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.

Adenoviral delivery of a leukocyte-type 12 lipoxygenase ribozyme inhibits effects of glucose and platelet-derived growth factor in vascular endothelial and smooth muscle cells.

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.

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.

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.

Transmyocardial fibrinolytic activity in patients with unstable angina pectoris.

OBJECTIVES: This study was performed to investigate local (transmyocardial) fibrinolytic activity in patients with unstable angina. BACKGROUND: Previous studies have reported decreased intrinsic fribrinolytic activity-increased systemic plasminogen activator inhibitor-1 (PAI-1) activity and/or decreased systemic tissue plasminogen activator (t-PA) activity-in patients with acute myocardial infarction. In contrast, the role of intrinsic fibrinolytic activity in patients with unstable angina is not well understood. METHODS: We studied 67 consecutive patients (52 men and 15 women, aged 38-82 years) undergoing cardiac catheterization for chest pain within 24 h of acute presentation: 17 with unstable angina, 33 with stable angina, and 17 with atypical chest pain with angiographically normal coronary arteries. In each, blood samples were obtained simultaneously from the aorta and coronary sinus for measurement of t-PA and PAI-1 activities. RESULTS: There was no difference in coronary sinus or systemic (aortic) t-PA activity among the three groups. The coronary sinus t-PA activity was 0.092 +/- 0.054, 0.088 +/- 0.038, and 0.080 +/- 0.050 IU/ml in the control, unstable angina, and stable angina groups, respectively [not significant (NS)], and the aortic t-PA activity was 0.114 +/- 0.053, 0.099 +/- 0.057, and 0.090 +/- 0.056 IU/ml in the control unstable angina, and stable angina groups, respectively (NS). Similarly, there was no difference in coronary sinus or systemic (aortic) PAI-1 activity among the three groups: the coronary sinus PAI-1 activity was 8.2 +/- 2.0, 7.4 +/- 2.0, and 8.0 +/- 2.5 AIU/ml in the control, unstable angina, and stable angina groups, respectively (NS). The aortic PAI-1 activity was 7.8 +/- 2.1, 7.2 +/- 1.4, and 8.0 +/- 1.8 AIU/ml in the control, unstable angina, and stable angina groups, respectively (NS). CONCLUSIONS: Although it has been suggested that alterations in local (transmyocardial) t-PA and PAI-1 activities may be of pathophysiologic importance in the genesis of unstable angina, our data show no difference in transmyocardial fibrinolytic activity in patients with unstable angina, stable angina, and noncardiac chest pain.

Gene therapy to restore prostacyclin presence to injured endothelium.

These preliminary studies demonstrate the feasibility of restoration of prostacyclin synthesis in mechanically-injured porcine carotid arteries following angioplasty. Our initial data suggest the possibility of inhibiting thrombus development by adenovirus-CMV-PGHS-1 therapy in the initial 10 days following angioplasty.

Southwestern Internal Medicine Conference: endothelial dysfunction and vascular disease.

Vascular endothelial cells, critically situated at the blood-tissue interface, exert important effects on vascular tone and permeability, regulate the coagulation and fibrinolytic systems, mediate translocation of inflammatory cells to the tissue compartment, and modulate proliferation of vascular smooth muscle cells. As the physiology of the endothelium has been defined, defects in endothelial function have been identified in association with human disease, and a syndrome of dysfunctional endothelium has been described. Although it remains debatable whether a coherent syndrome of endothelial dysfunction exists, disordered endothelial biology appears to contribute to the pathophysiology of human vascular disease. Identification of specific molecular mechanisms offers potential targets for novel therapeutic interventions, including genetic modification of endothelial cells in vivo.

Tissue plasminogen activator: from molecular biology to myocardial infarction.

The recent development of recombinant tissue plasminogen activator as a therapeutic agent during acute myocardial infarction is one of the most lucid examples of the potential impact of recombinant DNA technology in clinical medicine. This remarkable achievement would not have been possible without several key discoveries in molecular biology and clinical cardiology and exemplifies the synergistic relationship between basic and clinical research. This article chronicles this journey from molecular biology to myocardial infarction.

