Debra Saunders

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.

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.

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.

In vivo detection of c-Met expression in a rat C6 glioma model

The tyrosine kinase receptor, c‐Met, and its substrate, the hepatocyte growth factor (HGF), are implicated in the malignant progression of glioblastomas. In vivo detection of c‐Met expression may be helpful in the diagnosis of malignant tumours. The C6 rat glioma model is a widely used intracranial brain tumour model used to study gliomas experimentally. We used a magnetic resonance imaging (MRI) molecular targeting agent to specifically tag the cell surface receptor, c‐Met, with an anti‐c‐Met antibody (Ab) linked to biotinylated Gd (gadolinium)‐DTPA (diethylene triamine penta acetic acid)‐albumin in rat gliomas to detect overexpression of this antigen in vivo. The anti‐c‐Met probe (anti‐c‐Met‐Gd‐DTPA‐albumin) was administered intravenously, and as determined by an increase in MRI signal intensity and a corresponding decrease in regional T1 relaxation values, this probe was found to detect increased expression of c‐Met protein levels in C6 gliomas. In addition, specificity for the binding of the anti‐c‐Met contrast agent was determined by using fluorescence microscopic imaging of the biotinylated portion of the targeting agent within neoplastic and ‘normal’brain tissues following in vivo administration of the anti‐c‐Met probe. Controls with no Ab or with a normal rat IgG attached to the contrast agent component indicated no non‐specific binding to glioma tissue. This is the first successful visualization of in vivo overexpression of c‐Met in gliomas.

In vivo characterization of several rodent glioma models by 1H MRS

The assessment of metabolites by 1H MRS can provide information regarding glioma growth, and may be able to distinguish between different glioma models. Rat C6, 9 L/LacZ, F98 and RG2, and mouse GL261, cells were intracerebrally implanted into the respective rodents, and human U87 MG cells were implanted into athymic rats. Ethyl‐nitrosourea induction was also used. Glioma metabolites [e.g. total choline (tCho), total creatine (tCr), N‐acetylaspartate (NAA), lactate (Lac), glutamine (Gln), glutamate (Glu), aspartate (Asp), guanosine (Gua), mobile lipids and macromolecules (MMs)] were assessed from 1H MRS using point‐resolved spectroscopy (PRESS) [TE = 24 ms; TR = 2500 ms; variable pulse power and optimized relaxation delay (VAPOR) water suppression; 27‐μL and 8‐μL voxels in rats and mice, respectively] at 7 T. Alterations in metabolites (Totally Automatic Robust Quantitation in NMR, TARQUIN) in tumors were characterized by increases in lipids (Lip1.3: 8.8–54.5 m m for C6 and GL261) and decreases in NAA (1.3–2.0 m m for RG2, GL261 and C6) and tCr (0.8–4.0 m m for F98, RG2, GL261 and C6) in some models. F98, RG2, GL261 and C6 models all showed significantly decreased (p < 0.05) tCr, and RG2, GL261 and C6 models all exhibited significantly decreased (p < 0.05) NAA. The RG2 model showed significantly decreased (p < 0.05) Gln and Glu, the C6 model significantly decreased (p < 0.05) Asp, and the F98 and U87 models significantly decreased (p < 0.05) Gua, compared with controls. The GL261 model showed the greatest alterations in metabolites. 1H MRS was able to differentiate the metabolic profiles in many of the seven rodent glioma models assessed. These models are considered to resemble certain characteristics of human glioblastomas, and this study may be helpful in selecting appropriate models. Copyright

In Vivo Imaging of Immuno-Spin Trapped Radicals With Molecular Magnetic Resonance Imaging in a Diabetic Mouse Model

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.

Non-mammalian fat-1 gene prevents neoplasia when introduced to a mouse hepatocarcinogenesis model Omega-3 fatty acids prevent liver neoplasia

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.

Visualization of the protective ability of a free radical trapping compound against rat C6 and F98 gliomas with diffusion tensor fiber tractography

To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N‐tert‐butyl nitrone (PBN), on glioma‐affected neuronal fibers. The fiber tractography method was able to differentiate between different tumor types, such as the C6 and F98 rat glioma models.

In vivo MRS assessment of altered fatty acyl unsaturation in liver tumor formation of a TGFα/c-myc transgenic mouse model

Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in humans rely on visual confirmation of neoplastic formations. A more effective early detection method is needed. Using in vivo magnetic resonance spectroscopy (MRS), we show that alterations in the integral ratios of the bis-allyl to vinyl hydrogen protons in unsaturated lipid fatty acyl groups correlate with the development of neoplastic formations in vivo in a TGFalpha/c-myc mouse hepatocellular carcinoma (HCC) model. HPLC analysis of the TGFalpha/c-myc mice liver tissue revealed a significant increase in the amount of oleic acid, along with alterations in linoleic and gamma-linolenic acids, as compared with control CD1 mice. Electrospray ionization tandem mass spectrometry analysis indicated a significant increase in the abundance of specific glycerol phosphatidylcholine (GPCho) lipids containing palmitic and oleic acids between control CD1 and TGFalpha/c-myc mice liver tissue extracts. Western blot analysis of the mice liver tissue indicates alterations in the desaturase enzyme stearoyl CoA desaturase (SCD)1, responsible for palmitic and oleic acid formation. Microarray analysis detected alterations in several genes involved with fatty acid metabolism, particularly SCD2, in transgenic mouse liver tissue. In correlation with the HPLC, mass spectrometry, Western blot, and microarray analyses, we are able to confirm the ability of in vivo MRS to detect precancerous lesions in the mouse liver before visual neoplastic formations were detectable by MRI.

In vivo detection of free radicals using molecular MRI and immuno-spin trapping in a mouse model for amyotrophic lateral sclerosis.

Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin trapping and molecular magnetic resonance imaging, in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A)-transgenic (Tg) mice, a model for ALS. For this study, the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (ip) over 5 days before administration (iv) of an anti-DMPO probe (anti-DMPO antibody covalently bound to an albumin-gadolinium-diethylenetriamine pentaacetic acid-biotin MRI contrast agent) to trap free radicals. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p < 0.05) or anti-DMPO probe concentrations measured from T₁ relaxations (p < 0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescence-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via colocalization fluorescence microscopy. DMPO adducts were also confirmed in diseased/nondiseased tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p < 0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, noninvasive, in vivo diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models.