Joan Gretton

Myocardial Ischemic Memory Imaging With Molecular Echocardiography

Background— Diagnosing acute coronary syndrome in patients presenting with chest discomfort is a challenge. Because acute myocardial ischemia/reperfusion is associated with endothelial upregulation of leukocyte adhesion molecules, which persist even after ischemia has resolved, we hypothesized that microbubbles designed to adhere to endothelial selectins would permit echocardiographic identification of recently ischemic myocardium. Methods and Results— Lipid microbubbles (diameter, 3.3±1.7 &mgr;m) were synthesized. The selectin ligand sialyl Lewisx was conjugated to the microbubble surface (MBsLex). Control bubbles (MBCTL) bore surface Lewisx or sialyl Lewisc. Intravital microscopy of mouse cremaster muscle was performed after intravenous injection of MBsLex (n=11) or MBCTL (n=9) with or without prior intrascrotal tumor necrosis factor–&agr;. There was greater adhesion of MBsLex to inflamed versus noninflamed endothelium (P=0.0081). Rats (n=12) underwent 15 minutes of anterior descending coronary artery occlusion. After 30 minutes and 1 hour of reperfusion, high-mechanical-index nonlinear echocardiographic imaging was performed in which single frames were acquired at 3.5 and 4 minutes after intravenous injection of MBsLex or MBCTL. Video intensity at 4 minutes was subtracted from that at 3.5 minutes to derive target-specific acoustic signal. MBsLex caused greater opacification in postischemic versus nonischemic myocardium at both time points (P≤0.002). Immunostaining confirmed endothelial P-selectin expression in the ischemic bed. Conclusions— Echocardiographic identification of recently ischemic myocardium is possible using ultrasound contrast agents targeted to selectins. This may offer a new approach to the more timely and precise diagnosis of acute coronary syndrome in patients presenting with chest pain of uncertain cardiac origin.

Intratracheal injection of manganese superoxide dismutase (MnSOD) plasmid/liposomes protects normal lung but not orthotopic tumors from irradiation.

To determine whether intratracheal (IT) lung protective manganese superoxide–plasmid/liposomes (MnSOD–PL) complex provided ‘bystander’ protection of thoracic tumors, mice with orthotopic Lewis lung carcinoma-bacterial β-galactosidase gene (3LL-LacZ) were studied. There was no significant difference in irradiation survival of 3LL-LacZ cells irradiated, then cocultured with MnSOD–PL-treated compared with control lung cells (D0 2.022 and 2.153, respectively), or when irradiation was delivered 24 h after coculture (D0 0.934 and 0.907, respectively). Tumor-bearing control mice showed 50% survival at 18 days and 10% survival at 21 days. Mice receiving liposomes with no insert or LacZ–PL complex plus 18 Gy had 50% survival at 22 days, and a 20% and 30% survival at day 50, respectively. Mice receiving MnSOD–PL complex followed by 18 Gy showed prolonged survival of 45% at 50 days after irradiation (P < 0.001). nested rt-pcr assay for the human mnsod transgene demonstrated expression at 24 h in normal lung, but not in orthotopic tumors. decreased irradiation induction of tgf-β1, tgf-β2, tgf-β3, mif, tnf-α, and il-1 at 24 h was detected in lungs, but not orthotopic tumors from mnsod-pl-injected mice (p < 0.001). thus, pulmonary radioprotective mnsod–pl therapy does not provide detectable ‘bystander’ protection to thoracic tumors.

Mitochondrial Localization of Superoxide Dismutase is Required for Decreasing Radiation-Induced Cellular Damage

Abstract Epperly, M. W., Gretton, J. E., Sikora, C. A., Jefferson, M., Bernarding, M., Nie, S. and Greenberger, J. S. Mitochondrial Localization of Superoxide Dismutase is Required for Decreasing Radiation-Induced Cellular Damage. Radiat. Res. 160, 568–578 (2003). We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0 = 0.89 ± 0.01 and 1.08 ± 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0 = 1.15 ± 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0 = 2.1 ± 0.1 and 1.97 ± 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.

Radioprotective Gene Therapy

Control of cancer by irradiation therapy alone or in conjunction with combination chemotherapy is often limited by organ specific toxicity. Ionizing irradiation toxicity is initiated by damage to normal tissue near the tumor target and within the transit volume of radiotherapy beams. Irradiation-induced cellular, tissue, and organ damage is mediated by acute effects, which can be dose limiting. A latent period follows recovery from the acute reaction, then chronic irradiation fibrosis (late effects) pose a second cause of organ failure. We have developed the technology for radioprotective gene therapy using the transgene for the antioxidant manganese superoxide dismutase, delivered to specific target organs (lung, esophagus, oral cavity, oropharynx, and bladder) using gene transfer vectors including plasmid/liposomes (PL) and adenovirus. Irradiation protection by MnSOD transgene overexpression at the cellular level has been demonstrated to be localized to the mitochondrial membrane. Using MnSOD transgene constructs lacking the mitochondrial localization leader sequence, and in other experiments attaching this localization signal to otherwise non-radioprotective cytoplasmic Cu/ZnSOD, mitochondrial localization has been demonstrated to be critical to protection. Organ specific injection of MnSOD-PL prior to irradiation demonstrates transgene expression for 48-72 hours, and an associated decrease in ionizing irradiation-induced expression of inflammatory cytokine mRNA and protein. Significant reduction of organ specific tissue injury has been demonstrated in several organ systems in rodent models. Application of MnSOD-PL gene therapy in the setting of fractionated chemo-radiotherapy is being tested in clinical trials for prevention of esophagitis during treatment of non-small cell carcinoma of the lung, and in prevention of mucositis during combination therapy of carcinomas of the head and neck. Encouraging results in pre-clinical models suggest that radioprotective gene therapy may facilitate dose escalation protocols to allow increases in the therapeutic ratio of cancer radiotherapy.