Fabrication of resorbable microporous intravascular stents for gene therapy applications.

The authors have produced resorbable, microporous endoluminal stents from Poly-L-lactic acid (PLLA)/Poly epsilon-caprolactone (PCL) blends. Both helical and tube stent designs have been obtained by solvent casting and flotation-precipitation fabrication techniques. A range of PLLA/PCL blend ratios and process variables were employed to investigate their influence on mechanical properties, porosity, and degradation rate. Polymer blends with higher PLLA proportions exhibit higher elastic moduli and ultimate tensile strength, and lower elongation, porosity, and degradation rates than do materials with higher PCL content. Stents with suitable mechanical properties for deployment and support of the vessel wall were obtained. Poly(ethylene oxide) was incorporated into these devices using an acid swelling technique, opening the pore structure and improving the hydrophilic character, thereby enabling the uptake of recombinant adenoviral vectors. The 50:50 PLLA/PCL blended stents were impregnated with recombinant adenovirus (AdCMB beta Gal, encoding a nuclear localizing variant of Escherichia coli beta-galactosidase). Cultured CV-1 cells incubated with stents impregnated with the recombinant virus expressed nuclear localized beta-galactosidase activity, confirming that absorbed virus is released from the matrix in an infectious form, with kinetics suggesting that genetically enhanced endovascular devices of this design are feasible.

Improved bioresorbable microporous intravascular stents for gene therapy.

Drug imbibing microporous stents are under development at a number of centers to enhance healing of the arterial wall after balloon coronary angioplasty procedures. The authors improved the mechanical strength and reservoir properties of a biodegradable microporous stent reported to this Society in 1994. A combined tubular/helical coil stent is readily fabricated by flotation/precipitation and casting/ winding techniques. A two stage solvent swelling technique allows precise adjustment of the surface hydrophilic/hydrophobic balance. These developments permit seven-fold improvement in drug capacity without significantly altering mechanical properties. Stents modified in this manner retain tensile and compressive strength and are suitable for remote deployment. Elution kinetics of these modified stents suggest they are suitable for gene delivery. Successful gene transfer and transmural expression have been demonstrated after implantation of stents impregnated with a recombinant adenovirus carrying a nuclear localizing beta-galactosidase reporter gene into rabbit carotid arteries. These studies suggest that surface modified, bioresorbable polymer stents ultimately may be useful adjunctive devices for gene transfer during percutaneous transluminal revascularization.

Perinatal factors associated with early-onset intracranial hemorrhage in premature infants. A prospective study.

Serial ultrasound examinations were performed on 40 consecutive newborn infants less than 35 weeks' gestational age. Fifteen of 17 infants with intracranial hemorrhage (ICH) had evidence of hemorrhage on the first ultrasound examination (mean age, 1.9 +/- 0.2 hours post partum). Comparing the clinical course of these 15 infants with age- and weight-matched non-hemorrhage controls showed a significant association between the occurrence of early ICH and the pattern of labor. There was no correlation between ICH and the mode of delivery, the use of sodium bicarbonate, volume administration, or the initial BP. In nine of the 15 infants with early-onset ICH, the hemorrhage progressed in severity during the first three postpartum days in association with increasing ventilatory requirements. The results of this study suggest that the course of labor may be a precipitating factor in the onset and evolution of early ICH.

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster.

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.

Effect of up-regulating individual steps in the reverse cholesterol transport pathway on reverse cholesterol transport in normolipidemic mice.

