Valvuloplasty with a paclitaxel-eluting balloon prevents restenosis in an experimental animal model of aortic stenosis.

BACKGROUND AND AIM OF THE STUDY: Restenosis occurs invariably within 12 months following balloon valvuloplasty (BAV) in calcific aortic valve disease (CAVD), and is a limiting factor of this treatment. Cellular proliferation secondary to balloon injury is thought to play a pivotal role in the mechanism of restenosis. The study aim was to investigate the potential role of a paclitaxel-eluting valvuloplasty balloon to mitigate the progression of restenosis in an animal model of CAVD. METHODS: Fifty-three rabbits were fed with an aortic stenosis (AS)-inducing diet (cholesterol 0.5% plus vitamin D3 50,000 IU/day) for three months. The surviving animals (n = 40) underwent echocardiographic and invasive assessments, followed by valvuloplasty, randomly using either a paclitaxel-coated (3 µg/mm2) or a plain balloon. At one month after BAV, the surviving animals (n = 28) underwent repeat assessments, followed by histology and micro-computed tomography (MicroCT) analysis of the aortic valve. RESULTS: The baseline and post-BAV transvalvular gradients, aortic valve area (AVA), left ventricular stroke work loss (SWL) and aortic valve resistance (AVR) were similar between the groups (14 rabbits were assigned to paclitaxel-eluting, and 14 to plain balloon). Significant differences between the groups were observed at one-month post-BAV, which was suggestive of diminished restenosis in the paclitaxel-balloon group (mean maximum transvalvular pressure gradient 7.7 ± 7.7 versus 3.6 ± 3.7 mmHg, p = 0.08; AVA 0.91 ± 0.59 versus 0.55 ± 0.22 cm2, p = 0.04; SWL 3.5 ± 4.0 versus 8.6 ± 8.0%, p = 0.047; AVR 86 ± 71 versus 177 ± 137 dynes/s/cm(-5), p = 0.039). Histology demonstrated decreased leaflet thickness (0.60 ± 0.15 versus 0.71 ± 0.17 mm, p = 0.03), proliferating cell nuclear antigen (PCNA) staining (grade 1.53 ± 0.04 versus 2.24 ± 0.55, p = 0.049), and calcification in the paclitaxel-balloon group. CONCLUSION: Use of a paclitaxel-eluting valvuloplasty balloon in an animal model of AS resulted in attenuated restenosis, secondary to decrease in valve proliferation and calcification.

Evaluation of experimental myocardial infarction models via electromechanical mapping and magnetic resonance imaging.

The diagnostic characteristics of electromechanical mapping (EMM) were evaluated in porcine myocardial infarction (MI) models with the parallel application of cardiac magnetic resonance imaging (cMRI) from the aspect of different pathophysiology and localization. Balloon occlusion in the left anterior descending coronary artery (LAD balloon group) or coil deployment in the LAD (LAD coil group) or circumflex artery (Cx coil group) was applied percutaneously in 16 domestic pigs. Regional left ventricular viability data were captured via cMRI and EMM. The unipolar voltage (UV) value was significantly decreased in segments containing transmural and subendocardial late enhancement compared with viable segments in the LAD balloon, LAD coil, and Cx coil groups. Receiver operating characteristic analysis revealed area under the curve values of 0.809 and 0.691 in the LAD infarct territory, and 0.864 and 0.855 in the Cx infarct territory for the UV compared with cMRI viability results as transmural late enhancement or viable tissue and subendocardial late enhancement or viable tissue, respectively. In conclusion, the UV value detected the presence of scar tissue with differential transmural extent and which represented proper diagnostic features both in the reperfused and nonreperfused models. This data could provide additional benefit in the clinical use of EMM for diagnostic purposes.

Comparison of short- and long-term results of drug-eluting vs. bare metal stenting in the porcine internal carotid artery.

PURPOSE: To evaluate the development of neointimal hyperplasia after implantation of drug-eluting stents (paclitaxel) compared to bare metal stents in porcine internal carotid arteries (ICAs). METHODS: While drug-eluting stents have effectively reduced neointimal proliferation in porcine external carotid arteries, the porcine internal carotid artery (ICA) is more sensitive to shear stress and altered flow conditions. Thus, a study was conducted to evaluate bare vs. drug-eluting stents in porcine ICAs. Under general anesthesia, 18 domestic pigs were implanted with paclitaxel-eluting (n = 18) and bare (n = 18) stents in the left and right ICAs, respectively. After 1 and 3 months, control carotid angiography was performed, followed by histopathological and histomorphometric analyses of the stented ICA. RESULTS: Histopathological results (fibrin deposition, necrosis, inflammation) were similar in the groups at 1 and 3 months. Moreover, the injury score and rate of endothelialization did not differ between the groups. Histomorphometric analysis after 1 month revealed significantly (p<0.05) less neointimal hyperplasia after implantation of paclitaxel-eluting stents. The antiproliferative effect of paclitaxel-eluting stents were maintained during the 3-month follow-up: the neointimal area was 0.7 ± 0.5 vs. 1.2 ± 0.6 mm(2) (p<0.01), the area stenosis was 23.5% ± 13.9% vs. 37.8% ± 14.4% (p<0.01), the maximal neointimal thickness was 0.2 ± 0.1 vs. 0.2 ± 0.9 mm (p<0.05) in paclitaxel-eluting vs. bare stents, respectively. Implantation of paclitaxel-eluting and bare stents did not lead to edge restenosis or vessel remodeling in porcine ICAs at 1 or 3 months. CONCLUSION: Compared to bare metal stents, drug-eluting stents implanted in the porcine ICA produced significantly less neointimal hyperplasia.

