In Nordic countries 30-day mortality rate is half that estimated with EuroSCORE II in high-risk adult patients given aprotinin and undergoing mainly complex cardiac procedures.

Objectives. To describe current on- (isolated coronary arterty bypass grafting, iCABG) and off-label (non-iCABG) use of aprotinin and associated safety endpoints in adult patients undergoing high-risk cardiac surgery in Nordic countries. Design. Data come from 10 cardiac surgery centres in Finland, Norway and Sweden participating in the European Nordic aprotinin patient registry (NAPaR). Results. 486 patients were given aprotinin between 2016 and 2020. 59 patients (12.1%) underwent iCABG and 427 (87.9%) non-iCABG, including surgery for aortic dissection (16.7%) and endocarditis (36.0%). 89.9% were administered a full aprotinin dosage and 37.0% were re-sternotomies. Dual antiplatelet treatment affected 72.9% of iCABG and 7.0% of non-iCABG patients. 0.6% of patients had anaphylactic reactions associated with aprotinin. 6.4% (95 CI% 4.2%-8.6%) of patients were reoperated for bleeding. Rate of postoperative thromboembolic events, day 1 rise in creatinine >44µmol/L and new dialysis for any reason was 4.7% (95%CI 2.8%-6.6%), 16.7% (95%CI 13.4%-20.0%) and 14.0% (95%CI 10.9%-17.1%), respectively. In-hospital mortality and 30-day mortality was 4.9% (95%CI 2.8%-6.9%) and 6.3% (95%CI 3.7%-7.8%) in all patients versus mean EuroSCORE II 11.4% (95%CI 8.4%-14.0%, p < .01). 30-day mortality in patients undergoing surgery for aortic dissection and endocarditis was 6.2% (95%CI 0.9%-11.4%) and 6.3% (95%CI 2.7%-9.9%) versus mean EuroSCORE II 13.2% (95%CI 6.1%-21.0%, p = .11) and 14.5% (95%CI 12.1%-16.8%, p = .01), respectively. Conclusions. NAPaR data from Nordic countries suggest a favourable safety profile of aprotinin in adult cardiac surgery.

Levosimendan as a rescue drug in experimental propranolol-induced myocardial depression: a randomized study.

STUDY OBJECTIVE: Severe beta-blocker intoxication remains a clinical challenge despite a variety of treatment options. Because of its unique mechanism of action, the new calcium sensitizer levosimendan may provide more prominent cardiac support compared with current medications used to reverse negative inotropy. We hypothesize that levosimendan could reverse propranolol-induced severe negative inotropy in a porcine model of beta-blocker intoxication. METHODS: Twenty-four pigs were anesthetized and monitored. After severe propranolol intoxication was completed, animals were randomized into 3 groups. With a double-blind procedure, 9 animals received a 1.25-mg levosimendan bolus, followed by saline solution infusion, 9 animals received mean arterial pressure-targeted dobutamine infusion after saline solution bolus, and 6 animals received a saline solution bolus followed by saline solution infusion. Hemodynamic and laboratory data were collected during a follow-up period of 120 minutes. RESULTS: All 9 pigs in the levosimendan group survived. In contrast, 4 of 6 (67%) and 7 of 9 (78%) pigs died during the experiment in the placebo and the dobutamine groups, respectively. The levosimendan group showed improved change in the maximum positive slope of the left ventricular pressure, cardiac output, stroke volume, and mean arterial pressure compared with the dobutamine and the placebo groups. CONCLUSION: Levosimendan improved hemodynamic function and survival in this animal model of severe propranolol intoxication. The potential clinical application of levosimendan in propranolol intoxication warrants further investigation.

Nrf2 gene transfer induces antioxidant enzymes and suppresses smooth muscle cell growth in vitro and reduces oxidative stress in rabbit aorta in vivo.

