Jeffrey J. Rade

A Randomized, Double-Blind, Placebo-Controlled Trial of Ad5FGF-4 Gene Therapy and its Effect on Myocardial Perfusion in Patients with Stable Angina

OBJECTIVES The primary objective of this study was to determine whether intracoronary administration of the adenoviral gene for fibroblast growth factor (Ad5FGF-4) can improve myocardial perfusion compared with placebo. BACKGROUND Animal studies and observational clinical studies have shown improvement in perfusion of the ischemic myocardium using genes encoding angiogenic growth factors; however, randomized, double-blind data in humans are lacking. METHODS We performed a randomized, double-blind, placebo-controlled trial of intracoronary injection of 10(10) adenoviral particles containing a gene encoding fibroblast growth factor (Ad5FGF-4) to determine the effect on myocardial perfusion. Fifty-two patients with stable angina and reversible ischemia comprising >9% of the left ventricle on adenosine single-photon emission computed tomography (SPECT) imaging were randomized to gene therapy (n = 35) or placebo (n = 17). Clinical follow-up was performed, and 51 (98%) patients underwent a second adenosine SPECT scan after 8 weeks. RESULTS Overall (n = 52), the mean total perfusion defect size at baseline was 32.4% of the left ventricle, with 20% reversible ischemia and 12.5% scar. At eight weeks, Ad5FGF-4 injection resulted in a significant reduction of ischemic defect size (4.2% absolute, 21% relative; p < 0.001) and placebo-treated patients had no improvement (p = 0.32). Although the change in reversible perfusion defect size between Ad5FGF-4 and placebo was not significant (4.2% vs. 1.6%, p = 0.14), when a single outlier was excluded a significant difference was observed (4.2% vs. 0.8%, p < 0.05). Ad5FGF-4 was well tolerated and did not result in any permanent adverse sequelae. CONCLUSIONS Intracoronary injection of Ad5FGF-4 showed an encouraging trend for improved myocardial perfusion; however, further studies of therapeutic angiogenesis with Ad5FGF-4 will be necessary.

Focal modification of electrical conduction in the heart by viral gene transfer

Modern treatment of cardiac arrhythmias is limited to pharmacotherapy, radiofrequency ablation, or implantable devices. Antiarrhythmic medications suppress arrhythmias, but their systemic effects are often poorly tolerated and their proarrhythmic tendencies increase mortality. Radiofrequency ablation can cure only a limited number of arrhythmias. Implantable devices can be curative for bradyarrhythmias and lifesaving for tachyarrhythmias, but require a lifetime commitment to repeated procedures, are a significant expense, and may lead to severe complications. One possibility is the use of gene therapy as an antiarrhythmic strategy. As an initial attempt to explore this option, we focused on genetic modification of the atrioventricular node. First, we developed an intracoronary perfusion model for gene delivery, building on our previous work in isolated cardiac myocytes and hearts perfused ex vivo. Using this method, we infected porcine hearts with Adβgal (recombinant adenovirus expressing Escherichia coli β-galactosidase) or with AdGi (adenovirus encoding the Gαi2 subunit). We hypothesized that excess Gαi2 would mimic the effects of β-adreneric antagonists, in effect creating a localized β-blockade. Gαi2 overexpression suppressed baseline atrioventricular conduction and slowed the heart rate during atrial fibrillation without producing complete heart block. In contrast, expression of the reporter gene β-galactosidase had no electrophysiological effects. Our results demonstrate the feasibility of using myocardial gene transfer strategies to treat common arrhythmias.

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries.

We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10,000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.

Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity.

