modifications are needed to optimize direct gene transfer and minimize tissue injury.

Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol.

Clinical interventions that accelerate conversion of cholesterol to bile acids reduce circulating low density lipoprotein (LDL) cholesterol concentrations. The initial and rate-limiting step in the bile acid biosynthetic pathway is catalyzed by hepatic cholesterol 7 alpha-hydroxylase. To examine the effects of transient primary overexpression of this enzyme on sterol metabolism and lipoprotein transport, we constructed a recombinant adenovirus in which a cDNA encoding rat 7 alpha-hydroxylase is expressed from the human cytomegalovirus immediate-early promoter (AdCMV7 alpha). Syrian hamsters administered AdCMV7 alpha intravenously accumulated transgene-specific mRNA in the liver and demonstrated a dose-dependent increase in hepatic microsomal 7 alpha-hydroxylase activity. The increased conversion of cholesterol to bile acids resulted in a compensatory increase in hepatic cholesterol synthesis. In addition, overexpression of 7 alpha-hydroxylase reduced the rate of LDL cholesterol entry into the plasma space and, in animals maintained on a Western-type diet, restored hepatic LDL receptor expression. As a consequence, plasma LDL concentrations fell by approximately 60% in animals maintained on control diet and by approximately 75% in animals consuming a Western-type diet. Plasma high density lipoprotein cholesterol levels were reduced to a lesser degree. These results demonstrate that transient upregulation of bile acid synthesis by direct transfer of a 7 alpha-hydroxylase gene favorably alters circulating lipoprotein profiles and suggest one potential molecular target for genetic strategies aimed at reducing cardiovascular risk.

Adenovirus-mediated gene transfer

The introduction of foreign genetic material into somatic cells in intact organisms is an important investigational technique that holds considerable promise as a therapeutic tool. Although successful gene transfer has been achieved by the use of both cell-mediated and direct techniques, most strategies have been limited either by constraints on the type, accessibility, and growth state of the target cell population, or by the low efficiency of genetic modification. Among the available vectors for somatic cell gene transfer, recombinant adenoviruses have several properties that make them particularly attractive for direct, in vivo introduction of foreign genes into adult animals and people. Simple techniques for the efficient generation and propagation of recombinant adenoviruses have been developed, and early studies employing recombinant adenoviral vectors demonstrate their potential for broad experimental and eventual clinical application. To exploit this potential properly, a number of important issues, including the efficiency of genetic modification of a targeted cell population, stability of foreign gene expression, effects of host immune response, and cell-type specific targeting of gene transfer, remain to be addressed.

Adenovirus-mediated transfer of a gene encoding human apolipoprotein A-I into normal mice increases circulating high-density lipoprotein cholesterol.

BackgroundIn animal models of atherosclerosis, augmentation of circulating high-density lipoprotein (HDL) cholesterol exerts a protective effect against development of fatty streaks and promotes plaque regression. Methods and ResultsTo investigate the potential of gene transfer to increase HDL cholesterol, a fusion gene encoding human apolipoprotein A-I (apo A-I) under the control of the human cytomegalovirus (CMV) immediate-early promoter was packaged into a recombinant adenovirus (AdCMV apo A-I). BALB/c mice infected with AdCMV apo A-I by intravenous injection accumulate immunoreactive apo A-I in serum; levels 5 days after infection averaged 168 mg/dL. A 35% increase in HDL cholesterol and a 47% increase in total cholesterol were observed in mice infected with AdCMV apo A-I compared with control viruses. Analysis of size-fractionated lipoproteins revealed that human apo A-I is incorporated into murine HDL particles. Expression of human apo A-I declined to <10% of maximum after 12 days and mRNA encoding apo A-I, prevalent 5 days after infection, was undetectable in the livers of infected mice after 12 days. ConclusionsWe conclude that adenovirus-mediated transfer of a gene encoding apo A-I produces transient elevations of circulating HDL cholesterol of a magnitude correlated with important physiological effects. These observations suggest the potential for gene-based therapeutic strategies to reduce cardiovascular risk.

Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall

The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from (L-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation–precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing βGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous transluminal revascularization.

