Neurotoxic effects of dexmedetomidine in fetal cynomolgus monkey brains.

The neuroprotective effects of dexmedetomidine have been reported by many investigators; however its underlying mechanism to reduce neuronal injury during a prolonged anesthesia remains unclear. In this study, we investigated the neurotoxic effects of dexmedetomidine in fetal monkey brains. In the present study, we compare the neurotoxic effects of dexmedetomidine and ketamine, a general anesthetic with a different mechanism of action, in fetal cynomolgus monkeys. Twenty pregnant monkeys at approximate gestation day 120 were divided into 4 groups: non-treatment controls (Group 1); ketamine at 20 mg/kg intramuscularly followed by a 12-hr infusion at 20-50 mg/kg/hr (Group 2); dexmedetomidine at 3 µg/kg intravenously (i.v.) over 10 min followed by a 12-hr infusion at the human equivalent dose (HED) of 3 µg/kg/hr (Group 3); and dexmedetomidine at 30 µg/kg i.v. over 10 min followed by a 12-hr infusion at 30 µg/kg/hr, 10 times HED (Group 4). Blood samples from both dams and fetuses were measured for concentration of dexmedetomidine. Each fetus was perfusion-fixed, serial sections were cut through the frontal cortex, and stained to detect for apoptosis (activated caspase 3 and TUNEL) and neurodegeneration (silver stain). In utero treatment with ketamine resulted in marked apoptosis and degeneration primarily in layers I and II of the frontal cortex. In contrast, fetal brains from animals treated with dexmedetomidine showed none to minimal neuroapoptotic or neurodegenerative lesions at both low- and high-dose treatments. Plasma levels confirmed systemic exposure of dexmedetomidine in both dams and fetuses. In conclusion, these results demonstrate that dexmedetomidine at both low-dose (HED) and high-dose (10 times HED) does not induce apoptosis in the frontal cortex (layers I, II, and III) of developing brain of cynomolgus monkeys.

Quantitative radioimmunoPET imaging of EphA2 in tumor-bearing mice.

PURPOSE: EphA2 receptor tyrosine kinase is significantly overexpressed in a wide variety of cancer types. High EphA2 expression has been correlated with increased metastatic potential and poor patient survival. Although many recent reports have focused on blocking the EphA2 signaling pathway in cancer, the in vivo imaging of EphA2 has not yet been investigated. METHODS: We labeled 1C1, a humanized monoclonal antibody against both human and murine EphA2, with (64)Cu through the chelating agent 1,4,7,10-tetraazacyclododecane N,N',N'',N'''-tetraacetic acid (DOTA) and carried out positron emission tomography (PET) imaging of eight tumor models with different EphA2 expression levels. Western blotting of tumor tissue lysate was performed to correlate the EphA2 expression level with (64)Cu-DOTA-1C1 uptake in the tumors. Immunofluorescence staining and biodistribution studies were also carried out to validate the in vivo results. RESULTS: The radiolabeling yield was 88.9 +/- 9.5% (n = 7) and the specific activity of (64)Cu-DOTA-1C1 was 1.32 +/- 0.14 GBq/mg of 1C1 mAb. The antibody retained antigen-binding affinity/specificity after DOTA conjugation as measured by FACS analysis. The uptake of (64)Cu-DOTA-1C1 in CT-26 tumors was as high as 25.1 +/- 2.5 %ID/g (n = 3) at 18 h post injection. (64)Cu-DOTA-IgG, an isotype-matched control, exhibited minimal non-specific uptake in all eight tumor models. In vivo EphA2 specificity of (64)Cu-DOTA-1C1 was confirmed by successful blocking of CT-26 tumor uptake by unlabeled 1C1. Most importantly, the tumor uptake value obtained from PET imaging had excellent linear correlation with the relative tumor tissue EphA2 expression level measured by Western blot, where r (2) equals 0.90 and 0.92 at 18 h and 42 h post injection, respectively. CONCLUSION: The tumor uptake of (64)Cu-DOTA-1C1 measured by microPET imaging reflects tumor EphA2 expression level in vivo. This is, to our knowledge, the first report of quantitative radioimmunoPET imaging of EphA2 in living subjects. Future clinical investigation of (64)Cu-DOTA-1C1 is warranted.