Cholesterol acquired by extrahepatic tissues (from de novo synthesis or lipoproteins) is returned to the liver for excretion in a process called reverse cholesterol transport (RCT). We undertook studies to determine if RCT could be enhanced by up-regulating individual steps in the RCT pathway. Overexpression of 7alpha-hydroxylase, Scavenger receptor B1, lecithin:cholesterol acyltransferase (LCAT), or apoA-I in the liver did not stimulate cholesterol efflux from any extrahepatic tissue. In contrast, infusion of apoA-I.phospholipid complexes (rHDL) that resemble nascent HDL markedly stimulated cholesterol efflux from tissues into plasma. Cholesterol effluxed to rHDL was initially unesterified but by 24 h this cholesterol was largely esterified and had shifted to normal HDL (in mice lacking cholesteryl ester transfer protein) or to apoB containing lipoproteins (in cholesteryl ester transfer protein transgenic mice). Most of the cholesterol effluxed into plasma in response to rHDL came from the liver. However, an even greater proportion of effluxed cholesterol was cleared by the liver resulting in a transient increase in liver cholesterol concentrations. Fecal sterol excretion was not increased by rHDL. Thus, although rHDL stimulated cholesterol efflux from most tissues and increased net cholesterol movement from extrahepatic tissues to the liver, cholesterol flux through the entire RCT pathway was not increased.

Structured drug-loaded bioresorbable films for support structures.

Bioresorbable films can serve simultaneously as anatomic support structures and as drug delivery platforms. In the present study, bioresorbable PLLA films containing dexamethasone were developed through solution processing. The effect of processing parameters on the film morphology and the resulting mechanical properties was studied. A model describing the structuring of these films is suggested. Generally, the solvent evaporation rate determines the kinetics of drug and polymer crystallization and thus, both the mode of drug dispersion in the polymer and the resulting mechanical properties. Two types of structured films were studied: (1) a polymer film with drug located on its surface, obtained due to drug skin formation accompanied by a later polymer core formation; and (2) a polymer film with small drug particles and crystals distributed within the bulk, obtained by parallel solidification of the two components. A prototypical application of these films is an expandable biodegradable support structure (stent). which we have developed. This stent demonstrated good initial mechanical properties. The film structure has only a minor effect on the stent radial compression strength, but more significantly affects the tensile mechanical properties.

Overexpression of cholesterol 7alpha-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 7alpha-hydroxylase on low density lipoprotein (LDL) cholesterol transport in mice lacking LDL receptors (LDL receptor-/-). Primary overexpression of hepatic 7alpha-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 7alpha-hydroxylase activity necessary to achieve a 50% reduction in plasma LDL cholesterol concentrations was approximately 10-fold. In comparison, cholestyramine increased hepatic 7alpha-hydroxylase activity approximately 3-fold and reduced plasma LDL cholesterol concentrations by 17%. This study demonstrates that augmentation of hepatic 7alpha-hydroxylase expression is an effective strategy for lowering plasma LDL concentrations even in animals with a genetic absence of LDL receptors.

Feedback regulation of hepatic 7alpha-hydroxylase expression by bile salts in the hamster.

Hepatic 7alpha-hydroxylase activity appears to be regulated at the transcriptional level by the quantity of bile salts fluxing through the enterohepatic circulation. Whether bile salts directly suppress 7alpha-hydroxylase expression at the level of the hepatocyte or do so indirectly by promoting the release or absorption of an intestinal factor has not been resolved. We have investigated the ability of primary bile salts to suppress hepatic 7alpha-hydroxylase expression in bile-diverted hamsters. Biliary diversion was accompanied by derepression of both hepatic 7alpha-hydroxylase activity (4-5-fold) and bile salt secretion (approximately 3-fold). Derepression of hepatic 7alpha-hydroxylase expression could be prevented by several interventions that increase the availability of bile salts within the hepatocyte including 1) overexpression of an exogenous 7alpha-hydroxylase gene by adenovirus-mediated gene transfer, 2) obstruction of the common bile duct, and 3) intravenous infusions of taurocholate. In contrast, none of these interventions prevented derepression of hepatic cholesterol synthesis or significantly down-regulated hepatic low density lipoprotein receptor expression over the relatively short time course (24 h) of these studies. Together, these data indicate that primary bile salts contribute to the regulation of bile salt synthesis through feedback repression of 7alpha-hydroxylase expression at the level of the hepatocyte.

Adenovirus-mediated transfer of a gene encoding acyloxyacyl hydrolase (AOAH) into mice increases tissue and plasma AOAH activity.