BACKGROUND: Reactive oxygen species (ROS) play a major role in vascular inflammation and pathophysiology of many vascular diseases such as atherosclerosis and injury-induced neointima formation after balloon angioplasty. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor orchestrating antioxidant and cytoprotective responses on oxidative and electrophilic stress, and it has been shown to have antiinflammatory effects in vascular cells in vitro. We therefore postulated that Nrf2 gene transfer would have salutary effects on vascular inflammation after angioplasty. METHODS AND RESULTS: Transduction of vascular smooth muscle cells (VSMCs) with Nrf2-expressing adenovirus increased the expression of several antioxidant enzymes including heme oxygenase-1 (HO-1) compared with beta-galactosidase (AdLacZ)-transduced controls. Moreover, Nrf2 gene transfer also inhibited vascular smooth muscle cell (VSMC) proliferation, and the effect was partially reversed by the HO inhibitor Sn(IV) protoporphyrin. In vivo, adenoviral gene transfer effectively reduced oxidative stress determined by antibody staining against oxidized epitopes of LDL, as well as inhibited vascular inflammation assessed by the macrophage cell count and monocyte chemoattractant protein-1 (MCP-1) staining. However, the antiproliferative effects of Nrf2 in vivo were counterbalanced with diminished apoptosis in neointimal VSMCs, resulting in no change in neointimal hyperplasia. CONCLUSIONS: Nrf2 gene transfer or Nrf2-inducing drugs may have therapeutic applications in vascular diseases in which inflammation and oxidative stress play a role. However, the contrasting growth inhibitory and antiapoptotic effects of Nrf2 need to be considered in pathological conditions in which SMC proliferation plays a critical role.

Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta.

BACKGROUND: Restenosis is a frequent problem after invasive treatment of atherosclerotic vessels and is associated with intimal hyperplasia, which is primarily a result of proliferation and migration of smooth muscle cells, leading to the formation of neointima. Because there is no effective conventional medication for restenosis, gene therapy is a potential new treatment to prevent neointima formation. METHODS AND RESULTS: In the present study, we analyzed the effects of adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transfer (3x10(9) pfu/kg AdEC-SOD versus AdLacZ control virus) on neointima formation in balloon-denuded rabbit aortas. Local catheter-mediated gene transfer to the arterial wall reduced restenosis (P<0.001) and decreased the number of macrophages in the transduced segment (P<0.001) 2 weeks and 4 weeks after the gene transfer compared with AdLacZ controls. Transgene expression was detected in the arterial wall by RT-PCR 2 weeks after the procedure, and the production of superoxide anion was reduced after the gene transfer. Recovery of the endothelial layer was enhanced in EC-SOD-transduced rabbits compared with LacZ controls (P<0.001) 2 weeks after the gene transfer. The therapeutic effect was found to be extended, affecting the gene transfer site and flanking aortic segments from the renal arteries to the bifurcation. However, systemic AdEC-SOD gene transfer to liver did not have any effects on restenosis. CONCLUSIONS: The results suggest that EC-SOD gene transfer reduces restenosis and may be useful for the prevention of intimal hyperplasia after vascular manipulations.

Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart.

