Fred J. Clubb

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-α Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats

BACKGROUND Although patients with heart failure express elevated circulating levels of tumor necrosis factor-alpha (TNF-alpha) in their peripheral circulation, the structural and functional effects of circulating levels of pathophysiologically relevant concentrations of TNF-alpha on the heart are not known. METHODS AND RESULTS Osmotic infusion pumps containing either diluent or TNF-alpha were implanted into the peritoneal cavity of rats. The rate of TNF-alpha infusion was titrated to obtain systemic levels of biologically active TNF-alpha comparable to those reported in patients with heart failure (approximately 80 to 100 U/mL), and the animals were examined serially for 15 days. Two-dimensional echocardiography was used to assess changes in left ventricular (LV) structure (remodeling) and LV function. Video edge detection was used to assess isolated cell mechanics, and standard histological techniques were used to assess changes in the volume composition of LV cardiac myocytes and the extracellular matrix. The reversibility of cytokine-induced effects was determined either by removal of the osmotic infusion pumps on day 15 or by treatment of the animals with a soluble TNF-alpha antagonist (TNFR:Fc). The results of this study show that a continuous infusion of TNF-alpha led to a time-dependent depression in LV function, cardiac myocyte shortening, and LV dilation that were at least partially reversible by removal of the osmotic infusion pumps or treatment of the animals with TNFR:Fc. CONCLUSIONS These studies suggest that pathophysiologically relevant concentrations of TNF-alpha are sufficient to mimic certain aspects of the phenotype observed in experimental and clinical models of heart failure.

Swine as Models in Biomedical Research and Toxicology Testing

Swine are considered to be one of the major animal species used in translational research, surgical models, and procedural training and are increasingly being used as an alternative to the dog or monkey as the choice of nonrodent species in preclinical toxicologic testing of pharmaceuticals. There are unique advantages to the use of swine in this setting given that they share with humans similar anatomic and physiologic characteristics involving the cardiovascular, urinary, integumentary, and digestive systems. However, the investigator needs to be familiar with important anatomic, histopathologic, and clinicopathologic features of the laboratory pig and minipig in order to put background lesions or xenobiotically induced toxicologic changes in their proper perspective and also needs to consider specific anatomic differences when using the pig as a surgical model. Ethical considerations, as well as the existence of significant amounts of background data, from a regulatory perspective, provide further support for the use of this species in experimental or pharmaceutical research studies. It is likely that pigs and minipigs will become an increasingly important animal model for research and pharmaceutical development applications.

Cardiac-Specific Overexpression of Tumor Necrosis Factor-α Causes Oxidative Stress and Contractile Dysfunction in Mouse Diaphragm

BackgroundWe have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor-&agr; (TNF-&agr;). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF-&agr; in the peripheral circulation from cardiac spillover. Given that TNF-&agr; causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling. Methods and ResultsMuscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2′,7′-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm2;P <0.001); this weakness was associated with greater intracellular oxidant levels (P <0.025) and was partially reversed by 30-minute incubation with the antioxidant N-acetylcysteine 10 mmol/L (P <0.01). Exogenous TNF-&agr; 500 &mgr;mol/L increased oxidant production in diaphragm of wild-type mice and caused weakness that was inhibited by N-acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-&agr;. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis. ConclusionsElevated circulating levels of TNF-&agr; provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene.

BACKGROUND Prostacyclin is an important vasoprotective molecule. It inhibits platelet aggregation, monocyte interaction with endothelium, and smooth muscle cell lipid accumulation. Vascular cyclooxygenase-1 (COX-1) is the rate-limiting step in prostacyclin synthesis. The objective of this study was to determine whether adenovirus-mediated transfer of COX-1 could restore COX-1 activity, augment prostacyclin synthesis, and prevent thrombus formation in a porcine carotid angioplasty model. METHODS AND RESULTS Human COX-1 cDNA driven by a cytomegalovirus promoter was constructed into a replication-defective adenovirus 5 vector by homologous recombination. Recombinant adenovirus without a foreign gene (Ad-RR) and buffer were included as controls. Recombinant Ad-LacZ was used for marking the transfected cells in vivo. In the in vitro experiments, cultured human endothelial cells (ECs) and porcine arterial smooth muscle cells (SMCs) were incubated with Ad-COX-1 for 2 hours and 6-keto-PGF(1 alpha) level and the transgene expression were determined 72 hours after infection. In the in vivo experiments, recombinant adenoviruses were directly instilled into angioplasty-injured porcine carotid arteries for 30 minutes. Cyclic flow changes were monitored for 10 days and thrombus formation was examined histologically thereafter. Transgene expression and prostaglandin I2 (PGI2) synthesis by the injured arteries were determined. Cultured ECs infected with Ad-COX-1 produced a fivefold to eightfold increase in PGI2, and the transgene expression in cultured porcine SMCs was demonstrated by Northern analysis. Direct administration of Ad-COX-1 at a dose of 3 x 10(10) pfu completely inhibited carotid cyclic flow changes and thrombus formation accompanied by a fourfold increase in PGI2 synthesis by the injured arteries 10 days after infection, whereas Ad-COX-1 at a lower dose, 5 x 10(9) pfu, had no antithrombotic effects when compared with Ad-RR vector and buffer controls. CONCLUSIONS Adenovirus-mediated transfer of COX-1 to angioplasty-injured carotid arteries was efficacious in augmenting PGI2 synthesis and was associated with an inhibition of thrombosis when a relatively high titer of adenovirus was instilled.