Preclinical arterial gene transfer studies with adenoviral vectors are typically performed in laboratory animals that lack immunity to adenovirus. However, human patients are likely to have prior exposures to adenovirus that might affect: (a) the success of arterial gene transfer; (b) the duration of recombinant gene expression; and (c) the likelihood of a destructive immune response to transduced cells. We confirmed a high prevalence (57%) in adult humans of neutralizing antibodies to adenovirus type 5. We then used a rat model to establish a central role for the immune system in determining the success as well as the duration of recombinant gene expression after adenovirus-mediated gene transfer into isolated arterial segments. Vector-mediated recombinant gene expression, which was successful in naive rats and prolonged by immunosuppression, was unsuccessful in the presence of established immunity to adenovirus. 4 d of immunosuppressive therapy permitted arterial gene transfer and expression in immune rats, but at decreased levels. Ultraviolet-irradiated adenoviral vectors, which mimic advanced-generation vectors (reduced viral gene expression and relatively preserved capsid function), were less immunogenic than were nonirradiated vectors. A primary exposure to ultraviolet-irradiated (but not nonirradiated) vectors permitted expression of a recombinant gene after redelivery of the same vector. In conclusion, arterial gene transfer with current type 5 adenoviral vectors is unlikely to result in significant levels of gene expression in the majority of humans. Both immunosuppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to adenovirus-mediated arterial gene transfer.

Inhibitory G Protein Overexpression Provides Physiologically Relevant Heart Rate Control in Persistent Atrial Fibrillation

Background—The need for new treatment strategies for cardiac arrhythmias has motivated our continuing development of gene therapeutic options. Previously, we reported a decreased heart rate in an acute model of atrial fibrillation after atrioventricular nodal gene transfer. Here, we expand those observations to persistent atrial fibrillation and severe heart failure. Methods and Results—After 3 weeks of atrial fibrillation, domestic swine received atrioventricular nodal gene transfer with adenoviruses encoding &bgr;-galactosidase (&bgr;-gal), wild-type G&agr;i2 (wtGi), or constitutively active mutant (cGi). Heart rates in awake, alert animals were not altered by &bgr;-gal or wtGi. cGi caused a sustained 15% to 25% decrease in heart rate. The wtGi effect became evident with sedation. A tachycardia-induced cardiomyopathy was present before gene transfer. In the &bgr;-gal group, cardiomyopathy worsened over time. In the wtGi group, the condition improved slightly, and in the cGi group, ejection fraction was near normal at the end of the study. TUNEL staining results corroborated this finding. Conclusions—cGi overexpression in the porcine atrioventricular node causes physiologically relevant heart rate control in persistent atrial fibrillation. These data advance the development of gene therapy as a potential treatment for common cardiac arrhythmias.

Effects of Aspirin Responsiveness and Platelet Reactivity on Early Vein Graft Thrombosis After Coronary Artery Bypass Graft Surgery

OBJECTIVES The purpose of this study was to determine if an incomplete response to or inadequate antiplatelet effect of aspirin, or both, contribute to saphenous vein graft (SVG) occlusion after coronary artery bypass graft (CABG) surgery. BACKGROUND Thrombosis is the predominant cause of early SVG occlusion. Aspirin, which inhibits cyclooxygenase-1 activity and thromboxane generation in platelets, reduces early SVG occlusion by one-half. METHODS Aspirin responsiveness and platelet reactivity were characterized 3 days and 6 months after coronary artery bypass graft surgery in 229 subjects receiving aspirin monotherapy by platelet aggregation to arachidonic acid, adenosine diphosphate, collagen and epinephrine, Platelet Function Analyzer-100 (Siemens Healthcare Diagnostics, Newark, Delaware) closure time (CT) using collagen/epinephrine agonist cartridge and collagen/adenosine diphosphate (CADP) agonist cartridge, VerifyNow Aspirin assay (Accumetrics, Inc., San Diego, California), and urine levels of 11-dehydro-thromboxane B(2) (UTXB(2)). SVG patency was determined 6 months after surgery by computed tomography coronary angiography. RESULTS Inhibited arachidonic acid-induced platelet aggregation, indicative of aspirin-mediated cyclooxygenase-1 suppression, occurred in 95% and >99% of subjects 3 days and 6 months after surgery, respectively. Despite this, 73% and 31% of subjects at these times had elevated UTXB(2). Among tested parameters, only UTXB(2) and CADP CT measured 6 months after surgery correlated with outcome. By multivariate analysis, CADP CT of ≤88 s (odds ratio: 2.85, p = 0.006), target vessel diameter of ≤1.5 mm (odds ratio: 2.38, p = 0.01), and UTXB(2) of ≥450 pg/mg creatinine (odds ratio: 2.59, p = 0.015) correlated with SVG occlusion. CADP CT and UTXB(2) in combination further identified subjects at particularly high and low risk for SVG occlusion. CONCLUSIONS Aspirin-insensitive thromboxane generation measured by UTXB(2) and shear-dependent platelet hyper-reactivity measured by Platelet Function Analyzer-100 CADP CT are novel independent risk factors for early SVG thrombosis after coronary artery bypass graft surgery.