Optimization of drug-eluting balloon use for safety and efficacy: evaluation of the 2nd generation paclitaxel-eluting DIOR-balloon in porcine coronary arteries.

OBJECTIVES: The aim of this preclinical study was to optimize the use of drug-eluting balloon (DEB) DIOR(2nd) (generation) by measurements of tissue and plasma paclitaxel concentrations in porcine coronary artery overstretch and prove efficacy in inhibition of neointimal growth without complementary use of stent. BACKGROUND: The usually recommended DEB 60 sec inflation time causes prolonged ischemia and arterial injury. METHODS: Tissue, plasma, and balloon surface concentrations of paclitaxel were measured in pigs 45 min and 12 hr after balloon inflation times of 15, 20, 30, 45, and 60 sec. Extent of neointimal hyperplasia was compared using DIOR(2nd) (generation) or noncoated balloon at two-week follow-up. Paclitaxel was replaced by fluorescent paclitaxel derivative in DEB and DES to demonstrate the distribution of the drug in arterial wall. RESULTS: DIOR(2nd) (generation) DEB provided 29 ± 3 µM/L, 52 ± 6 µM/L, 196 ± 44 µM/L, 202 ± 36 µM/L, and 184 ± 59 µM/L paclitaxel to the vessel wall after 15, 20, 30, 45, and 60 sec of dilation, reaching plateau at 30 sec inflation time. Paclitaxel penetrated up to 2 mm tissue deepness. Measurable plasma paclitaxel level (45 ± 28 ng/mL) was found only after 60 sec balloon inflation time. At follow-up, the dilated arterial segment neointimal area and maximal neointimal thickness were significantly smaller with DIOR vs. uncoated balloon use. Fluorescence images of DIOR showed a homogenous distribution of the drug on the vessel, in contrast with DES. CONCLUSION: Using the DIOR(2nd) (generation) DEB, a maximal balloon inflation time of 30-45 sec is optimal, reducing effectively the neointimal hyperplasia.

Attainment of local drug delivery with paclitaxel-eluting balloon in porcine coronary arteries.

OBJECTIVE: Our purpose was to confirm the local drug delivery of a paclitaxel-eluting balloon by percutaneous intervention of single arterial segments or bifurcations of porcine coronary arteries. METHODS: Eight domestic pigs were subjected to 2 x 30 s Dior balloon dilatation of the mid left anterior descending, left circumflex and proximal right coronary arteries. Bifurcation intervention was performed in six arteries. The dilated, and the distal and proximal reference segments were prepared for tissue paclitaxel concentration measurement. Tissue samples were harvested at mean 1.5, 12, 24 and 48 h after balloon dilatation and plasma samples were taken at various time points. RESULTS: The tissue paclitaxel concentration of the single dilated segment was at 1.5 h postdilatation 1.82+/-1.60 micromol/l, which decreased significantly to 0.73+/-0.27 (P=0.032), 0.62+/-0.34 and 0.44+/-0.31 micromol/l at 12, 24 and 48 h. The bifurcation intervention resulted in 5.10+/-1.80 micromol/l tissue paclitaxel amount in the main branch, which at 12 h had diminished to 1.41+/-1.23 micromol/l (P=0.004). The bifurcation side contained 7.00+/-4.80 micromol/l paclitaxel at 1.5 h postdilatation, which lowered to 2.72+/-0.40 micromol/l (P=0.034). The mean paclitaxel concentration of the reference segments decreased gradually from 0.84+/-0.99 to 0.34+/-0.36 micromol/l (P=0.09), 0.28+/-0.16 and 0.19+/-0.18 micromol/l tissue at 1.5, 12, 24 and 48 h postdilatation, respectively. No paclitaxel was found in the peripheral blood at any time point. CONCLUSION: Short exposure of the coronary artery to paclitaxel with a coated balloon is sufficient for the attainment of an adequate tissue concentration of paclitaxel, which is known to be efficient in inhibiting neointimal growth.

Cross-sectional imaging assisted selection for heart performance in pigs.

A two-way selection procedure was performed on a Landrace pig line. First the cardiac output (CO) values (6.89 +/- 1.27 L/min) were estimated by ECG-gated magnetic resonance imaging (MRI) to quantify the heart performance of the boars (n = 30), then total body skeletal muscle volume (MVTB; 48.7 +/- 5.17 dm3) was determined applying spiral computerised tomography (CT). The relative cardiac output value (RCO) was developed, expressing the relationship between CO and MVTB. Four superior [RCO value below 6.7 dm3 / (L x min(-1))] and one minus variant [(8.97 dm3 / (L x min(-1))] boars were taken into breeding. The best fifteen percentage of the first generation males (n = 30) was characterised according to their RCO values. Significant difference was found between the developed performance measure of the progenies of minus and plus variant boars [8.2 +/- 0.65 and 5.63 +/- 0.70 dm3 / (L x min(-1)), n = 7 and 23], respectively. Comparing the existing five progeny groups, all of the 'HP+' groups differed significantly from the 'HP-' one, where the largest heart performance contrast found was 3.2 dm3 / (L x min(-1)). Based upon the results, the selection is being continued on a larger population.

