Rheal A. Towner

Nitrones as Therapeutics

Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones, including alpha-phenyl-tert-butylnitrone (PBN), have been shown to have potent biological activity in many experimental animal models. Many diseases of aging, including stroke, cancer development, Parkinson disease, and Alzheimer disease, are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development for stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also, in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic-noise-induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes cannot readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our studies and others in which the use of selected nitrones as novel therapeutics has been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases.

Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling.

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

Early myocardial dysfunction in streptozotocin-induced diabetic mice: a study using in vivo magnetic resonance imaging (MRI)

BackgroundDiabetes is associated with a cardiomyopathy that is independent of coronary artery disease or hypertension. In the present study we used in vivo magnetic resonance imaging (MRI) and echocardiographic techniques to examine and characterize early changes in myocardial function in a mouse model of type 1 diabetes.MethodsDiabetes was induced in 8-week old C57BL/6 mice with two intraperitoneal injections of streptozotocin. The blood glucose levels were maintained at 19–25 mmol/l using intermittent low dosages of long acting insulin glargine. MRI and echocardiography were performed at 4 weeks of diabetes (age of 12 weeks) in diabetic mice and age-matched controls.ResultsAfter 4 weeks of hyperglycemia one marker of mitochondrial function, NADH oxidase activity, was decreased to 50% of control animals. MRI studies of diabetic mice at 4 weeks demonstrated significant deficits in myocardial morphology and functionality including: a decreased left ventricular (LV) wall thickness, an increased LV end-systolic diameter and volume, a diminished LV ejection fraction and cardiac output, a decreased LV circumferential shortening, and decreased LV peak ejection and filling rates. M-mode echocardiographic and Doppler flow studies of diabetic mice at 4 weeks showed a decreased wall thickening and increased E/A ratio, supporting both systolic and diastolic dysfunction.ConclusionOur study demonstrates that MRI interrogation can identify the onset of diabetic cardiomyopathy in mice with its impaired functional capacity and altered morphology. The MRI technique will lend itself to repetitive study of early changes in cardiac function in small animal models of diabetic cardiomyopathy.

Immuno-spin trapping of protein and DNA radicals: “Tagging” free radicals to locate and understand the redox process

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.

Glioma morphology and tumor‐induced vascular alterations revealed in seven rodent glioma models by in vivo magnetic resonance imaging and angiography

To evaluate the added value of non–contrast‐enhanced MR angiography (MRA) to conventional MR imaging for a detailed characterization of different rodent glioma models.

Sepsis-associated encephalopathy: a magnetic resonance imaging and spectroscopy study

Brain dysfunction is frequently observed in sepsis as a consequence of changes in cerebral structure and metabolism, resulting in worse outcome and reduced life-quality of surviving patients. However, the mechanisms of sepsis-associated encephalopathy development and a better characterization of this syndrome in vivo are lacking. Here, we used magnetic resonance imaging (MRI) techniques to assess brain morphology and metabolism in a murine sepsis model (cecal ligation and puncture, CLP). Sham-operated and CLP mice were subjected to a complete MRI session at baseline, 6 and 24 h after surgery. Accumulation of vasogenic edematic fluid at the base of the brain was observed in T2-weighted image at 6 and 24 h after CLP. Also, the water apparent diffusion coefficients in both hippocampus and cortex were decreased, suggesting a cytotoxic edema in brains of nonsurvival septic animals. Moreover, the N-acetylaspartate/choline ratio was reduced in brains of septic mice, indicating neuronal damage. In conclusion, noninvasive assessment by MRI allowed the identification of new aspects of brain damage in sepsis, including cytotoxic and vasogenic edema as well as neuronal damage. These findings highlight the potential applications of MRI techniques for the diagnostic and therapeutic studies in sepsis.

Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects ≈500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

Translational research involving oxidative stress and diseases of aging.

There is ample mounting evidence that reactive oxidant species are exacerbated in inflammatory processes, many pathological conditions, and underlying processes of chronic age-related diseases. Therefore there is increased expectation that therapeutics can be developed that act in some fashion to suppress reactive oxidant species and ameliorate the condition. This has turned out to be more difficult than at first expected. Developing therapeutics for indications in which reactive oxidant species are an important consideration presents some unique challenges. We discuss important questions including whether reactive oxidant species should be a therapeutic target, the need to recognize the fact that an antioxidant in a defined chemical system may be a poor antioxidant operationally in a biological system, and the importance of considering that reactive oxidant species may accompany the disease or pathological system rather than being a causative factor. We also discuss the value of having preclinical models to determine if the processes that are important in causing the disease under study are critically dependent on reactive oxidant species events and if the therapeutic under consideration quells these processes. In addition we discuss measures of success that must be met in commercial research and development and in preclinical and clinical trials and discuss as examples our translational research effort in developing nitrones for the treatment of acute ischemic stroke and as anti-cancer agents.

Anti-cancer activity of nitrones and observations on mechanism of action.

The nitrone compound PBN, α-phenyl-tert-butylnitrone, and closely related nitrones have anti-cancer activity in several experimental cancer models. The three experimental models most extensively studied include A) the rat choline deficiency liver cancer model, B) the rat C6 glioma model and C) the mouse APC(Min/+) colon cancer model. The two PBN-nitrones mostly studied are PBN and a PBN derivative 2,4-disulfophenyl-tert-butylnitrone, referred as OKN-007. OKN-007 is a proprietary compound that has had extensive commercial development (designated as NXY-059) for another indication, acute ischemic stroke, and after extensive clinical studies was shown to lack efficacy for this indication but was shown to be very safe for human use. This compound administered orally in the rat glioma model has potent activity in treating fully formed gliomas. In this report observations made on the PBN-nitrones in experimental cancer models will be summarized. In addition the experimental results will be discussed in the general framework of the properties of the compounds with a view to try to understand the mechanistic basis of how the PBN-nitrones act as anti-cancer agents. Possible mechanisms related to the suppression of NO production, S-nitrosylation of critical proteins and inhibition of NF-κB activation are discussed.

ELTD1, a potential new biomarker for gliomas

BACKGROUND Glioblastoma multiforme (GBM), a high-grade glioma, is characterized by being diffuse, invasive, and highly angiogenic and has a very poor prognosis. Identification of new biomarkers could help in the further diagnosis of GBM. OBJECTIVE To identify ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) as a putative glioma-associated marker via a bioinformatic method. METHODS We used advanced data mining and a novel bioinformatics method to predict ELTD1 as a potential novel biomarker that is associated with gliomas. Validation was done with immunohistochemistry, which was used to detect levels of ELTD1 in human high-grade gliomas and rat F98 glioma tumors. In vivo levels of ELTD1 in rat F98 gliomas were assessed using molecular magnetic resonance imaging. RESULTS ELTD1 was found to be significantly higher (P = .03) in high-grade gliomas (50 patients) compared with low-grade gliomas (21 patients) and compared well with traditional immunohistochemistry markers including vascular endothelial growth factor, glucose transporter 1, carbonic anhydrase IX, and hypoxia-inducible factor 1α. ELTD1 gene expression indicates an association with grade, survival across grade, and an increase in the mesenchymal subtype. Significantly high (P < .001) in vivo levels of ELTD1 were additionally found in F98 tumors compared with normal brain tissue. CONCLUSION Results of this study strongly suggests that associative analysis was able to accurately identify ELTD1 as a putative glioma-associated biomarker. The detection of ELTD1 was also validated in both rodent and human gliomas and may serve as an additional biomarker for gliomas in preclinical and clinical diagnosis of gliomas.