Identification and characterization of cells with high angiogenic potential and transitional phenotype in calcific aortic valve.

Recent data suggest that angiogenesis plays an important role in the pathogenesis of valvular disease. However, the cellular mechanisms underlying this process remain unknown. This study aimed at identifying and characterizing the cellular components responsible for pathological neovascularization in calcific aortic valves (CAV). Immunohistochemical analysis of uncultured CAV tissues revealed that smooth muscle alpha-actin (alpha-SMA)-positive cells, which coexpressed Tie-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), can be identified prior to the initiation of capillary-like tube formation. In a second step, leaflets of CAV and non-calcific aortic valves (NCAV) were cultured and the cells involved in capillary-like tube formation were isolated. The majority of these cells displayed the same phenotype as non-cultured cells identified in CAV tissues, i.e., expression of alpha-SMA, Tie-2, and VEGFR-2. In comparison to cells isolated from cultures of NCAV leaflets, these cells showed enhanced angiogenic activity as demonstrated by migration and tube assays. The coexpression of VEGFR-2 and Tie-2 together with alpha-SMA suggests both endothelial and mesenchymal properties of the angiogenically activated cells involved in valvular neovascularization. Hence, our findings might provide new insights into the process of pathological angiogenesis in cardiac valves.

Angiogenic activation of valvular endothelial cells in aortic valve stenosis.

Here, we demonstrate the angiogenic response of valvular endothelial cells to aortic valve (AV) stenosis using a new ex vivo model of aortic leaflets. Histological analysis revealed neovascularization within the cusps of stenotic but not of non-stenotic aortic valves. Correspondingly, the number of capillary-like outgrowth in 3D collagen gel was significantly higher in stenotic than in non-stenotic valves. Capillary-like sprouting was developed significantly faster in stenotic than in non-stenotic valves. New capillary sprouts from stenotic aortic valves exhibited the endothelial cell markers CD31, CD34 and von-Willebrand factor (vWF) as well as carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), Tie-2 and angiogenesis inhibitor endostatin. Western blot analyses revealed a significant increase of CEACAM1 and endostatin in stenotic aortic valve tissue. Electron microscopic examinations demonstrate that these capillary-like tubes are formed by endothelial cells containing Weibel-Palade bodies. Remarkably, inter-endothelial junctions are established and basement membrane material is partially deposited on the basal side of the endothelial tubes. Our data demonstrate the capillary-like sprout formation from aortic valves and suggest a role of angiogenesis in the pathogenesis of aortic valve stenosis. These data provide new insights into the mechanisms of valvular disorders and open new perspectives for prevention and early treatment of calcified aortic stenosis.

CEACAM1 enhances invasion and migration of melanocytic and melanoma cells.

Expression of the cell adhesion molecule CEACAM1 in melanomas is an independent factor for the risk of metastasis with a predictive value superior to that of tumor thickness. We have previously shown that CEACAM1 co-localizes at the tumor-stroma interface of invading melanoma masses with integrin beta(3) and that these two adhesion molecules interact via the CEACAM1 cytoplasmic domain. To address the functional consequences of CEACAM1 expression, we investigated invasion and migration of melanocytic and melanoma cells that stably express CEACAM1 using two different in vitro systems. Here, we demonstrate that CEACAM1 expression markedly enhances cell invasion and migration. The enhanced invasion and migration of CEACAM1-transfected cells was dependent on the presence of Tyr-488 within the full-length cytoplasmic CEACAM1 domain. Treatment with anti-CEACAM monoclonal antibodies blocked CEACAM1-enhanced cell invasion and cell migration in a dose-dependent manner. Furthermore, the enhanced invasion and migration of CEACAM1-transfected melanoma cells was blocked by integrin-antagonizing RGD peptides. Expression of integrin beta(3) induces the up-regulation of CEACAM1 in melanocytic MEL6 cells. These results strengthen the view that CEACAM1 and alpha(v)beta(3) integrin are functionally interconnected with respect to the invasive growth of melanomas.