Leptin enhances the recruitment of endothelial progenitor cells into neointimal lesions after vascular injury by promoting integrin-mediated adhesion.

The adipocytokine leptin modulates vascular remodeling and neointima formation. Because endothelial progenitor cells (EPCs) participate in vascular repair, we analyzed the effects of leptin on human EPC function in vitro and in vivo. After 7 days in culture, EPCs expressed the leptin receptor and responded to leptin stimulation with increased STAT3 phosphorylation. Incubation of EPCs with leptin (at concentrations between 1 and 100 ng/mL) increased the number of EPCs adhering to vitronectin and fibronectin in a receptor-specific manner. It also enhanced the capacity of EPCs to incorporate into a monolayer of human endothelial cells and the adherence of these cells to activated platelets. Leptin upregulated alphavbeta5 and alpha4 integrin expression in EPCs, and the effects of leptin on EPC function could be prevented, at least in part, by RGD peptides and function-blocking antibodies. Intravenous injection of fluorescently labeled human EPCs into athymic nude mice shortly after vascular injury revealed that preincubation of EPCs with leptin augmented their accumulation within intimal lesions, accelerating reendothelialization and decreasing neointima formation in an alphavbeta5 and alpha4 integrin-dependent manner. Our findings suggest that leptin specifically modulates the adhesive properties and the homing potential of EPCs and may thus enhance their capacity to promote vascular regeneration in vivo.

Potassium channels as tumour markers.

An increasing number of ion channels are being found to be causally involved in diseases, giving rise to the new field of "channelopathies". Cancer is no exception, and several ion channels have been linked to tumour progression. Among them is the potassium channel EAG (Ether-a-go-go). Over 75% of tumours have been tested positive using a monoclonal antibody specific for EAG, while inhibition of this channel decreased the proliferation of EAG expressing cells. The inhibition of EAG is accomplished using RNA interference, functional anti-EAG1 antibodies, or (unspecific) EAG channel blockers. Fluorescently labelled recombinant Fab fragments recognizing EAG allow the distribution of EAG to be visualized in an in vivo mouse tumour model.

Hybrid system for simultaneous fluorescence and x-ray computed tomography.

A hybrid imaging system for simultaneous fluorescence tomography and X-ray computed tomography (XCT) of small animals has been developed and presented. The system capitalizes on the imaging power of a 360 ( degrees )-projection free-space fluorescence tomography system, implemented within a microcomputed tomography scanner. Image acquisition is based on techniques that automatically adjust a series of imaging parameters to offer a high dynamic range dataset. Image segmentation further allows the incorporation of structural priors in the optical reconstruction problem to improve the imaging performance. The functional system characteristics are showcased, and images from a brain imaging study are shown, which are reconstructed using XCT-derived priors into the optical forward problem.

Fast automatic segmentation of anatomical structures in x-ray computed tomography images to improve fluorescence molecular tomography reconstruction.

The recent development of hybrid imaging scanners that integrate fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) allows the utilization of x-ray information as image priors for improving optical tomography reconstruction. To fully capitalize on this capacity, we consider a framework for the automatic and fast detection of different anatomic structures in murine XCT images. To accurately differentiate between different structures such as bone, lung, and heart, a combination of image processing steps including thresholding, seed growing, and signal detection are found to offer optimal segmentation performance. The algorithm and its utilization in an inverse FMT scheme that uses priors is demonstrated on mouse images.

Semiautomatic landmark-based two-dimensional-three-dimensional image fusion in living mice: correlation of near-infrared fluorescence imaging of Cy5.5-labeled antibodies with flat-panel volume computed tomography.

Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice. Here, we present a semiautomated approach to correlate two-dimensional (2D) noninvasive near-infrared fluorescence (NIRF) imaging with three-dimensional (3D), high-resolution, flat-panel volume computed tomography (fpVCT). We developed an algorithm to colocalize fluorescence signals of NIRF-labeled antibodies directed against matriptase and urokinase plasminogen activator receptor (uPAR) to orthotopic carcinomas in mice visualized by fpVCT. For this purpose, mice were anesthetized and fixed on a multimodality animal bed containing fiducial markers filled with iodine-containing contrast agent and fluorescent dye. After intravenous administration of contrast agent and Cy5.5-labeled antibodies, NIRF and fpVCT images were obtained, without repositioning the mice. Binding of Cy5.5-labeled matriptase-specific antibody to pancreatic tumors and Cy5.5-labeled uPAR-specific antibody to mammary carcinomas was assessed by time-domain NIRF imaging measuring the location of fluorescence intensity and its lifetime. In summary, we developed a novel 2D-3D registration technique for image fusion with NIRF imaging and fpVCT to provide complementary information in tumor models on the in vivo association of functional information with anatomic structures. The combination of fpVCT with NIRF imaging will now allow targeted and effective monitoring of preclinical tumor therapies.

Morphologic changes of mammary carcinomas in mice over time as monitored by flat-panel detector volume computed tomography.

Noninvasive methods are strongly needed to detect and quantify not only tumor growth in murine tumor models but also the development of vascularization and necrosis within tumors. This study investigates the use of a new imaging technique, flat-panel detector volume computed tomography (fpVCT), to monitor in vivo tumor progression and structural changes within tumors of two murine carcinoma models. After tumor cell inoculation, single fpVCT scans of the entire mice were performed at different time points. The acquired isotropic, high-resolution volume data sets enable an accurate real-time assessment and precise measurements of tumor volumes. Spreading of contrast agent-containing blood vessels around and within the tumors was clearly visible over time. Furthermore, fpVCT permits the identification of differences in the uptake of contrast media within tumors, thus delineating necrosis, tumor tissues, and blood vessels. Classification of tumor tissues based on the decomposition of the underlying mixture distribution of tissue-related Hounsfield units allowed the quantitative acquisition of necrotic tissues at each time point. Morphologic alterations of the tumor depicted by fpVCT were confirmed by histopathologic examination. Concluding, our data show that fpVCT may be highly suitable for the noninvasive evaluation of tumor responses to anticancer therapies during the course of the disease.

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands.

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.

Flat-panel detector-based volume computed tomography: a novel 3D imaging technique to monitor osteolytic bone lesions in a mouse tumor metastasis model.

Skeletal metastasis is an important cause of mortality in patients with breast cancer. Hence, animal models, in combination with various imaging techniques, are in high demand for preclinical assessment of novel therapies. We evaluated the applicability of flat-panel volume computed tomography (fpVCT) to noninvasive detection of osteolytic bone metastases that develop in severe immunodeficient mice after intracardial injection of MDA-MB-231 breast cancer cells. A single fpVCT scan at 200-microm isotropic resolution was employed to detect osteolysis within the entire skeleton. Osteolytic lesions identified by fpVCT correlated with Faxitron X-ray analysis and were subsequently confirmed by histopathological examination. Isotropic three-dimensional image data sets obtained by fpVCT were the basis for the precise visualization of the extent of the lesion within the cortical bone and for the measurement of bone loss. Furthermore, fpVCT imaging allows continuous monitoring of growth kinetics for each metastatic site and visualization of lesions in more complex regions of the skeleton, such as the skull. Our findings suggest that fpVCT is a powerful tool that can be used to monitor the occurrence and progression of osteolytic lesions in vivo and can be further developed to monitor responses to antimetastatic therapies over the course of the disease.