Expandable Bioresorbable Endovascular Stent. I. Fabrication and Properties

AbstractA bioresorbable, expandable poly(L-lactic acid) stent has been designed, based on a linear, continuous coil array principle, by which multiple furled lobes convert to a single lobe upon balloon expansion, without heating. Stent strength and compliance are sufficient to permit deployment by a conventional balloon angioplasty catheter. Several multiple lobe configurations were investigated, with expansion ratios ranging from 1.4 to 1.9 and expanded diameters ranging from 2.3 to 4.7 mm. Compression resistance of the expanded stent is dependent on fiber coil density and fiber ply. A range sufficient for endovascular service was obtained, with less than 4% elastic recoil in six day saline incubation studies. Surface plasma treatment with di(ethylene glycol) vinyl ether significantly reduced platelet adhesion in a 1 h porcine arteriovenous shunt model. Patency was maintained in one week implant studies in the porcine common femoral artery. However, a strong inflammatory response, and significant reduction of the vascular lumen were observed following two weeks implantation. The design principles and fabrication techniques for this bioresorbable stent are sufficiently versatile that a broad range of applications can be addressed. Much work remains to be done, including long-term evaluation of the inflammatory response, and of polymer degradation. The results of this study demonstrate the feasibility of expandable biodegradable stent design and deployment by conventional means.

Overexpression of Cholesterol 7α-Hydroxylase (CYP7A) in Mice Lacking the Low Density Lipoprotein (LDL) Receptor Gene LDL TRANSPORT AND PLASMA LDL CONCENTRATIONS ARE REDUCED

This study was undertaken to determine the effect of transient overexpression of hepatic cholesterol 7α-hydroxylase on low density lipoprotein (LDL) cholesterol transport in mice lacking LDL receptors (LDL receptor−/−). Primary overexpression of hepatic 7α-hydroxylase in LDL receptor−/− mice was accompanied by a dose-dependent decrease in the rate of LDL cholesterol appearance in plasma (whole body LDL cholesterol transport) and a corresponding reduction in circulating LDL cholesterol levels. The increase in hepatic 7α-hydroxylase activity necessary to achieve a 50% reduction in plasma LDL cholesterol concentrations was ∼10-fold. In comparison, cholestyramine increased hepatic 7α-hydroxylase activity ∼3-fold and reduced plasma LDL cholesterol concentrations by 17%. This study demonstrates that augmentation of hepatic 7α-hydroxylase expression is an effective strategy for lowering plasma LDL concentrations even in animals with a genetic absence of LDL receptors.

Relation of plasma lipoprotein(a) to infarct artery patency in survivors of myocardial infarction.

BACKGROUND In the minutes to days after myocardial infarction, endogenous lysis of an occlusive coronary arterial thrombus occurs in most subjects. Compared with those in whom thrombolysis does not occur, those with antegrade flow in the infarct artery have improved left ventricular performance, less left ventricular dilatation, and improved survival. This study was performed to assess intrinsic hemostasis and fibrinolysis in survivors of myocardial infarction with or without antegrade perfusion of the infarct artery. METHODS AND RESULTS In 105 survivors of infarction (75 men, 30 women; age, 30 to 80 years) not given thrombolytic therapy, coronary angiography revealed a patent (group 1, n = 52) or occluded (group 2, n = 53) infarct artery. Plasma concentrations of plasminogen, fibrinogen, tissue plasminogen activator activity, infarct artery. Plasma concentrations of plasminogen, fibrinogen, tissue plasminogen activator activity, plasminogen activator inhibitor activity, cholesterol, triglycerides, and lipoproteins, including lipoprotein(a) (Lp[a]), were measured in blood procured 23 +/- 13 (mean +/- SD) months after infarction. Groups 1 and 2 were similar in age, sex, race, cardioactive medications, infarct artery, extent of coronary artery disease, and left ventricular performance. Of the plasma constituents assayed, the groups were similar except that Lp(a) averaged 18.5 +/- 21.7 mg/dL in group 1 and 49.1 +/- 44.8 mg/dL in group 2 (P < .001). This difference was evident in both Caucasian (n = 65) (P = .009) and African American (n = 40) (P = .01) subjects. CONCLUSIONS Survivors of myocardial infarction who failed to recanalize the infarct artery have higher plasma Lp(a) concentrations than those with a patent infarct artery. Lp(a) may inhibit intrinsic fibrinolysis.