Bruce D. Klugherz

HEMODYNAMIC EFFECTS OF SILDENAFIL IN MEN WITH SEVERE CORONARY ARTERY DISEASE

BACKGROUND The cardiovascular effects of sildenafil are important because of the frequent presence of underlying cardiac disease in men with erectile dysfunction and reports indicating serious cardiac events temporally associated with the use of this drug. METHODS We assessed the systemic, pulmonary, and coronary hemodynamic effects of oral sildenafil (100 mg) in 14 men (mean [+/-SD] age, 61+/-11 years) with severe stenosis of at least one coronary artery (stenosis of >70 percent of the vessel diameter) who were scheduled to undergo percutaneous coronary revascularization. Blood-flow velocity and flow reserve were assessed with a Doppler guidewire in 25 coronary arteries, including 13 severely diseased arteries (mean stenosis, 78+/-7 percent) and 12 arteries without stenosis, used as a reference; maximal hyperemia was induced (to assess flow reserve) with the intracoronary administration of adenosine both before and after sildenafil. RESULTS Oral sildenafil produced only small decreases (<10 percent) in systemic arterial and pulmonary arterial pressures, and it had no effect on pulmonary-capillary wedge pressure, right atrial pressure, heart rate, or cardiac output. There were no significant changes in average peak coronary flow velocity, coronary-artery diameter, volumetric coronary blood flow, or coronary vascular resistance. Coronary flow reserve at base line was lower in the stenosed arteries (1.26+/-0.26) than in the reference arteries (2.19+/-0.44) and increased about 13 percent in both groups of arteries combined after the administration of sildenafil (from 1.70+/-0.59 to 1.92+/-0.72, P=0.003). The ratio of coronary flow reserve in coronary arteries with stenosis to that in the reference arteries (0.57+/-0.14) was not affected by sildenafil. CONCLUSIONS No adverse cardiovascular effects of oral sildenafil were detected in men with severe coronary artery disease.

Gene delivery from a DNA controlled-release stent in porcine coronary arteries.

Expandable intra-arterial stents are widely used for treating coronary disease. We hypothesized that local gene delivery could be achieved with the controlled release of DNA from a polymer coating on an expandable stent. Our paper reports the first successful transfection in vivo using a DNA controlled-release stent. Green fluorescent protein (GFP) plasmid DNA within emulsion-coated stents was efficiently expressed in cell cultures (7.9% ± 0.7% vs. 0.6% ± 0.2% control, p < 0.001) of rat aortic smooth muscle cells. In a series of pig stent-angioplasty studies, GFP expression was observed in all coronary arteries (normal, nondiseased) in the DNA-treated group, but not in control arteries. GFP plasmid DNA in the arterial wall was confirmed by PCR, and GFP presence in the pig coronaries was confirmed by immunohistochemistry. Thus, DNA-eluting stents are capable of arterial transfection, and could be useful as delivery systems for candidate vectors for gene therapy of cardiovascular diseases.

Twenty-eight-day efficacy and phamacokinetics of the sirolimus-eluting stent.

BackgroundIn-stent restenosis is caused by neointimal hyperplasia. Sirolimus (rapamycin; Wyeth Research, Radnor, Pennsylvania, USA) inhibits vascular smooth muscle cell proliferation and we evaluated the efficacy of sirolimus in reducing neointimal formation in a rabbit iliac model and in-vivo pharmacokinetics in the porcine coronary model. DesignRandomized, blinded, prospective animal study. MethodsBilateral rabbit iliac artery stent implantation was performed using crossflex stents (Cordis Corporation, Warren, New Jersey, USA) coated with sirolimus incorporated in a nonerodable polymer. Arteries were randomized to one of four stent groups: uncoated stents (n  = 8); polymer control stents (n  = 10); low-dose sirolimus-eluting stents (n  = 9); and high-dose sirolimus-eluting stents (n  = 10). Histomorphometry was performed at 28 days. Arterial tissue and stents were retrieved at 8, 14 and 28 days and blood samples were obtained daily during the first week. ResultsTreatment with low-dose sirolimus was associated with a 23% (P  = NS) reduction in neointimal area and treatment with high-dose sirolimus with a 45% (P  < 0.05) reduction. Sustained drug release from the stent and prolonged intramural arterial deposition were confirmed for up to 28 days. No detectable sirolimus was found in the blood after 2 days. ConclusionControlled-release local delivery of a cell-cycle inhibitor from a nonerodable polymer-coated stent reduced neointimal formation in rabbit iliac arteries in a dose-dependent manner and represents a promising strategy for preventing restenosis.