BACKGROUND: It is unclear what is the most efficient vector and growth factor for induction of therapeutic vascular growth in the heart. Furthermore, the histological nature of angiogenesis and potential side effects caused by different vascular endothelial growth factors (VEGFs) in myocardium have not been documented. METHODS AND RESULTS: Adenoviruses (Ad) at 2 doses (2x10(11) and 2x10(12) viral particles) or naked plasmids (1 mg) encoding LacZ control, VEGF-A165, or the mature, soluble form of VEGF-D (VEGF-D(DeltaNDeltaC)) were injected intramyocardially with the NOGA catheter system into domestic pigs. AdVEGF-D(DeltaNDeltaC) gene transfer (GT) induced a dose-dependent myocardial protein production, as measured by ELISA, resulting in an efficient angiogenic effect 6 days after the injections. Also, AdVEGF-A165 produced high gene transfer efficacy, as demonstrated with immunohistochemistry, leading to prominent angiogenesis effects. Despite the catheter-mediated approach, angiogenesis induced by both AdVEGFs was transmural, with maximal effects in the epicardium. Histologically, strongly enlarged alpha-smooth muscle actin-positive microvessels involving abundant cell proliferation were found in the transduced regions, whereas microvessel density did not change. Myocardial contrast echocardiography and microspheres showed marked increases in perfusion in the transduced areas. VEGF-D(DeltaNDeltaC) but not matrix-bound VEGF-A165 was detected in plasma after adenoviral GT. A modified Miles assay demonstrated myocardial edema resulting in pericardial effusion with the higher AdVEGF doses. All effects returned to baseline by 3 weeks. Naked plasmid-mediated GT did not induce detectable protein production or vascular effects. CONCLUSIONS: Like AdVEGF-A165, AdVEGF-D(DeltaNDeltaC) GT using the NOGA system produces efficient transmural angiogenesis and increases myocardial perfusion. AdVEGF-D(DeltaNDeltaC) could be useful for the induction of therapeutic vascular growth in the heart.

Oral imatinib mesylate (STI571/gleevec) improves the efficacy of local intravascular vascular endothelial growth factor-C gene transfer in reducing neointimal growth in hypercholesterolemic rabbits.

BACKGROUND: Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach. METHODS AND RESULTS: To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg(-1) per d(-1)), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15x10(10) pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point. CONCLUSIONS: Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.

Intravascular adenovirus-mediated lipoprotein-associated phospholipase A2 gene transfer reduces neointima formation in balloon-denuded rabbit aorta.

Postangioplasty restenosis is a multifactorial process and involves mechanisms such as inflammation and stimulation of the expression of growth factors. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) can modify inflammatory responses by hydrolyzing phospholipids with shortened and/or oxidized sn-2 residues. In this study, we tested a hypothesis that adenovirus-mediated Lp-PLA(2) gene transfer can reduce restenosis in rabbits. Aortas of cholesterol-fed NZW rabbits were balloon-denuded and intra-arterial gene transfer was performed using Dispatch catheter with Lp-PLA(2) or LacZ adenoviruses (1.15 x 10(10)pfu). Intima/media ratio (I/M), histology and cell proliferation were analyzed. Two weeks after the gene transfer I/M in the LacZ-transduced control group was 0.45+/-0.05 but Lp-PLA(2) gene transfer reduced I/M to 0.25+/-0.03. At four weeks time point I/M in the Lp-PLA(2) group (0.34+/-0.05) was also lower than in the LacZ group (0.53+/-0.06). Plasma Lp-PLA(2) activity was increased in the Lp-PLA(2) group (48.2+/-4.2) as compared to the LacZ group (33.6+/-3.51) at two weeks time point. Transgene expression was detected in the arterial wall two and four weeks after the procedure. Apoptosis was higher in the control vessels than in the Lp-PLA(2) group at two weeks time point. In conclusion, local adenovirus-mediated Lp-PLA(2) gene transfer resulted in a significant reduction in neointima formation in balloon-denuded rabbit aorta and may be useful for the prevention of restenosis after arterial manipulations.

Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo.

Osteoporosis is a major problem in elderly population. We tested the hypothesis whether vascular endothelial growth factor (VEGF-A) gene transfer is an appropriate way to enhance bone formation and recruitment of osteoblasts in vivo. Adenovirus vectors containing VEGF-A or lacZ cDNAs (1.4x10(10) pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyses. X-Gal staining showed that up to 20% of the bone marrow cells were transfected although gene transfer also resulted in biodistribution of the vector and expression of the transgene in liver and spleen. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to analyze the effect of gene transfer on bone turnover. When compared with unilateral lacZ transfected trabecular bone at one-week and three-week time points, VEGF-A gene transfer significantly increased bone formation parameters, such as osteoblast number, osteoid volume, and bone volume. Also, bone resorption surface was greatly reduced. It is concluded that injection of adenovirus vector can transfect bone marrow cells in vivo with a relatively high efficiency. Our results suggest that adenovirus-mediated VEGF-A gene transfer induces bone formation via increasing osteoblast activity and may be useful for the treatment of osteoporosis and other diseases that require efficient osteogenic therapy.

Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model.

Previous studies have shown that fibroblast growth factor (FGF)-1, FGF-2, and FGF-5 induce therapeutic angiogenesis. Here, we investigated the potential of FGF-4 for therapeutic neovascularization in comparison to vascular endothelial growth factor (VEGF), using adenoviral gene transfer in a novel rabbit hind limb ischemia model, with ischemia restricted to the calf. Magnetic resonance imaging and a modified Miles assay showed that both AdFGF-4 and AdVEGF given intramuscularly (i.m.) resulted in increases in vascular permeability and edema in transduced muscles 6 days after the gene transfer. In contrast, recombinant FGF-4 protein injected in the rabbit skin did not induce acute vascular permeability. Injections (i.m.) of AdFGF-4 and AdVEGF, but not intra-arterially administered AdVEGF, increased collateral growth, popliteal blood flow, and muscle perfusion compared with controls. The angiogenesis response consisted mainly of the enlargement of pre-existing vessels rather than an increase in capillary density. Adenoviral FGF-4 overexpression up-regulated endogenous VEGF, which may explain many of the effects thought to be specific for VEGF such as the increase in vascular permeability. This study demonstrates for the first time that FGF-4 induces vascular permeability, therapeutic angiogenesis, and arteriogenesis comparable to that of VEGF and could be useful for the treatment of peripheral vascular disease.

Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, and interleukin receptors.

OBJECTIVE: Inflammatory cells play an important role in atherogenesis. However, more information is needed about their gene expression profiles in human lesions. METHODS AND RESULTS: We used laser microdissection (LMD) to isolate macrophage-rich shoulder areas from human lesions. Gene expression profiles in isolated cells were analyzed by cDNA array and compared with expression patterns in normal intima and THP-1 macrophages. Upregulation of 72 genes was detected with LMD and included 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, interferon regulatory factor-5 (IRF-5), colony stimulating factor (CSF) receptors, CD11a/CD18 integrins, interleukin receptors, CD43, calmodulin, nitric oxide synthase (NOS), and extracellular superoxide dismutase (SOD). Several of these changes were also present in PMA-stimulated THP-1 macrophages in vitro. On the other hand, expression of several genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C-I and C-II, decreased. CONCLUSIONS: Overexpression of HMG-CoA reductase in macrophage-rich lesion areas may explain some beneficial effects of statins, which can also modulate increased expression of CD11a/CD18 and CD43 found in microdissected cells. We also found increased expression of CSF receptors, IRF-5, and interleukin receptors, which could become useful therapeutic targets for the treatment of atherosclerotic diseases.

Vascular endothelial growth factor-D expression in human atherosclerotic lesions.

OBJECTIVE: Vascular endothelial growth factor-D (VEGF-D) is a recently characterized member of the VEGF family, but its expression in atherosclerotic lesions remains unknown. We studied the expression of VEGF-D and its receptors (VEGFR-2 and VEGFR-3) in normal and atherosclerotic human arteries, and compared that to the expression pattern of VEGF-A. METHODS: Human arterial samples (n=39) obtained from amputation operations and fast autopsies were classified according to the stage of atherosclerosis and studied by immunohistochemistry. The results were confirmed by in situ hybridization and RT-PCR. RESULTS: We found that while VEGF-A expression increased during atherogenesis, VEGF-D expression remained relatively stable only decreasing in complicated lesions. In normal arteries and in early lesions VEGF-D was mainly expressed in smooth muscle cells, whereas in complicated atherosclerotic lesions the expression was most prominent in macrophages and also colocalized with plaque neovascularization. By comparing the staining profiles of different antibodies, we found that proteolytic processing of VEGF-D was efficient in the vessel wall. VEGFR-2, but not VEGFR-3, was expressed in the vessel wall at every stage of atherosclerosis. CONCLUSIONS: Our results suggest that in large arteries VEGF-D is mainly expressed in smooth muscle cells and that it may have a role in the maintenance of vascular homeostasis. However, in complicated lesions it was also expressed in macrophages and may contribute to plaque neovascularization. The constitutive expression of VEGFR-2 in arteries suggests that it may be one of the principal mediators of the VEGF-D effects in large arteries.