Prevention of Radiation-Induced Oral Cavity Mucositis by Plasmid/Liposome Delivery of the Human Manganese Superoxide Dismutase (SOD2) Transgene

Abstract Guo, H., Seixas-Silva, Jr., J. A., Epperly, M. W., Gretton, J. E., Shin, D. M., Bar-Sagi, D., Archer, H. and Greenberger, J. S. Prevention of Radiation-Induced Oral Cavity Mucositis by Plasmid/Liposome Delivery of the Human Manganese Superoxide Dismutase (SOD2) Transgene. Radiat. Res. 159, 361–370 (2003). Oral cavity mucositis is a major toxicity of radiation therapy for head and neck cancer. In the present mouse model studies, we evaluated intraoral administration of SOD2-PL complexes 24 h before single-fraction 30-Gy irradiation for the prevention of oral cavity mucositis. Expression of the human SOD2 transgene in the oral cavity of C3H/HeNsd mice was demonstrated by nested reverse transcriptase polymerase chain reaction (RT-PCR). Mice treated intraorally with bacterial β-galactosidase gene-plasmid/liposome (LacZ-PL) or hemagglutinin (HA)-manganese superoxide dismutase-plasmid/liposome (HA-SOD2-PL) demonstrated LacZ or HA-SOD2 expression, respectively, 24 h after injection. In a second strain of mouse, SOD2-PL-treated female athymic nude mice demonstrated significantly decreased ulceration at day 5 after 30 Gy, compared to LacZ-PL-injected, irradiated mice or irradiated controls. No further reduction in radiation-induced ulceration was detected in mice treated with both SOD2-PL and 10 mg/kg of amifostine (WR-2721) 30 min before 30 Gy compared to SOD2-PL alone. No significant protection of orthotopically transplanted murine squamous cell carcinoma (SCC-VII) tumors was detected in mice that received SOD2-PL treatment before 18 Gy. Thus overexpression of human SOD2 in the oral cavity mucosa can prevent radiation-induced mucositis with no detectable compromise in the therapeutic response of orthotopically transplanted tumors.

Manganese Superoxide Dismutase-Plasmid/ Liposome (MnSOD-PL) Administration Protects Mice from Esophagitis Associated with Fractionated Radiation

Intraesophageal administration of manganese superoxide dismutase‐plasmid/liposome (MnSOD‐PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD‐PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD‐PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial β‐galactosidase gene)‐plasmid/liposome (LacZ‐PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope‐tagged MnSOD (HA‐MnSOD). In fractionation protocols, mice receiving MnSOD‐PL, but not LacZ‐PL (200 μl of plasmid/liposomes containing 200 μg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD‐PL administration. The HA‐MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase‐plasmid/liposome (GPX‐PL), or liposomes containing MnSOD protein, vitamin E, co‐enzyme Q10, or 21‐aminosteroid. Thus, MnSOD‐PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD‐PL treatment into human esophageal radiation protection.

Overexpression of the transgene for manganese superoxide dismutase (MnSOD) in 32D cl 3 cells prevents apoptosis induction by TNF-α, IL-3 withdrawal, and ionizing radiation

OBJECTIVE Stabilization of the mitochondria in IL-3-dependent hematopoietic progenitor cell line 32D cl 3 by overexpression of the transgene for manganese superoxide dismutase (MnSOD) prior to ionizing radiation prevents apoptosis. We now demonstrate that overexpression of the MnSOD transgene also protects 32D cl 3 cells from apoptosis caused by exposure to tumor necrosis factor-alpha (TNF-alpha) or withdrawal of interleukin (IL)-3. MATERIALS AND METHODS The hematopoietic progenitor cell line, 32D cl 3, and subclones overexpressing the human MnSOD transgene, 1F2 or 2C6, were radiated to 1000 cGy or were exposed to TNF-alpha (0 to 100 etag/mL) or were subjected to IL-3 withdrawal. The cells were then examined at several time points for DNA strand breaks using a comet assay, depolarization of the mitochondrial membrane, activation of caspase-3, PARP cleavage, and apoptosis, and also for changes in cell cycle distribution. RESULTS Overexpression of the transgene for MnSOD resulted in increased survival following exposure to radiation, exposure to TNF-alpha, or IL-3 withdrawal. The cell lines overexpressing MnSOD (1F2 or 2C6) displayed decreased radiation-induced, TNF-alpha-induced, or IL-3 withdrawal-induced mitochondrial membrane permeability, caspase-3 and PARP activation, and apoptosis. CONCLUSIONS Overexpression of the human MnSOD transgene in 32D cl 3 cells results in stabilization of the mitochondria and reduction in radiation-, TNF-alpha-, or IL-3 withdrawal-induced damage. Thus, MnSOD stabilization of the mitochondrial membrane is relevant to reduction of apoptosis by several classes of oxidative stress inducers.