Gene Delivery to Pig Coronary Arteries from Stents Carrying Antibody-Tethered Adenovirus

Deployment of coronary stents to relieve atherosclerotic obstruction has benefitted millions of patients. However, gene therapy to prevent in-stent restenosis, while promising in experimental studies, remains a challenge. Conventional strategies for viral vector administration utilize catheters that deliver infusions of viral suspensions, which result in suboptimal localization and potentially dangerous distal spread of vector. Stent-based gene delivery may circumvent this problem. We hypothesized that site-specific delivery of adenoviral gene vectors from a stent could be achieved through a mechanism involving anti-viral antibody tethering. Stents were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication defective adenoviruses through highly specific antigen-antibody affinity. We report for the first time successful stent-based gene delivery using antibody-tethered adenovirus encoding green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in both cell culture and pig coronary arteries. Overall arterial wall transduction efficiency in pigs was 5.9 +/- 1.1% of total cells. However, neointimal transduction was more than 17% of total cells in this region. Importantly, when specific antibody was used to tether adenovirus, no distal spread of vector was detectable by PCR, in either distal organs, or in the downstream segments of the stented arteries. Control adenovirus stents, with nonspecific antibody plus adenovirus, demonstrated only a few isolated foci of transduction, and poor site-specific transduction with distal spread of vector. We conclude that a vascular stent is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using stent-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies.

DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection

We previously demonstrated that DNA–polylactic–polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (β-galactosidase) transfection than SMC grown on native collagen (18.3±1.2 vs 1.0±0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-αvβ3 integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by αvβ3 integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-αvβ3 antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0±1.8 vs 3.02±0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen–PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-αvβ3 antibody. Pig coronary studies comparing denatured-collagen–PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

Three-year clinical follow-up after Palmaz-Schatz stenting

OBJECTIVES Our goals were to examine late clinical outcome in a cohort of patients who electively received Palmaz-Schatz intracoronary stents, to identify specific predictors of outcome and to determine the time course of the development of ischemic cardiac events after stenting. BACKGROUND Short-term results of Palmaz-Schatz intracoronary stenting have been promising, with a reduction in both angiographic restenosis and clinical cardiac events up to 1 year after stenting. METHODS We analyzed the clinical outcomes in 65 consecutive patients who underwent stenting at least 3 years before analysis. Demographic, clinical and procedural predictors of survival and event-free survival, defined as freedom from myocardial infarction, stent-site percutaneous transluminal coronary angioplasty, coronary artery bypass graft surgery or death, were analyzed at a mean follow-up of 39 +/- 17 months. RESULTS Absolute survival and event-free survival at 3 years were 88% and 56%, respectively. Three-year freedom from stent-site revascularization was 66%. Predictors of decreased long-term survival (p < 0.05) included diabetes and a high angina score (Canadian Cardiovascular Society class III/IV) at 6 and 12 months after stenting. Predictors of decreased event-free survival (p < 0.05) included a high angina score at 3, 6 and 12 months after stenting, smaller stent deployment balloon size and greater number of stents implanted. Freedom from adverse events by 6 months after stenting also correlated with long-term event-free survival. Eighty-five percent of stent-site revascularizations occurred within 1 year. During late follow-up (>24 months), no patients had stent-site stenoses requiring revascularization, whereas 11% of patients required revascularization in nonstented coronary segments. CONCLUSIONS Clinical predictors of worse long-term outcome included diabetes mellitus, higher angina score at follow-up, smaller stent deployment balloon size and greater number of stents at implantation. During follow-up, the majority of adverse events and stent-site revascularizations occurred early after stenting, and disease progression in nonstented vessels accounted for the majority of late revascularization events.

Trial of a novel prostacyclin analog, UT-15, in patients with severe intermittent claudication

Prostacyclin is an endothelially derived vasodilator and inhibitor of platelet aggregation. Despite its therapeutic potential for peripheral arterial disease, the short half-life and chemical instability are barriers to routine therapy. Accordingly, prostacyclin analogs are being evaluated in patients with peripheral arterial disease. State-of-the-art non-invasive ultrasonography allows for serial testing of the hemodynamic effects of vasoactive drugs. The safety, efficacy and hemodynamic effects of UT-15, a novel, long-acting prostacyclin analog, were studied in patients with severe intermittent claudication. A total of eight patients with stable severe intermittent claudication, Fontaine classes IIb-III, were admitted to the hospital for intravenous infusion of UT-15. A symptom-limited, dose-escalation protocol was instituted, beginning with placebo and then with increasing dosage at 60-min intervals, followed by a 2-h period of maintenance dose at the maximum well-tolerated infusion rate. The hemodynamic response in the lower limbs was assessed with serial ultrasonography, segmental arterial pressures and pulse volumes. Blood flow in the common femoral artery increased 29% (p = 0.003) by the end of the maintenance period and remained above baseline throughout the washout period (p = 0.044). Blood velocity in the lower limb increased in most of the peripheral arteries. These increases achieved statistical significance in the common femoral artery (p = 0.025) and anterior tibial artery (p = 0.019), and approached significance in the popliteal artery (p=0.062). In two of four patients in whom blood flow was undetectable before the infusion, arterial blood flow at the ankle level became apparent on ultrasonography during maintenance infusion. UT-15 infusion improved the pulse volume recording (p = 0.016) but the ankle/brachial index did not change significantly. Common side effects at peak dose included headache and nausea. There were no serious adverse events attributable to UT-15 treatment. In most patients, the optimal infusion rate was 10-20 ng/kg per min. In conclusion, ultrasonography is a novel approach for assessing the hemodynamic response to vasoactive agents. UT-15 is well tolerated when given for up to 2 h and increases arterial blood flow and velocity in patients with severe intermittent claudication.