Bioglue surgical adhesive impairs aortic growth and causes anastomotic strictures

BACKGROUND BioGlue surgical adhesive (CryoLife, Inc, Kennesaw, GA) is currently being used to secure hemostasis at cardiovascular anastomoses in adults. Interference with vessel growth would preclude its use during congenital heart surgery. The purpose of this study was to determine if BioGlue reinforcement of aortic anastomoses impairs vessel growth and causes strictures. METHODS Ten 4-week-old piglets (8.0 +/- 1.4 kg) underwent primary aorto-aortic anastomoses. Five piglets were randomly assigned to anastomotic reinforcement with BioGlue. After a 7-week growth period, the aortas were excised for morphometric analysis and histopathology. RESULTS Weight gains were similar in both groups. In BioGlue animals, however, aortic circumference increased only 1.5 +/- 0.8 mm (versus 2.7 +/- 0.8 mm in controls; p = 0.054). BioGlue animals developed a 33.9% stenosis of the aortic lumen area (versus 3.7% in controls, p = 0.038). Adventitial changes reflecting tissue injury and fibrosis were present in all BioGlue animals versus none of the control animals (p = 0.008). CONCLUSIONS BioGlue reinforcement impairs vascular growth and causes stricture when applied circumferentially around an aorto-aortic anastomosis. This adhesive should not be used on cardiovascular anastomoses in pediatric patients.

Multilayer vascular grafts based on collagen-mimetic proteins.

A major roadblock in the development of an off-the-shelf, small-caliber vascular graft is achieving rapid endothelialization of the conduit while minimizing the risk of thrombosis, intimal hyperplasia, and mechanical failure. To address this need, a collagen-mimetic protein derived from group A Streptococcus, Scl2.28 (Scl2), was conjugated into a poly(ethylene glycol) (PEG) hydrogel to generate bioactive hydrogels that bind to endothelial cells (ECs) and resist platelet adhesion. The PEG-Scl2 hydrogel was then reinforced with an electrospun polyurethane mesh to achieve suitable biomechanical properties. In the current study, initial evaluation of this multilayer design as a potential off-the-shelf graft was conducted. First, electrospinning parameters were varied to achieve composite burst pressure, compliance, and suture retention strength that matched reported values of saphenous vein autografts. Composite stability following drying, sterilization, and physiological conditioning under pulsatile flow was then demonstrated. Scl2 bioactivity was also maintained after drying and sterilization as indicated by EC adhesion and spreading. Evaluation of platelet adhesion, aggregation, and activation indicated that PEG-Scl2 hydrogels had minimal platelet interactions and thus appear to provide a thromboresistant blood contacting layer. Finally, evaluation of EC migration speed demonstrated that PEG-Scl2 hydrogels promoted higher migration speeds than PEG-collagen analogs and that migration speed was readily tuned by altering protein concentration. Collectively, these results indicate that this multilayer design warrants further investigation and may have the potential to improve on current synthetic options.