HIF-VEGF-VEGFR-2, TNF-alpha and IGF pathways are upregulated in critical human skeletal muscle ischemia as studied with DNA array.

Critical lower limb ischemia is a common cause for amputation. To develop new therapeutic strategies, more information is needed about molecular mechanisms of tissue responses to ischemic stress and factors inducing angiogenesis. Using a DNA array of 8400 genes, gene expression patterns in human skeletal muscle samples collected from lower limbs amputated due to acute-on-chronic or chronic critical lower limb ischemia, were compared with the control samples collected from the same limb. The results were confirmed by RT-PCR and immunohistochemistry. In acute-on-chronic ischemia, 291 genes were significantly upregulated and 174 genes were downregulated (change in 5.5% of all genes) as compared to control samples. Significant induction of the hypoxia-inducible angiogenic pathway involving hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, vascular endothelial growth factor (VEGF) and its angiogenic receptor VEGFR-2, as well as tumor necrosis factor-alpha (TNF-alpha) with its downstream signaling machinery promoting inflammation and cell death, were found in acute-on-chronic ischemia. In chronic critical ischemia, gene expression changes were much less striking than in acute-on-chronic ischemia, with 74 genes significantly upregulated and 34 genes downregulated (change in 1.3% of all genes). In the chronic situation, the anabolic and survival factors, insulin-like growth factor-1 (IGF-1) and IGF-2, were upregulated in atrophic and regenerating myocytes together with attenuated HIF, VEGF, and VEGFR-2 expression in the same cells. In conclusion, acute-on-chronic and chronic human skeletal muscle ischemia result in distinct gene expression patterns. These findings may be of importance in the design of novel therapies, such as therapeutic vascular growth, for patients suffering from lower limb ischemia.

Gene therapy for restenosis: current status.

Atherosclerosis is a major cause of morbidity and mortality in Western world. Vascular occlusion caused by atherosclerosis usually requires invasive treatment, such as surgical bypass or angioplasty. However, bypass graft failure and restenosis limit the usefulness of these procedures, with 20% of patients needing a new revascularisation procedure within 6 months of angioplasty. Numerous pharmacological agents have been investigated for the prevention of restenosis but none has shown undisputed efficacy in clinical medicine. Gene transfer offers a novel approach to the treatment of restenosis because of easy accessibility of vessels and already existing gene delivery methods. It can be used to overexpress therapeutically important proteins locally without high systemic toxicity, and the therapeutic effect can be targeted to a particular pathophysiological event. Promising results have been obtained from many pre-clinical experiments using therapeutic genes or oligonucleotides to prevent restenosis. Early clinical trials have shown that plasmid- and adenovirus-mediated vascular gene transfers can be conducted safely and are well tolerated. Ex vivo gene therapy with E2F-decoy succeeded in reducing graft occlusion rate after surgical bypass in a randomised, double-blind clinical trial. In the future, further development of gene delivery methods and vectors is needed to improve the efficacy and safety of gene therapy. Also, better knowledge of vascular biology at the molecular level is needed to find optimal strategies and gene combinations to treat restenosis. Provided that these difficulties can be solved, gene therapy offers an enormous potential for clinical medicine in the future.

Regional variation and outcome of out-of-hospital cardiac arrest (ohca) in Finland - the Finnresusci study.