Combination therapy with clopidogrel and aspirin after coronary stenting

Combination antiplatelet therapy using aspirin and ticlopidine has been the standard of care for prevention of subacute thrombosis following coronary stent implantation. However, the use of ticlopidine is associated with a significant risk of adverse hematologic side effects. Clopidogrel is an inhibitor of ADP‐induced platelet aggregation that has a better safety profile than ticlopidine. We examined the 30‐day clinical outcome following coronary stent implantation in 253 consecutive patients treated with clopidogrel and aspirin. Follow‐up was achieved in 99% of patients and four adverse events were documented. Two patients had angiographically confirmed subacute stent thrombosis (0.8%), and both of these patients underwent successful repeat angioplasty at the stent site. There were two patient deaths during follow‐up (0.8%). One was sudden within 1 week of stent placement and the other occurred in a patient with multisystem organ failure after an extensive myocardial infarction that antedated the stent procedure, with no clinical evidence for stent thrombosis. The combined frequency of subacute stent thrombosis and death was 1.6%. This is comparable to prior studies using the combination of ticlopidine and aspirin following stenting. Therefore, clopidogrel in combination with aspirin appears to be a safe and effective therapy in the prevention of subacute thrombosis following coronary stent implantation. Cathet. Cardiovasc. Intervent. 50:276–279, 2000.

Adenovirus-catheter compatibility increases gene expression after delivery to porcine myocardium.

Endomyocardial injection of adenoviral gene vectors enables localized delivery to comprised myocardial tissue. However, many materials used in endomyocardial delivery catheters may not be compatible with adenoviral gene vectors. In this study, a series of catheter-based endocardial and epicardial (direct visualization) procedures were performed to assess catheter-adenovirus compatibility in an in vivo model. A standard Nitinol-stainless steel (Ni-SS) catheter was compared with a novel Stiletto catheter designed for improved biocompatibility. In 4 animals 40 endocardial injections of adenovirus encoding beta-galactosidase (beta-Gal) were performed with the 2 catheters. After sectioning of the hearts only 8 of 20 Ni-SS beta-Gal+ sites could be identified (40% retrieval) whereas 16 of the 20 Stiletto injection sites were identified (80%). Within these areas successful transfection was observed (12.2 +/- 4.0 beta-Gal+ cells/high-power field [HPF] in the Ni-SS group vs. 30.1 +/- 6.8 beta-Gal+ cells/HPF in the Stiletto group; p = 0.03). After epicardial delivery to distinct areas of the myocardium adenoviral delivery as assayed by beta-galactosidase protein activity was >21 +/- 16-fold (range, 5 to >43-fold) greater than after Stiletto delivery. In conclusion, this study highlights the importance of adenovirus-material compatibility in gene delivery to the myocardium. Efficiency was greater when using the catheter designed to enhance biocompatibility.

Predictors of clinical outcome following percutaneous intervention for in-stent restenosis

Percutaneous intervention for the first episode of in-stent restenosis was performed in 177 patients 5.4 +/- 0.3 months after native coronary stent implantation. Medical records were reviewed and patients contacted 13.3 +/- 1.2 months after in-stent intervention to ascertain the subsequent clinical course. The effects of demographic, procedural, and angiographic variables on clinical outcomes were determined. At 2 years, Kaplan-Meier estimated survival was 93 +/- 3% and freedom from death, myocardial infarction, and a third target artery revascularization (TAR) was 67 +/- 4%. The actuarial frequency of a third TAR was 26 +/- 4% at 1 year. Stratification of outcomes according to timing of in-stent intervention revealed an approximate twofold higher frequency of adverse events among patients with early (</=3 months) in-stent restenosis. Advanced age (p = 0.019), prior coronary bypass (p = 0. 017), and early in-stent intervention (p = 0.006) independently predicted increased mortality at follow-up. Systemic hypertension (p = 0.004), diabetes mellitus (p = 0.044), and early in-stent intervention (p <0.0001) independently predicted a third TAR. These variables (p = 0.007, p = 0.027, and p <0.0001, respectively) also independently predicted a composite end point consisting of death, myocardial infarction, and a third TAR. No angiographic variable predicted outcome after in-stent intervention. Thus, long-term outcome following in-stent intervention was favorable. Early in-stent intervention, advanced age, hypertension, and diabetes predicted adverse outcomes.