Citation Details

Journal Article

IVIS, Xen29, Xen 29, Staphylococcus aureus Xen29, Animals; Blood Glucose/metabolism; Cell Adhesion/drug effects; Cell Count; Cell Movement/drug effects; Chemokine CXCL2/pharmacology; Diabetes Mellitus, Type 1/blood/complications/*immunology/microbiology; Diabetes Mellitus, Type 2/blood/complications/*immunology/microbiology; Diet, High-Fat/adverse effects; Disease Models, Animal; Hyperglycemia/chemically induced/complications; Inflammation/blood/complications/immunology/microbiology; Leukocytes/cytology/drug effects/*immunology/microbiology; Male; Mice; Mice, Inbred C57BL; Phagocytes/cytology/drug effects/microbiology; Staphylococcus aureus/physiology

BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62+/-18% and 85+/-21%, respectively) and high fat diet-induced (77+/-25% and 86+/-17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3+/-1.0) compared to control (8.0+/-1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.

Journal Article

MDA-MB-231-D3H1, MDA-MB-231-luc-D3H1, IVIS, Bioware, Breast Cancer

Abstract Purpose: This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with (125)I-labeled 5-iodo-2 -deoxyuridine ((125)IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner. Materials and methods: In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix carbon scaffold. Cultures were pulse-labeled for 3 h with (125)IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2'-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT((R)) EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with (125)IdU/EdU. Additional aliquots were used to determine the mean (125)I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured. Results: Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells. Conclusions: These studies demonstrate the capacity of (125)IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype.

Journal Article

MDA-MB-231-D3H2Ln, IVIS, Bioluminescence

We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition (EMT). To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation as well as induced epithelial marker expression. These results implicate ZEB in induction of the EMT as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase (CDK) inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

Journal Article

Aaa; abdominal aortic aneurysm; FX Pro Kodak molecular Imaging System; ImageJ software; in vivo imaging; jugular vein injection; mice; MMPSense 680; ProSense 750; tail vein injection; thoracic aorta; vascular

<AbstractText Label=“OBJECTIVE” NlmCategory=“OBJECTIVE”>To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs).</AbstractText> <AbstractText Label=“METHODS AND RESULTS” NlmCategory=“RESULTS”>MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)-filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P<0.05), whereas both models showed a significant 2-fold decrease in aortic cyclic strain (P<0.05). Differences in patterns of elastin degradation and localization of fluorescent signal from protease-activated probes were also observed.</AbstractText> <AbstractText Label=“CONCLUSIONS” NlmCategory=“CONCLUSIONS”>The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.</AbstractText>

Journal Article

OsteoSense, Animals; Anti-Inflammatory Agents/pharmacology; Antioxidants/pharmacology; Atherosclerosis/etiology/genetics/metabolism/pathology/*prevention & control; Bone Morphogenetic Protein Receptors, Type I/metabolism; Bone Morphogenetic Proteins/*antagonists & inhibitors/metabolism; Cardiovascular Agents/*pharmacology; Cholesterol, LDL/blood; Diet, High-Fat; Disease Models, Animal; Endothelial Cells/drug effects/metabolism; Fatty Liver/etiology/metabolism/prevention & control; Female; Hep G2 Cells; Humans; Lipoproteins, LDL/metabolism; Liver/drug effects/metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles/*pharmacology; Pyrimidines/*pharmacology; Reactive Oxygen Species/metabolism; Receptors, LDL/deficiency/genetics; Recombinant Fusion Proteins/metabolism; Signal Transduction/*drug effects; Time Factors; Vascular Calcification/etiology/genetics/metabolism/pathology/*prevention &; control

OBJECTIVE: The expression of bone morphogenetic proteins (BMPs) is enhanced in human atherosclerotic and calcific vascular lesions. Although genetic gain- and loss-of-function experiments in mice have supported a causal role of BMP signaling in atherosclerosis and vascular calcification, it remains uncertain whether BMP signaling might be targeted pharmacologically to ameliorate both of these processes. METHODS AND RESULTS: We tested the impact of pharmacological BMP inhibition on atherosclerosis and calcification in LDL receptor-deficient (LDLR-/-) mice. LDLR-/- mice fed a high-fat diet developed abundant vascular calcification within 20 weeks. Prolonged treatment of LDLR-/- mice with the small molecule BMP inhibitor LDN-193189 was well-tolerated and potently inhibited development of atheroma, as well as associated vascular inflammation, osteogenic activity, and calcification. Administration of recombinant BMP antagonist ALK3-Fc replicated the antiatherosclerotic and anti-inflammatory effects of LDN-193189. Treatment of human aortic endothelial cells with LDN-193189 or ALK3-Fc abrogated the production of reactive oxygen species induced by oxidized LDL, a known early event in atherogenesis. Unexpectedly, treatment of mice with LDN-193189 lowered LDL serum cholesterol by 35% and markedly decreased hepatosteatosis without inhibiting HMG-CoA reductase activity. Treatment with BMP2 increased, whereas LDN-193189 or ALK3-Fc inhibited apolipoprotein B100 secretion in HepG2 cells, suggesting that BMP signaling contributes to the regulation of cholesterol biosynthesis. CONCLUSION: These results definitively implicate BMP signaling in atherosclerosis and calcification, while uncovering a previously unidentified role for BMP signaling in LDL cholesterol metabolism. BMP inhibition may be helpful in the treatment of atherosclerosis and associated vascular calcification.