Wall Tension Is a Potent Negative Regulator of In Vivo Thrombomodulin Expression

Abstract— Thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is a major contributor to vascular thromboresistance. We previously found that TM protein expression is dramatically reduced in autologous vein grafts during the first two weeks after implantation, coincident to a local inflammatory response, and remains suppressed for at least 6 weeks. To determine the proximate cause of TM loss, in vivo gene expression was quantified by real-time PCR. TM gene expression in vein grafts declined >85% during the first postoperative week and remained suppressed >55% at 6 weeks, accounting for the observed changes in protein expression. The effects of vein graft inflammation were evaluated in animals rendered leukopenic with vinblastine before graft implantation. Abrogating the local inflammatory response affected neither TM protein nor gene expression. To determine how hemodynamic forces might modulate TM expression, the surgical protocol was modified to alter blood flow and pressure-induced vessel distension. TM protein and gene expression did not correlate to changes in shear stress but highly correlated to changes in wall tension, both acutely and over time. We conclude that the primary stimulus for altered TM expression in vein grafts is the exposure to arterial pressure. Furthermore, these data identify strain as a novel and important pathway for in vivo TM gene regulation.

Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure.

Platelet Function in Patients with Diabetes Mellitus: From a Theoretical to a Practical Perspective

Patients with diabetes mellitus have an increased prevalence of vascular disease. Pathologic thrombosis associated with atherosclerotic plaque rupture is a major cause of morbidity and mortality. Platelets are intimately involved in the initiation and propagation of thrombosis. Evidence suggests that platelets from patients with type 2 diabetes have increased reactivity and baseline activation compared to healthy controls. We review the pathophysiology of platelet hyperreactivity in DM patients and its implications in clinical practice, with particular focus on acute coronary syndromes, percutaneous coronary intervention, and novel antiplatelet agents.

Hemodynamic Modulation of Endocardial Thromboresistance

Background— Patients with heart failure are at increased risk for thromboembolic events, including stroke. Historically attributed to blood stasis, little is known about the adverse effects of elevated chamber filling pressure on endocardial function, which could predispose to intracardiac thrombus formation. Methods and Results— We investigated changes in the expression of thrombomodulin, a key component of the anticoagulant protein C pathway, in rats subjected to acute atrial pressure overload caused by aortic banding. Acute elevation of left atrial filling pressure, without an associated decline in ventricular systolic function, caused a 70% inhibition of atrial endocardial thrombomodulin expression and resulted in increased local thrombin generation. Targeted restoration of atrial thrombomodulin expression with adenovirus-mediated gene transfer successfully reduced thrombin generation to baseline levels. In vitro co-culture studies revealed that thrombomodulin downregulation is caused by the paracrine release of transforming growth factor-&bgr; from cardiac connective tissue in response to mechanical stretch. This was confirmed in vivo by administration of a neutralizing transforming growth factor-&bgr; antibody, which effectively prevented thrombomodulin downregulation during acute pressure overload. Conclusions— These findings suggest that increased hemodynamic load adversely affects endocardial function and is a potentially important contributor to thromboembolus formation in heart failure.