BACKGROUND: Despite the efforts of the modern Emergency Medical Service Systems (EMS), survival rates for sudden out-of-hospital cardiac arrest (OHCA) have been poor as approximately 10% of OHCA patients survive hospital discharge. Many aspects of OHCA have been studied, but few previous reports on OHCA have documented the variation between different sizes of study areas on a regional scale. The aim of this study was to report the incidence, outcomes and regional variation of OHCA in the Finnish population. METHODS: From March 1st to August 31st, 2010, data on all OHCA patients in the southern, central and eastern parts of Finland was collected. Data collection was initiated via dispatch centres whenever there was a suspected OHCA case or if a patient developed OHCA before arriving at the hospital. The study area includes 49% of the Finnish population; they are served by eight dispatch centres, two university hospitals and six central hospitals. RESULTS: The study period included 1042 cases of OHCA. Resuscitation was attempted on 671 patients (64.4%), an incidence of 51/100,000 inhabitants/year. The initial rhythm was shockable for 211 patients (31.4%). The survival rate at one-year post-OHCA was 13.4%. Of the witnessed OHCA events with a shockable rhythm of presumed cardiac origin (n=140), 64 patients (45.7%) were alive at hospital discharge and 47 (33.6%) were still living one year hence. Surviving until hospital admission was more likely if the OHCA occurred in an urban municipality (41.5%, p=0.001). CONCLUSIONS: The results of this comprehensive regional study of OHCA in Finland seem comparable to those previously reported in other countries. The survival of witnessed OHCA events with shockable initial rhythms has improved in urban Finland in recent decades.

Comparing the effect of hydroxyethyl starch 130/0.4 with balanced crystalloid solution on mortality and kidney failure in patients with severe sepsis (6S--Scandinavian Starch for Severe Sepsis/Septic Shock trial): study protocol, design and rationale for a double-blinded, randomised clinical trial.

BACKGROUND: By tradition colloid solutions have been used to obtain fast circulatory stabilisation in shock, but high molecular weight hydroxyethyl starch (HES) may cause acute kidney failure in patients with severe sepsis. Now lower molecular weight HES 130/0.4 is the preferred colloid in Scandinavian intensive care units (ICUs) and 1st choice fluid for patients with severe sepsis. However, HES 130/0.4 is largely unstudied in patients with severe sepsis. METHODS/DESIGN: The 6S trial will randomize 800 patients with severe sepsis in 30 Scandinavian ICUs to masked fluid resuscitation using either 6% HES 130/0.4 in Ringer's acetate or Ringer's acetate alone. The composite endpoint of 90-day mortality or end-stage kidney failure is the primary outcome measure. The secondary outcome measures are severe bleeding or allergic reactions, organ failure, acute kidney failure, days alive without renal replacement therapy or ventilator support and 28-day and 1/2- and one-year mortality. The sample size will allow the detection of a 10% absolute difference between the two groups in the composite endpoint with a power of 80%. DISCUSSION: The 6S trial will provide important safety and efficacy data on the use of HES 130/0.4 in patients with severe sepsis. The effects on mortality, dialysis-dependency, time on ventilator, bleeding and markers of resuscitation, metabolism, kidney failure, and coagulation will be assessed. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00962156.

Effect of levosimendan in experimental verapamil-induced myocardial depression.

BACKGROUND: Calcium antagonist overdose can cause severe deterioration of hemodynamics unresponsible to treatment with beta adrenergic inotropes. The aim of the study was to evaluate in an experimental model the effects of levosimendan during severe calcium antagonist intoxication. METHODS: Twelve landrace-pigs were intoxicated with intravenous verapamil at escalating infusion rates. The infusion containing 2.5 mg/ml verapamil was used aiming to a reduction of cardiac output by 40% from the baseline value. Intoxicated pigs were randomized into two groups: control (saline) and levosimendan (intravenous bolus). Inotropic effect was measured as a change in a maximum of the positive slope of the left ventricular pressure (LV dP/dt). The survival and hemodynamics of the animals were followed for 120 min after the targeted reduction of cardiac output. RESULTS: In the control group, five out of six pigs died during the experiment. In the levosimendan group, one pig died before completion of the experiment (p = 0.04). In the levosimendan group a change in LV dP/dt was positive in four out of six pigs compared to one out of six pigs in the control group (p = ns). CONCLUSIONS: In this experimental model, the use of levosimendan was associated with improved survival.