Cell phenotype specific kinetics of expression of intratracheally injected manganese superoxide dismutase–plasmid/liposomes (MnSOD–PL) during lung radioprotective gene therapy

Intratracheal (IT) injection of manganese superoxide dismutase–plasmid/liposome (MnSOD-PL) complexes prior to whole lung irradiation of C57BL/6J mice provides significant protection from acute and chronic irradiation damage. We determined the duration of increased MnSOD biochemical activity and differential expression of a hemagglutinin (HA) epitope-tagged MnSOD transgene. HA–MnSOD–PL was IT injected at doses of 0–1000 μg, and mice were killed 1,2,3 or 4 days later. Other groups of mice were irradiated to 20 Gy to the pulmonary cavity 24 h after injection and killed at the same time points as non-irradiated mice. Both non-irradiated and irradiated groups of mice showed increased MnSOD biochemical activity with plasmid dose that plateaued at 100 μg of MnSOD plasmid DNA. In control mice, MnSOD biochemical activity decreased at 2, 3 or 4 days after injection. In irradiated mice, MnSOD biochemical activity decreased at day 2 but increased on days 3 and 4. HA–MnSOD expression decreased in broncheoalveolar macrophages and alveolar type-II cells 3 days after injection in non-irradiated and irradiated mice, but remained elevated in endothelial and epithelial cells past 4 days. The data provide a rationale for every second-day administration of intrapulmonary MnSOD–PL in clinical trials of radioprotective gene therapy. This should be sufficient to provide radioprotection during radiation treatments.

Overexpression of the human manganese superoxide dismutase (Mnsod) Transgene prevents irradiation apoptosis of 32D cl 3 hematopoietic progenitor cells by stabilization of the mitochondria

Abstract Subclones of 32D cl 3, 32D-Bcl-xl, 1F2 and 2C6 which demonstrate overexpression of the human MnSOD transgene had increased radioresistance and decreased apoptosis compared to 32D cl 3. Comet assay demonstrated that DNA strand breaks were repaired in all cell lines by 30 minutes (mins) following 200, 400 or 600 cGy, and all had increased migration of Bax and SAP kinases p38 and Jnk1 to the mitochondria 3 hrs after irradiation. Only 32D cl 3 had increased cytochrome-C release, caspase-3 biochemical activity, and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6 hrs. At 6 hrs, 10.6% of 32D cl 3 cells showed a decrease in mitochondrial membrane potential compared to 2.9, 1.7 and 1.8% for 1F2, 2C6 and 32D-Bcl-xl, respectively. 32D c1 3 showed a decrease in mitochondrial complex I and III activity, and decreased levels of adenosine triphosphate (ATP) compared to 1F2 and 2C6. 32D cl 3, 1F2, 2C6 or 32D-Bcl-xl were incubated with 5,5-dimethyl-1-pyroline N-oxide (DMPO) spin-trap chemical and irradiated. Electron paramagnetic (spin) resonance (EPR) spectroscopy of spin-trapped adducts demonstrated that 32D cl 3 or 32D-Bcl-xl cells had a higher concentration of free radicals following irradiation than 1F2 or 2C6. Peroxidation of mitochondrial-specific diphosphatidyl-glycerol (DPG) was increased in 32D cl 3 cells compared to 1F2. Thus, the irradiation protective role of MnSOD is due to stabilization of the mitochondria.

Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (Mnsod-pl) Protects normal lung but not orthotopic thoracic tumors from irradiation

Abstract Major complications following total body irradiation (TBI) prior to bone marrow transplantation (BMT) include pneumonitis and lung fibrosis. Our laboratory previously demonstrated that intratracheal (IT) injection of C57BL/6J mice with MnSOD-PL complex 24 hours before irradiation reduces organizing alveolitis/fibrosis and increases survival. To demonstrate that overexpression of the human MnSOD transgene in the lung would not protect lung tumors, C57BL/6J mice were injected at the carina with 5×10 5 orthotopic 3LL Lewis lung carcinoma cells 10 days before MnSOD-PL injection, and sacrificed 24 hrs later. Lungs were removed, orthotopic tumor isolated, RNA extracted, and nested RT-PCR performed with primers specific for the human MnSOD transgene. Expression of the human MnSOD transgene was detected in lung cells but not in orthotopic tumors. Tumor bearing mice that received MnSOD-PL + 1800 cGy showed a significant increase in survival compared to LacZ-PL + 1800 cGy or 1800 cGy alone. Pulmonary MnSOD-PL treatment may compliment TBI by not only protecting the lungs from irradiation but also by increasing tumor control.