Although the host response to gram-negative bacterial infection follows largely from the interactions of bacterial lipopolysaccharides (LPS or endotoxin) with host cells, little information is available concerning the mechanisms by which the host eliminates or detoxifies LPS. Acyloxyacyl hydrolase (AOAH) is an enzyme, found in phagocytic cells, that catalyzes the enzymatic deacylation of the lipid A moiety of LPS. Enzymatically deacylated LPS is much less potent than LPS at inducing responses in human cells, and it can antagonize the ability of LPS to activate human macrophages, neutrophils, and endothelial cells. Despite these observations, the physiologic role of LPS deacylation remains undefined. To investigate the ability of AOAH to carry out LPS deacylation in vivo, we produced a recombinant adenovirus carrying a gene encoding (AOAH) (Ad.CMV-AOAH) and employed this vector to elicit transient overexpression of AOAH in mice. Mice infected with Ad.CMV-AOAH expressed high levels of the enzyme in plasma, liver, spleen, and kidney. Although adenovirus-induced hepatitis reduced hepatic uptake of intravenously injected [3H]LPS, animals expressing the transgene deacylated a larger fraction of the [3H]LPS taken up by their livers than did mice infected with a control adenovirus. These studies indicate that AOAH can catalyze the deacylation of LPS in vivo, and they provide evidence that the rates of hepatic LPS uptake and deacylation are not closely linked.

Alpha 1-adrenergic stimulation of rat myocardial cells increases protein synthesis.

The effects of adrenergic stimulation on the rates of protein synthesis, degradation, and accumulation were examined in primary cultures of neonatal rat heart cells. Treatment of myocardial cells with norepinephrine increased total cellular protein content and the rate of incorporation of radiolabeled tyrosine into trichloroacetic acid insoluble protein. alpha 1-Adrenergic, but not alpha 2- or beta-adrenergic blockade, inhibited these norepinephrine induced increases. The rate of protein synthesis estimated from the kinetics of equilibrium labeling and from combined equilibrium and pulse labeling was increased by norepinephrine stimulation, whereas protein degradation estimated by release of previously incorporated radiolabeled tyrosine or in pulse-chase experiments was unaffected. To determine whether alpha 1-adrenergic stimulation produced similar effects on the turnover of myofibrillar proteins, rates of synthesis and degradation were estimated for a myofibrillar-enriched protein fraction and for myosin heavy chain and actin. Norepinephrine treatment produced increases in the synthesis of myofibrillar protein without significantly altering degradation rates. These experiments suggest that alpha 1-adrenergic stimulation increases myocardial cell protein content by accelerating protein synthesis.

Kinetic characteristics and regulation of HDL cholesteryl ester and apolipoprotein transport in the apoA-I-/- mouse.

The concentration dependence and tissue distribution of high density lipoprotein (HDL) cholesteryl ester and apolipoprotein (apo) transport were determined in apoA-I knockout mice (apoA-I-/-) that lack normal HDL in plasma. Rates of HDL cholesteryl ester clearance were highly sensitive to plasma HDL cholesteryl ester concentrations with clearance rates falling by 80% in the liver and by 95% in the adrenal glands when plasma HDL cholesteryl ester concentrations were acutely raised to levels normally seen in control mice (approximately 50 mg/dl). With the exception of the brain, saturable HDL cholesteryl ester uptake was demonstrated in all tissues of the body, with the adrenal glands and liver manifesting the highest maximal transport rates (Jm). The plasma concentration of HDL cholesteryl ester necessary to achieve half-maximal transport (Km) equaled 4 mg/dl in the adrenal glands and liver; as a consequence, HDL cholesteryl ester uptake by these organs is maximal (saturated) at normal plasma HDL concentrations in the mouse. When expressed per whole organ, the liver was the most important site of HDL cholesteryl ester clearance accounting for approximately 72% of total HDL cholesteryl ester turnover at normal plasma HDL concentrations. HDL cholesteryl ester transporter activity and scavenger receptor type B1 (SR-BI) protein and mRNA levels were not up-regulated in any organ of apoA-I-/- mice even though these animals lack normal HDL.