INCREASED ARTERY WALL STRESS POST-STENTING LEADS TO GREATER INTIMAL THICKENING

Since the first human procedure in the late 1980s, vascular stent implantation has been accepted as a standard form of treatment of atherosclerosis. Despite their tremendous success, these medical devices are not without their problems, as excessive neointimal hyperplasia can result in the formation of a new blockage (restenosis). Clinical data suggest that stent design is a key factor in the development of restenosis. Additionally, computational studies indicate that the biomechanical environment is strongly dependent on the geometrical configuration of the stent, and, therefore, possibly involved in the development of restenosis. We hypothesize that stents that induce higher stresses on the artery wall lead to a more aggressive pathobiologic response, as determined by the amount of neointimal hyperplasia. The aim of this investigation was to examine the role of solid biomechanics in the development of restenosis. A combination of computational modeling techniques and in vivo analysis were employed to investigate the pathobiologic response to two stent designs that impose greater or lesser levels of stress on the artery wall. Stent designs were implanted in a porcine model (pigs) for approximately 28 days and novel integrative pathology techniques (quantitative micro-computed tomography, histomorphometry) were utilized to quantify the pathobiologic response. Concomitantly, computational methods were used to quantify the mechanical loads that the two stents place on the artery. Results reveal a strong correlation between the computed stress values induced on the artery wall and the pathobiologic response; the stent that subjected the artery to the higher stresses had significantly more neointimal thickening at stent struts (high-stress stent: 0.197±0.020 mm vs low-stress stent: 0.071±0.016 mm). Therefore, we conclude that the pathobiologic differences are a direct result of the solid biomechanical environment, confirming the hypothesis that stents that impose higher wall stresses will provoke a more aggressive pathobiological response.

Active oxygen species play a role in mediating platelet aggregation and cyclic flow variations in severely stenosed and endothelium-injured coronary arteries.

A canine model with cyclic flow variations (CFVs) in stenosed and endothelium-injured coronary arteries was used to examine the role of active oxygen species in platelet aggregation in vivo. We studied 90 anesthetized dogs in which the pericardial cavity was opened and the heart was exposed. The velocity of blood flow in the left anterior descending coronary artery (LAD) was monitored by a pulsed Doppler flow probe. In 67 dogs, the LADs were stenosed by applying external constrictors at the site where the endothelium was mechanically injured. CFVs developed in all 67 dogs. Treatment with the antioxidants recombinant human copper-zinc superoxide dismutase (r-h-CuZnSOD), recombinant human manganese superoxide dismutase (r-h-MnSOD), and catalase eliminated platelet aggregation-associated coronary CFVs in 63%, 62%, and 64% of animals, respectively. Intravenous infusion of epinephrine restored CFVs in most dogs. Ketanserin, a serotonin (5-hydroxytryptamine2) receptor antagonist, abolished epinephrine-restored CFVs and eliminated CFVs in dogs in which CFVs had not been eliminated by free radical scavengers. In an additional 23 dogs, the LADs were stenosed but not mechanically injured. For control studies, saline was infused into the LADs of 5 dogs. Xanthine/xanthine oxidase was infused into the LADs of 8 dogs and induced CFVs in 4. Hydrogen peroxide was infused into the other 10 dogs and induced CFVs in 9. Histological analysis of the coronary artery revealed that the intima was significantly injured by the infusion. In ex vivo platelet aggregation studies, the in vivo treatment with r-h-CuZnSOD, r-h-MnSOD, and catalase significantly inhibited platelet aggregation induced by platelet-activating factor. Thus, active oxygen species are involved in mediating platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries in vivo.

New Gianturco-Grifka Vascular Occlusion Device Initial Studies in a Canine Model

BACKGROUND Transcatheter closure of cardiovascular defects remains a challenge. Several occlusion devices are available, but each device has limitations. The purpose of this study was to evaluate the new Gianturco-Grifka vascular occlusion device (GGVOD) in a canine model. METHODS AND RESULTS A total of 26 GGVODs were implanted as part of short- and long-term studies. In the short-term study, 1 GGVOD was implanted in each of 11 systemic arteries from 3.2 to 9.0 mm in diameter. All 11 arteries were occluded immediately. In the long-term study, an aortopulmonary shunt was placed in 10 dogs (9, Gore-tex graft; 1, subclavian artery) followed by GGVOD implantation; additionally, a GGVOD was implanted in 5 subclavian arteries. The dogs were boarded for 3 to 6 months, then recatheterized and euthanatized. Immediately after implantation, the 5 subclavian arteries and 9 Gore-tex shunts were occluded completely; the 1 subclavian artery shunt had a small residual leak. At recatheterization, all 10 shunts and 5 subclavian arteries were occluded completely. Necropsy revealed all shunts to be occluded, with the aortic and pulmonic orifices covered with a neointimal layer. The mean fluoroscopic time needed for GGVOD implantation was 9 minutes (range, 3 to 22 minutes). CONCLUSIONS (1) In a canine model, the GGVOD is effective for transcatheter occlusion of arteries and aortopulmonary shunts from 3 to 9 mm in diameter. Possible indications in children include aortopulmonary collateral vessels, long patent ductus arteriosus, systemic-pulmonary shunts, AV malformations, and arteries supplying tumors. (2) GGVOD implantation requires a short fluoroscopic time.