Extracellular superoxide dismutase accelerates endothelial recovery and inhibits in-stent restenosis in stented atherosclerotic Watanabe heritable hyperlipidemic rabbit aorta.

OBJECTIVES: This study examined whether local gene therapy with extracellular superoxide dismutase (EC-SOD) could inhibit in-stent restenosis in atherosclerotic Watanabe heritable hyperlipidemic rabbits. BACKGROUND: Stenting causes an acute increase in superoxide anion production and oxidative stress; EC-SOD is a major component of antioxidative defense in blood vessels and has powerful cardioprotective effects in ischemic myocardium. METHODS: Endothelial denudation and stenting were done in 36 adult (15 to 18 months old) rabbits. Catheter-mediated intramural delivery of clinical good manufacturing practice-grade adenoviruses encoding rabbit EC-SOD were done simultaneously with stenting. Control animals received adenovirus-encoding nuclear-targeted beta-galactosidase (AdLacZ). Circulating markers for oxidative stress (nonesterified 8-iso-prostaglandin F2 alpha) were measured. Analysis of 6-day, 28-day, and 90-day vessel histology, radical production, oxidation-specific epitopes, and expression studies were performed. RESULTS: The EC-SOD treatment reduced oxidant production in stented vessels compared with control vessels. Early systemic recovery of total SOD activity was observed in the treated rabbits. The EC-SOD significantly accelerated endothelial recovery (67.4% +/- 10.8% vs. 24.2.1% +/- 4.6% at 6 days, p < 0.05; 89.3% +/- 3.7% vs. 45.1% +/- 9.6% at 28 days, p < 0.05), and the beneficial effect involved increased proliferation of regenerating endothelium. The EC-SOD group showed a 61.3% lower (p < 0.05) neointimal formation at 28 days, with a similar, albeit nonsignificant trend at 90 days (1.20 +/- 0.32 mm2 vs. 1.88 +/- 0.24 mm2, p = 0.06). CONCLUSIONS: The results suggest a central pathogenetic role of oxidation sensitive signaling processes in endothelial recovery and developing in-stent restenosis in atherosclerotic vessels. Local therapy against oxidative stress represents a promising therapeutic strategy in stent-induced vascular injury.

Vascular endothelial growth factor C-induced collateral formation in a model of myocardial ischemia.

BACKGROUND: Besides being a known lymphangiogenic activator, vascular endothelial growth factor (VEGF)-C may express angiogenic potential by proteolytic cleavage and activation of endothelial cells. We assessed myocardial collateral formation and functional changes after adenovirus-mediated VEGF-C gene transfer in an ischemic porcine model. METHODS: Fifteen Landrace piglets underwent Ameroid-induced gradual occlusion of the left circumflex artery (LCx) and consequent progressive myocardial ischemia. Three weeks after Ameroid placement, the animals underwent gated 99mTc SPECT during rest and stress, in vivo angiography and 18FDG PET. Pigs were randomized to intramyocardial injections of adenoviruses encoding vascular endothelial growth factor (VEGF-C; n = 7) or control beta-galactosidase (LacZ; n = 5). Four weeks later, the examinations were repeated and histology was analyzed. RESULTS: Angiography showed significant progression of LCx stenosis in both groups during the treatment period. Left ventricular wall thickening (LVWT) at the LCx area in gated 99mTc SPECT remained unchanged in the VEGF-C group, indicating that VEGF-C prevented progression of myocardial ischemia, whereas LVWT deteriorated in the LacZ group (p = 0.042). Semi-quantitative assessment of 18FDG PET suggests more reduction in ischemia in the adVEGF-C group than in controls (p = 0.052). Angiography showed significant clustering of collaterals in the adVEGF-C gene transfer area compared that in LacZ (p = 0.004). von Willebrand factor staining revealed a significantly (p = 0.03) greater number of microvessels in the adVEGF-C-treated myocardium. CONCLUSIONS: This appears to be the first large-animal study in which, during progressive ischemia, functional and metabolic benefits of intramyocardial VEGF-C gene transfer were apparent. VEGF-C-induced collateral formation occurred at the site of gene transfer. The angiogenic potency of VEGF-C deserves further study as a therapeutic option.

Tissue inhibitor of metalloproteinase 1 adenoviral gene therapy alone is equally effective in reducing restenosis as combination gene therapy in a rabbit restenosis model.

Neointimal formation is a common feature after angioplasty, bypass grafting and stenting. Angioplasty damages endothelium, causing pathological changes in arteries which lead to smooth muscle cell proliferation, synthesis of extracellular matrix components and eventually restenosis formation. Adenoviruses offer an efficient transgene expression in the vascular system. In this study, we compared the effects of different gene combinations. We wanted to find out whether adenoviral catheter-mediated delivery of an additive combination of the vascular endothelial growth factor (VEGF)-A with VEGF-C is more effective than the combination of tissue inhibitor of metalloproteinase 1 (TIMP-1) alone or with VEGF-C in a rabbit balloon denudation model. Additionally, we wanted to clarify whether the combination therapy prolongs the treatment effect. It was found that TIMP-1 alone prevents restenosis and that the combination of VEGF-A and VEGF-C has a similar effect at the 2-week time point. However, the combination of VEGF-A and VEGF-C lost the treatment effect at the 4-week time point due to the catch-up growth of neointima. On the other hand, TIMP-1 and the combination of TIMP-1 with VEGF-C still had an extended treatment effect at the 4-week time point. When considering the gene combination used in this study, it is concluded that gene therapy with adenoviral TIMP-1 alone is sufficient in reducing restenosis and that combination gene therapy does not bring any significant advantages.

Gene transfer for therapeutic vascular growth in myocardial and peripheral ischemia.

Therapeutic vascular growth in the treatment of peripheral and myocardial ischemia has not yet fulfilled its expectations in clinical trials. Randomized, double-blinded placebo-controlled trials have predominantly shown the safety and feasibility but not the clear-cut clinically relevant efficacy of angiogenic gene or recombinant growth factor therapy. It is likely that growth factor levels achieved with single injections of recombinant protein or naked plasmid DNA are too low to induce any relevant angiogenic effects. Also, the route of administration of gene transfer vectors has not been optimal in many cases leading to low gene-transfer efficacy. Animal experiments using intramuscular or intramyocardial injections of adenovirus encoding vascular endothelial growth factor (VEGF, VEGF-A), the mature form of VEGF-D, and fibroblast growth factors (FGF-1, -2, and -4) have shown high angiogenic efficacy. Adenoviral overexpression of VEGF receptor-2 ligands, VEGF-A and the mature form of VEGF-D, enlarge the preexisting capillaries in skeletal muscle and myocardium via nitric oxide(NO)-mediated mechanisms and via proliferation of both endothelial cells and pericytes, resulting in markedly increased tissue perfusion. VEGF also enhances collateral growth, which is probably secondary to increased peripheral capillary blood flow and shear stress. As a side effect of VEGF overexpression and rapid microvessel enlargement, vascular permeability increases and may result in substantial tissue edema and pericardial effusion in the heart. Because of the transient adenoviral gene expression, the majority of angiogenic effects and side effects return to baseline by 2 weeks after the gene transfer. In contrast, VEGF overexpression lasting over 4 weeks has been shown to induce the growth of a persistent vascular network in preclinical models. To improve efficacy, the choice of the vascular growth factor, gene transfer vector, and route of administration should be optimized in future clinical trials. This review is focused on these issues.