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.

Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model.

BACKGROUND: The purpose of this study was to determine the efficacy of stent-based delivery of sirolimus (SRL) alone or in combination with dexamethasone (DEX) to reduce in-stent neointimal hyperplasia. SRL is a potent immunosuppressive agent that inhibits SMC proliferation by blocking cell cycle progression. METHODS AND RESULTS: Stents were coated with a nonerodable polymer containing 185 microgram SRL, 350 microgram DEX, or 185 microgram SRL and 350 microgram DEX. Polymer biocompatibility studies in the porcine and canine models showed acceptable tissue response at 60 days. Forty-seven stents (metal, n=13; SRL, n=13; DEX, n=13; SRL and DEX, n=8) were implanted in the coronary arteries of 16 pigs. The tissue level of SRL was 97+/-13 ng/artery, with a stent content of 71+/-10 microgram at 3 days. At 7 days, proliferating cell nuclear antigen and retinoblastoma protein expression were reduced 60% and 50%, respectively, by the SRL stents. After 28 days, the mean neointimal area was 2.47+/-1.04 mm(2) for the SRL alone and 2.42+/-1.04 mm(2) for the combination of SRL and DEX compared with the metal (5.06+/-1.88 mm(2), P<0.0001) or DEX-coated stents (4.31+/-3.21 mm(2), P<0.001), resulting in a 50% reduction of percent in-stent stenosis. CONCLUSIONS: Stent-based delivery of SRL via a nonerodable polymer matrix is feasible and effectively reduces in-stent neointimal hyperplasia by inhibiting cellular proliferation.

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.

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 (

Local delivery of a hydrophobic heparin reduces neointimal hyperplasia after balloon injury in rat carotid but not pig coronary arteries.

BACKGROUND: Intimal hyperplasia following percutaneous interventional vascular procedures is a major cause of restenosis. Although heparin inhibits intimal hyperplasia, it has not proven clinically useful in part due to an inadequate duration of intramural drug residence. This study was designed to evaluate the efficacy of local delivery of hydrophobic heparin (PTIR-RS-1), exhibiting increased intramural binding, on neointimal hyperplasia after angioplasty injury. METHODS AND RESULTS: PTIR-RS-1 was delivered locally into rat carotid arteries at three doses: 0.1 mM (440 IU), 0.3 mM (1320 IU), or 1.0 mM (4400 IU). Animals were killed at 14 days. In the pig, the doses tested were the low dose in the rat and a high dose 1 log higher. Animals were killed 28 days later. Morphometric analysis was performed to evaluate the intima: media ratio in rats and the normalized neointimal area in pigs. In rats a significant reduction in neointimal to medial area ratio from 0.73 +/- 0.15 for control vs 0.80 +/- 0.27 for sodium heparin (P = NS) and 0.15 +/- 0.07 for the 0.1 mM PTIR-RS-1 dose (P < 0.008). In pigs, PTIR-RS-1 the high dose reduced the normalized neointimal area by 16%, a difference that was not statistically significant. CONCLUSIONS: Increased hydrophobicity of heparin reduced neointimal area following balloon overstretch injury in the rat carotid but not the pig coronary artery model. This study attests to the importance of performing studies evaluating the pharmacologic effect of local delivery of a medication in at least two animal models of restenosis.

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.

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.

Regulation of the endothelial cell urokinase-type plasminogen activator receptor. Evidence for cyclic AMP-dependent and protein kinase C-dependent pathways.

Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.