Matrix Metalloproteinase-2 Impairs Homing of Intracoronary Delivered Mesenchymal Stem Cells in a Porcine Reperfused Myocardial Infarction: Comparison With Intramyocardial Cell Delivery.

BACKGROUND: Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs. METHODS: Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 106) or intramyocardially (group IM, 9.88 ± 1.44 × 106). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image. RESULTS: AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm2, p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p = 0.046) at the 1-month follow up. CONCLUSION: Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.

Platelet activation and high tissue factor level predict acute stent thrombosis in pig coronary arteries: prothrombogenic response of drug-eluting or bare stent implantation within the first 24 hours.

Increased thrombogenicity of drug-eluting stents (DESs) has recently been reported. The aim of the present study was to investigate the prothrombogenic effect of DESs and Bare stents, and determine factors predictive of acute stent thrombosis (AST) in preclinical experiments using new stent design or coating. Circulating pre- and post-stenting parameters of platelet activation (mean platelet volume, MPV; platelet distribution width, platelet large cell ratio), thrombin activation (thrombin-antithrombin complex, TAT and prothrombin fragments, F1+2), tissue factor antigen (TF-ag) and -activity (TF-act) and plasminogen activator inhibitor-1 (PAI-1) were measured in 141 consecutive pigs. Stent implantations were performed after pretreatment with aspirin and clopidogrel with unfractionated heparin anticoagulation. Nineteen pigs (groups AST-DES, n = 12; and AST-Bare, n = 7) died mean 6.3 +/- 2.9 h after stent implantation from AST. The remaining 122 control (C) pigs (groups C-DES, n = 76, and C-Bare, n = 46) survived the 1-month follow-up. Non-significantly elevated levels of post-stent F1+2 and TAT were measured in AST groups. Post-stenting MPV was increased significantly in the groups ASTDES and AST-Bare as compared with the groups C-DES and C-Bare (11.73 +/- 1.12 and 11.6 +/- 0.68 vs. 8.85 +/- 0.78 and 9.04 +/- 0.81 fL; p < 0.001), similarly to TF-ag (189.1 +/- 87.5 and 127 +/- 34.9 vs. 42.5 +/- 24.6 and 35.3 +/- 37.6 pg/ml; p < 0.001, respectively), Tfact (3.23 +/- 0.95 and 2.73 +/- 1.68 vs. 1.43 +/- 1.12 and 1.61 +/- 1.31 pM; p < 0.01, respectively) and PAI-1 (99.1 +/- 15.8 and 99 +/- 14.7 vs.53.4 +/- 40.2 and 46.9 +/- 42.4 ng/ml;p < 0.01, respectively). Multivariate analysis revealed elevated post-stenting plasma levels of TF-ag (p = 0.016) and MPV (p = 0.001) as independent risk factors for developing AST within the first 24 h in a porcine coronary stent model.

Preclinical randomised safety, efficacy and physiologic study of the silicon dioxide inert-coated Axetis and bare metal stent: short-, mid- and long-term outcome.

AIMS: To evaluate the short-, mid- and long-term safety, efficacy and vascular physiology of Axetis silicon dioxide (SiO2, abrading the micropores) inert-coated stent implantation in a randomised preclinical setting. METHODS AND RESULTS: Coronary arteries of domestic pigs were randomised to receive either Axetis or BMS (same design) stents with one-, three- and six-month follow-up (FUP), controlled by coronary angiography, optical coherence tomography (OCT), intravascular ultrasound (IVUS) and histology (n=32). The time-dependent vasomotor reaction of coronary arteries to stenting was measured using modified myography (n=12). Complete endothelialisation of the Axetis stent was confirmed by OCT, IVUS and histology at one-month FUP. Histopathology revealed continuous healing of the vessel wall with a gradual reduction of inflammation and fibrin score during the six-month FUP in both stent types. Significantly smaller neointimal area and %area stenosis were measured in Axetis stents compared with BMS at each FUP time point. Vascular reactivity measurements showed significantly better endothelium-dependent vasodilation of stented arteries with Axetis implantation. CONCLUSIONS: Implantation of the Axetis SiO2-coated stent resulted in a significantly better safety, efficacy and vessel physiology profile compared with BMS of the same design with a continuous decrease in vessel inflammation during the six-month FUP.

Long-acting beneficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction.

The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 × 10(9) apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC (n = 8) or medium solution (control; n = 8) injected intramyocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression profiling of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC significantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 ± 5.9% vs. 37.4 ± 8.9%, p = 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes (p < 0.05) in APOSEC-treated pigs. In conclusion, overexpression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors.